Res Report 2.Teil App - leibniz-fmp.de · duktion/Molekularer Genetik, Strukturbiologie und Chemi-scher Biologie geführt hat. Kennzeichnend für die wissenschaftliche Arbeit am FMP

RESEARCH REPORT2003 I 2004

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FORSCHUNGSINSTITUT

FÜR MOLEKULARE PHARMAKOLOGIE

IM FORSCHUNGSVERBUND BERLIN E.V.

FORSCHUNGSINSTITUT

FÜR MOLEKULARE PHARMAKOLOGIE

IM FORSCHUNGSVERBUND BERLIN E.V.

FORSCHUNGSINSTITUT FÜR

MOLEKULARE PHARMAKOLOGIE

Campus Berlin-Buch

Robert-Rössle-Str. 10

D-13125 Berlin

fon: +49-(0)30-94793-100

fax: +49-(0)30-94793-109

e-mail: [email protected]

Outstation:

Department of Molecular Genetics

FEM Steglitz

Krahmerstr. 6-10

D-12207 Berlin

fon: +49-(0)30-8437-1910

fax: +49-(0)30-8437-1922

The Forschungsinstitut für Molekulare

Pharmakologie (FMP) evolved out of the

Institut für Wirkstoffforschung (IWF) of the

Academy of Sciences of the GDR. Together

with seven other research institutions, the

institute is administratively organized

within the Forschungsverbund Berlin e.V.

The FMP is a member of the Leibniz

Association.

Das Forschungsinstitut für Molekulare

Pharmakologie ist aus dem Institut für

Wirkstoffforschung (IWF) der Akademie

der Wissenschaften der DDR hervor-

gegangen. Es ist mit sieben weiteren

Forschungsinstituten im Forschungs-

verbund Berlin e.V. zusammengeschlossen.

Das FMP ist ein Mitglied der

Leibniz-Gemeinschaft.

RESEARCH REPORT 2003 / 2004

Editorial Board:

Walter Rosenthal, Michael Bienert,

Ivan Horak, Hartmut Oschkinat

Compilation & Editing:

Björn Maul

Cover Design and Layout:

Hoch3 GmbH, Berlin

Photos:

Bernhard Schurian, Thomas Oberländer,

Björn Maul, Dana Hausbeck

FMP

Print:

Druckhaus Berlin-Mitte

This Research Report is also available at

www.fmp-berlin.de

Cover: The G-Protein coupled receptor

GPR 109: binding site with ligand

(nicotinic acid)

SCIENTIFIC ADVISORY BOARDWISSENSCHAFTLICHER BEIRAT

Professor Dr. Rudolf BallingGBF - Gesellschaft für Biotechnologische Forschung mbHMascheroder Weg 138124 Braunschweig

Professor Dr. Annette G. Beck-SickingerInstitut für Biochemie der Universität LeipzigBrüderstr. 3404103 Leipzig

Professor Dr. Matthias BräutigamSchering AGMüllerstr. 170-17813342 Berlin

Professor Dr. Christian GriesingerMax-Planck-Institut für Biophysikalische ChemieAm Faßberg 1137077 Göttingen

Professor Dr. Reinhard Jahn (Vorsitz)Max-Planck-Institut für Biophysikalische ChemieAm Faßberg 1137077 Göttingen

Professor Dr. Hans-Georg JoostDeutsches Institut für ErnährungsforschungArthur-Scheunert-Allee 11414558 Potsdam-Rehbrücke

Professor Dr. Frauke MelchiorUniversitätsklinikum der Georg-August-UniversitätGöttingenZentrum für Biochemie und Molekulare ZellbiologieHumboldt-Allee 2337073 Göttingen

Professor Dr. Herbert WaldmannMax-Planck-Institut für Molekulare PhysiologieOtto-Hahn Str. 1144202 Dortmund

The Scientific Advisory Board of the FMP formally consti-tuted itself at its meeting on November 6, 1992. At theboard meeting on November 16, 2000 Professor ReinhardJahn was elected chairman.

Der Wissenschaftliche Beirat des FMP konstituierte sichanläßlich seiner Sitzung am 6. November 1992. Auf der Sit-zung am 16. November 2000 wurde Professor Dr. ReinhardJahn zum Vorsitzenden gewählt.

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The FMP undertakes research in the field of molecularpharmacology. In accordance with this objective, the FMPstrives to make contributions of fundamental significance.In this context, it is engaged in elucidating the structure,functions and interactions of proteins and in developingnew concepts relating to their pharmacological impact.Thus, the research activity of the FMP is at the forefront ofdrug development. The FMP pursues an interdisciplinaryresearch approach, which has brought together cellularsignal transduction/molecular genetics, structural biolo-gy and chemical biology. Characteristic for the scientificwork at the FMP is the close interrelationship of chemi-stry and biology.

MILESTONES 2003/2004

The years 2003/4 were characterized by the expansion ofthe chemistry department. With the appointment of JörgRademann as Professor for Medicinal Chemistry (jointappointment by the FMP and the Free University Berlin), anew important area of competence, combinatorial chemi-

stry, was established alongside peptide chemistry. At thesame time a Screening Unit was built up, making substan-ce libraries and screening technology available. JensPeter von Kries could be recruited to direct it. The FMP isthus the first academic institution in Germany which, usinghigh throughput methods, can identify small moleculesthat bind to specific proteins and develop a biologicaleffect (pages 92-95). These small molecules representimportant tools for research. At the same time they canalso be regarded as prototypes of novel pharmaceuticals.FMP scientists are centrally involved in building up a newnational network (ChemBioNet; www.chembionet.de),through which academic groups can obtain access to sub-stance libraries and screening technology.

With the establishment of the research group Solid StateNMR and the appointment of group leader Bernd Reif asProfessor for Drug Design (joint appointment of the FMPand the Charité – University Medicine Berlin), the staff ofthe NMR Center of the FMP was greatly expanded. An out-standing event was also the first-time operation of the 900MHz spectrometer, which represents the current state-of-

Das FMP betreibt Forschung auf dem Gebiet der Moleku-laren Pharmakologie. Dabei ist es bestrebt, Beiträge vongrundsätzlicher Bedeutung zu erbringen. In diesem Rah-men versucht es, die Struktur, Funktionen und Interaktio-nen von Eiweißen (Proteinen) aufzuklären und neue Kon-zepte zu ihrer pharmakologischen Beeinflussung zuentwickeln. Damit ist die Forschungstätigkeit des FMP imVorfeld der Entwicklung von Arzneimitteln angesiedelt.Das FMP verfolgt einen interdisziplinären Forschungsan-satz, der zur Zusammenführung von Zellulärer Signaltrans-duktion/Molekularer Genetik, Strukturbiologie und Chemi-scher Biologie geführt hat. Kennzeichnend für diewissenschaftliche Arbeit am FMP ist die enge Verknüp-fung von Chemie und Biologie.

MEILENSTEINE 2003/2004

Die Jahre 2003/2004 waren durch den Ausbau der Chemiegeprägt. Durch die Berufung von Jörg Rademann auf eineProfessur für Medizinische Chemie (gemeinsame Beru-fung von FMP und Freier Universität Berlin) wurde nebender Peptidchemie ein neuer Schwerpunkt „Kombina-torische Chemie“ geschaffen. Gleichzeitig wurde eine

Screening Unit aufgebaut, die über Substanzbibliothekenund Screening-Technologie verfügt. Als Leiter konnte Jens Peter von Kries gewonnen werden. Damit ist dasFMP die erste akademische Einrichtung in Deutschland,die kleine Moleküle, die an bestimmte Proteine binden undeine biologische Wirkung entfalten, im Hochdurchsatzver-fahren identifizieren kann. Diese kleinen Moleküle stellenwichtige Werkzeuge für die Forschung dar. Zugleich sindsie aber auch als Prototypen neuartiger Pharmaka zubetrachten. Wissenschaftler des FMP sind auch in zentra-ler Weise am Aufbau eines nationalen Netzwerks (Chem-BioNet; www.chembionet.de) beteiligt, durch das akade-mische Gruppen Zugang zu Substanzbibliotheken undScreening-Technologie erhalten sollen.

Mit der Einrichtung der Arbeitsgruppe Festkörper-NMRund der Berufung eines Leiters, Bernd Reif, auf eine Pro-fessur für Drug Design (gemeinsame Berufung von FMPund Charité – Universitätsmedizin Berlin) wurde das NMR-Zentrum des FMP personell stark erweitert. Ein heraus-ragendes Ereignis war auch die Inbetriebnahme eines900 MHz-Spektrometers, das den aktuellen Stand der Ent-wicklung repräsentiert. Weltweit sind erst wenige Gerätedieses Typs verfügbar. Die erforderlichen Mittel (insge-

FOREWORD

samt 4,9 Mio €) wurden vom BMBF im Rahmen der Pro-jektförderung und der Berliner Senatsverwaltung für Wis-senschaft, Forschung und Kultur (EFRE-Mittel) zur Verfü-gung gestellt. Damit ist ein gerätemäßig und personellhervorragend ausgestattetes, international ausstrahlen-des Zentrum für die strukturbiologisch orientierte NMR-Spektroskopie etabliert.

Im Juni 2003 wurde gemeinsam mit dem Max-Delbrück-Centrum für Molekulare Medizin (MDC) im Beisein desRegierenden Bürgermeisters von Berlin der Grundstein fürden Neubau „Medizinische Genomik“ gelegt. Das mar-kante, von dem Architektenbüro Staab entworfene Gebäu-de bietet 120 Mitarbeitern Labor- und Büroarbeitsplätze.Der Bezug des neuen Gebäudes wird im Herbst 2005 erfol-gen. Von den 230 Mitarbeitern des FMP werden ca. 40 imneuen Gebäude tätig sein.

PERSPEKTIVEN

Arzneimittel entfalten ihre Wirkung durch Interaktion ihrerInhaltsstoffe (Wirkstoffe) mit körpereigenen Strukturen.Bei diesen Andockstellen im Organismus (Zielstrukturen,„targets“) handelt es sich fast ausnahmslos um Proteine(> 99%). Bisher ist erst ein sehr kleiner Teil der Eiweiße desmenschlichen Körpers als Zielstruktur für einen Wirkstofferschlossen (ca. 500). Sicher kommen nicht alle 100.000Eiweiße des Organismus als „targets“ für Wirkstoffe inFrage. Man geht aber davon aus, dass zumindest einigetausend geeignet sind. Damit stellt sich die Aufgabe, diederzeit schmale Basis der Arzneimitteltherapie durchIdentifizierung neuer Zielstrukturen zu erweitern und sobestehende Therapiekonzepte zu verbessern und neue zuetablieren. Hier sieht das FMP ein zentrales Tätigkeitsfeld.Zu den Proteingruppen, die gegenwärtig am FMP bearbei-tet werden, gehören als klassische „targets“ Membran-proteine wie Rezeptoren und Kanalproteine, aber auchProteine, die bisher kaum als „targets“ in Betracht gezo-gen wurden. Zu dieser Gruppe gehören Transkriptions-faktoren, die Gene regulieren, Interaktionsdomänen vonProteinen und sogenannte Ankerproteine, die Proteine in

FOREWORD

the-art development. Worldwide only a few devices of thistype are in existence. The necessary funding (totaling 4.9million €) was provided by the BMBF (Federal Ministry ofEducation and Research) in the framework of a projectgrant and the Berlin Senate Department for Science,Research and Culture (EFRE funds). Thus an outstandinglywell-equipped and well-staffed center for structural bio-logy oriented NMR spectroscopy has been created thathas an international reputation.

In June 2003 at a ceremony which included the governingmayor of Berlin, the cornerstone was laid for the newMedicinal Genomics Building. It is a joint initiative of theFMP and the Max Delbrück Center for Molecular Medicine(MDC). The striking building, designed by the Staab archi-tectural bureau, provides lab and office space for a staff of120. The move into the new building will take place inautumn 2005. Of the 230 employees on the staff of the FMP,about 40 will work in the new building.

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PERSPECTIVES

Drugs achieve their effect through interaction of their con-tents (substances) with the body’s own structures. Atthese docking sites in the organism (targets), this almostwithout exception involves proteins (> 99%). Until now,drugs have been developed for only a small number of theproteins of the human body (approx. 500).

To be sure, not all 100,000 proteins of the organism areappropriate as a target for substances. However, it isassumed that at least several thousand are suitable. The-refore, the task is to expand the present narrow base ofdrug therapy by identifying new target structures and thusto improve existing therapy concepts and establish newones. Here the FMP sees a central field of activity. The pro-tein groups that are currently being processed at the FMPinclude as classical targets membrane proteins such asreceptors and channel proteins, but also proteins that uptill now have hardly been considered targets. To this groupbelong transcription factors, which regulate genes, inter-action domains of proteins and so-called anchor proteins,

which assemble the proteins in the cell to larger functio-nal complexes, as well as extracellular fibrils (which e.g.are increasingly observed with Alzheimer’s disease).

Pharmaceutical companies are withdrawing more andmore out of research preparatory to a “proof of principle“,i.e. from “high-risk” research which precedes drug deve-lopment. Moreover, for a long time it has been observedthat the research departments of pharmaceutical compa-nies have been relocating abroad. Due to the strengthe-ning of chemical research and particularly due to the esta-blishment of the Screening Unit, the FMP has considerablyexpanded its competence within the value creation chainthat leads to new drugs with regard to application, yetwithout relinquishing its firm anchoring in basic research.Now it can combine suggestions for new “targets” withsuggestions for substance candidates and possibly pre-sent a first pharmacological characterization of potential“targets”. We hope that the extension of the platform willhelp close the gap between pharmacological basicresearch in the research institutes and drug developmentin the pharmaceutical industry, and thus that the environ-

der Zelle zu größeren funktionellen Komplexen zusammen-fügen, sowie extrazelluläre Fibrillen. (die z. B. bei der Alz-heimerschen Erkrankung vermehrt auftreten).

Pharmazeutische Firmen ziehen sich immer mehr aus derForschung im Vorfeld eines „proof of principle“, d. h. ausder „high-risk“ Forschung, die der Arzneimittelentwick-lung vorausgeht, zurück. Außerdem ist seit langem eineVerlagerung der Forschungsabteilungen pharmazeu-tischer Firmen ins Ausland zu beobachten. Durch die Stär-kung der chemischen Forschung und besonders durch dieEinrichtung der Screening Unit hat das FMP seine Kompe-tenz innerhalb der Wertschöpfungskette, die zu neuenArzneimitteln führt, erheblich in Richtung Anwendungerweitert, ohne allerdings seine feste Verankerung in derGrundlagenforschung aufzugeben. Es ist kann nun Vor-schläge zu neuen „targets“ mit Vorschlägen zu Wirkstoff-kandidaten verbinden und evtl. auch eine erste pharma-kologische Charakterisierung potentieller „targets“präsentieren. Wir hoffen, dass die Erweiterung der Platt-form dazu beiträgt, die Lücke zwischen pharmako-logischer Grundlagenforschung in den Forschungsein-richtungen und der Arzneimittelentwicklung in derpharmazeutischen Industrie zu schließen und damit das

Umfeld für Unternehmen des Pharma-Branche attraktiverzu gestalten. Diesem Ziel sollen auch die verschiedenenNetzwerkinitiativen dienen, an denen das FMP maßgeb-lich beteiligt ist. Beispiele sind die Etablierung eines Netz-werks „Arzneimittelentwicklung in Berlin und Branden-burg“ und die bereits erwähnte nationale Initiative„ChemBioNet“.

Wie in den vergangenen Jahren stehen Beiträge derArbeitsgruppen über ihre wissenschaftliche Arbeit im Zen-trum dieses Berichtes 2003/2004. Er enthält aber auch dieLeistungsbilanz für den Berichtszeitraum. So wird diePublikationstätigkeit, die Einwerbung von Drittmitteln unddie Beteiligung des FMP an der Ausbildung von Studieren-den verschiedener Disziplinen dargestellt.

Der Erfolg der letzten Jahre ist den Mitarbeiterinnen undMitarbeiter des FMP zuzuschreiben, bei denen ich michherzlich für ihr Engagement bedanken möchte. Mein Dankrichtet sich auch an den wissenschaftlichen Beirat, derdas FMP konstruktiv-kritisch begleitet hat.

Ich wünsche den Lesern einen aufschlussreichen undinteressanten Streifzug durch das FMP, wie es sich 12 Jahre nach seiner Neugründung darstellt.

Walter Rosenthal, DirektorApril 2005

FOREWORD

ment for companies in the pharma sector will becomemore attractive. Various network initiatives in which theFMP plays a major role shall also serve this goal. Exam-ples are the establishment of a network “Drug Develop-ment in Berlin und Brandenburg” and the already mentio-ned national initiative “ChemBioNet”.

As in past years, the contributions of the various researchgroups about their scientific work stand in the center ofthis research report 2003/2004. However, it also containsa performance account for the period of the report. Thus,publication activity, solicitation of third-party funds and theparticipation of the FMP in the education and training ofstudents of the various disciplines are also presented.

The success of the last years is due to the colleagues ofthe FMP, whom I would like to thank very much for theirdedication. My thanks also go to the Scientific AdvisoryBoard for its constructive and critical support of the FMP.

I would like to invite readers to take an informative andinteresting tour through the pages of this report to see howthe FMP has developed since its founding 12 years ago.

Walter Rosenthal, Director

Cont

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CONTENTS / INHALT

Scientific Advisory Board / Wissenschaftlicher Beirat ......................................................................................................... 2Foreword / Vorwort ..................................................................................................................................................................... 3

Section Structural Biology / Bereich Strukturbiologie Introduction ......................................................................................................................................................................... 10Protein Structure ................................................................................................................................................................ 12Solution NMR....................................................................................................................................................................... 16Structural Bioinformatics .................................................................................................................................................. 20Molecular Modelling .......................................................................................................................................................... 24Solid State NMR ................................................................................................................................................................. 28Protein Engineering ............................................................................................................................................................ 31

Section Cellular Signalling / Molecular Genetics / Bereich Signaltransduktion/ Molekulare Genetik

Introduction ......................................................................................................................................................................... 36Protein Trafficking .............................................................................................................................................................. 39Anchored Signalling ........................................................................................................................................................... 42Cellular Imaging .................................................................................................................................................................. 46Molecular Cell Physiology ................................................................................................................................................ 49Biochemical Neurobiology ................................................................................................................................................ 52Biophysics ........................................................................................................................................................................... 56Cytokine Signaling .............................................................................................................................................................. 59Molecular Myelopoiesis .................................................................................................................................................... 62Mouse Models ..................................................................................................................................................................... 64Cellular Signal Processing ................................................................................................................................................ 65

Section Chemical Biology / Bereich Chemische Biologie Introduction ......................................................................................................................................................................... 70Peptide Synthesis ............................................................................................................................................................... 72Peptide Lipid Interaction/Peptide Transport .................................................................................................................. 75Peptide Biochemistry ......................................................................................................................................................... 80Mass Spectrometry ............................................................................................................................................................ 83Synthetic Organic Biochemistry ...................................................................................................................................... 86Medicinal Chemistry .......................................................................................................................................................... 89Screening Unit .................................................................................................................................................................... 92

Scientific and technical services / Wissenschaftlicher und technischer ServiceMicrodialysis ....................................................................................................................................................................... 98DNA Sequencing .............................................................................................................................................................. 101Public Relations and the Media / Presse- und Öffentlichkeitsarbeit ........................................................................ 102Administration / Verwaltung ........................................................................................................................................... 106Computer Services / Computer-Service ........................................................................................................................ 108

Appendix / Anhang

Peer reviewed Articles 2003 / Originalarbeiten ............................................................................................................ 110 Peer reviewed Articles 2004 / Originalarbeiten ............................................................................................................ 114Reviews 2003/2004 / Übersichtsarbeiten ...................................................................................................................... 121Contributions in Monographs 2003/2004 / Beiträge zu Sammelwerken ....................................................................121 Monographs 2003/2004 / Monographien ...................................................................................................................... 122Memberships in Editorial Boards 2003/2004 / Mitgliedschaften in Editorial Boards ..............................................123 Invited Talks 2003/2004 / Eingeladene Vorträge .......................................................................................................... 123External funding 2003/2004 / Drittmittel .......................................................................................................................... 128 Participation in Research Networks 2003/2004 / Beteiligung an Netzwerken und Verbundprojekten ................ 130 Cooperations with contract 2003/2004 / Vertragliche Kooperationen....................................................................... 132 Meetings, Workshops, Symposia 2003/2004 / Wissenschaftliche Veranstaltungen ............................................... 134 Work in Panels 2003/2004 / Gremienarbeit .................................................................................................................... 135 Review Activities 2003/2004 / Gutachtertätigkeit ......................................................................................................... 135 Academic Teaching 2003 / 2004 / Lehre.......................................................................................................................... 138 Calls for Appointments 2003/2004 / Rufe........................................................................................................................ 141 Post-Doctoral Lecture Qualifications 2003/2004 / Habilitationen .............................................................................. 141 Graduations 2003/2004 / Promotionen ........................................................................................................................... 142 Diploma theses 2003/2004 / Diplomarbeiten ................................................................................................................. 143 Internships 2003/2004 / Praktikanten ............................................................................................................................. 144 Guest Scientists 2003/2004 / Gastwissenschaftler ...................................................................................................... 148 Lectures at the FMP 2003 / Kolloquien und Seminare am FMP .................................................................................. 149 Lectures at the FMP 2004 / Kolloquien und Seminare am FMP ................................................................................. 153 Technology Transfer 2003/2004 / Technologietransfer ................................................................................................ 156 Structure of the Forschungsinstitut für Molekulare Pharmakologie (FMP) / Organigramm .................................. 158 Index / Namensregister ................................................................................................................................................... 160 Maps / Lagepläne ............................................................................................................................................................. 164

Flip-book Daumenkino

STRUCTURAL BIOLOGY

INTRODUCTION

SECTION STRUCTURAL BIOLOGYProf. Hartmut Oschkinat,Department Head: NMR-supported Structural Biology (Secretary: Andrea Steuer)

The research themes of the section cover structural andfunctional studies of receptors and proteins involved inintracellular signalling, and projects on the biophysicaldescription of protein aggregate formation. Strong empha-sis is thereby put on a structural characterization of mem-brane-integrated receptors, channels and fibril-formingsystems, in combination with exploring the potential ofsolid-state magic angle spinning NMR for studying suchsystems. As a special theme with regard to intracellularsignalling, non-catalytical protein domains that mediateinteractions with linear peptide segments in target pro-teins are analyzed with respect to specificity and affinity-determining sequence features. A growing part of theresearch is the systematic derivation of small organiccompounds as modulators of protein-protein interactions

and amyloid formation. There are strong links to thescreening facility of the FMP through the molecularmodellling and NMR aspects of small-molecule inhibitordevelopment.

The department hosts expertise in the areas of structuralbioinformatics, molecular biology, solution and solid-stateNMR spectroscopy, and molecular modelling. NMRspectroscopy is used as the major tool for determiningstructures of biological macromolecules in solution, andof quasi-solid preparations which are difficult to crystalli-ze, like amyloid fibrils and membrane proteins in native-like lipids. It is particularly well suited for studying thedynamics of protein structures and interactions with veryweakly binding ligands. The various skills are contained in

BEREICH STRUKTURBIOLOGIE Prof. Hartmut Oschkinat,Abteilungsleiter: NMR-unterstützte Strukturforschung (Sekretariat: Andrea Steuer)

Der Bereich Strukturbiologie erforscht Struktur und Funk-tion von Rezeptoren und Proteinen, die an der intrazel-lulären Signalübertragung beteiligt sind, sowie die Ent-stehung von Proteinaggregaten. Im Mittelpunkt desInteresses steht die strukturelle Charakterisierung vonRezeptoren, Kanälen und fibrillenbildenen Systemen inKombination mit der Erforschung des Potentials der„magic-angle-spinning“ Festphasen-NMR für das Studiumsolcher Systeme. Im Hinblick auf die intrazelluläre Signal-übertragung wird besonderes Augenmerk auf die spezifi-täts- und affinitätsbestimmenden Eigenschaften nicht-katalytischer Proteindomänen gelegt. Diese vermitteln dieInteraktionen mit linearen Peptidsegmenten in Zielprotei-nen. Zunehmend rückt die systematische Derivatisierungkleiner organischer Moleküle als Modulatoren von Pro-tein-Protein-Interaktionen und der Amyloid-Bildung in denMittelpunkt des Interesses. Wichtige inhaltliche Brückezum Screening-Labor des FMP sind die Molekülmodel-

lierung und NMR-Aspekte der Entwicklung niedermoleku-larer Hemmstoffe.

Der Bereich kann Expertise auf den Gebieten strukturelleBioinformatik, Molekularbiologie, Flüssigkeits- und Fest-körper-NMR-Spektroskopie sowie Molekülmodellierungvorweisen. Hauptsächlich mittels NMR-Spektroskopiewerden Strukturen biologischer Makromoleküle in Lösungund in Präparationen an der Festkörperphase bestimmt(Amyloidfibrillen und Membranproteine in natürlichenLipiden). NMR-Spektroskopie ist besonders geeignet fürStudien zur Dynamik von Proteinen und Interaktionen mitsehr schwach bindenden Liganden. Gegenwärtig sechsArbeitsgruppen bieten wissenschaftliche Kompetenz inProtein Engineering (Christian Freund), Molekülmodellie-rung (Ronald Kühne), Struktureller Bioinformatik (GerdKrause), Proteinstruktur (Hartmut Oschkinat), Festkörper-NMR (Bernd Reif) und Lösungs-NMR (Peter Schmieder).

currently six research groups, Protein Engineering(Christian Freund), Molecular Modelling (Ronald Kühne),Structural Bioinformatics (Gerd Krause), Protein Structure(Hartmut Oschkinat), Solid State NMR (Bernd Reif) andSolution NMR (Peter Schmieder). The instrumentationincludes a variety of solution and solid-state NMR spectro-meters ranging from 400 to 900 MHz, and equipment foranalytical ultracentrifugation and isothermal calorimetry.In the past two years, the direction of research has beenshifted stronger towards solid-state NMR and its applica-tions, resulting in the installation of a new group focusedon solid-state NMR and amyloid-forming systems. Further-more, a 700 MHz wide bore NMR spectrometer was pur-chased.

Der Bereich ist mit einer Vielzahl von Lösungs- und Fest-körper-NMR-Spektrometern (400 bis 900 MHz) sowie Gerä-ten zur analytischen Ultrazentrifugation und isothermi-schen Kalorimetrie ausgestattet. In den zurückliegendenzwei Jahren hat sich der Fokus der Forschung stärker inRichtung Festkörper-NMR und deren Anwendungen ver-schoben. Dies führte zur Ansiedlung einer neuen Arbeits-gruppe, die sich mit Festkörper-NMR und Amyloid-bilden-den Systemen befasst. Außerdem wurde ein 700MHz-„wide bore“-NMR-Spektrometer in Betrieb genom-men.

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PROTEIN STRUCTUREGroup Leader: Prof. Hartmut Oschkinat

STRUCTURAL CHARACTERIZATION OF PROTEIN-PROTEIN-INTERACTIONS

The main focus of our group is the structural characteri-zation of protein-protein interactions responsible for thereception and transduction of signals in biologicalsystems. This research concerns protein domains whichrecognise specific peptides and on the long run investiga-tions on membrane integrated receptors and receptor-ligand complexes. It is embedded into efforts towardsstructural genomics of soluble and membrane proteinsand supported by NMR-technical developments. Thetechnical developments aim at higher throughput andtowards concepts for structure determination of membra-ne proteins by solid-state NMR.

Structure and function of non-catalytic proteindomains

Non-catalytic protein domains mediate protein-proteininteractions through the recognition of peptide segmentsin a highly specific manner. Such interactions govern thereversible assembly of macromolecular signalling comple-xes which finally form a logical network of interacting pro-teins, thereby transmitting information. Our investigationson non-catalytic protein domains typically involve a com-bination of structural and functional studies, e.g. by usingpeptide libraries, NMR spectroscopy and a variety of otherbiophysical techniques, aiming at an understanding ofmolecular recognition and drug design.

We have determined the structures of a number of WWdomain-peptide complexes, and the structure of an SH3domain in complex with a canonical and a non-canonicalpeptide revealing two different binding sites. Intense stu-dies with PDZ and WW domains involving NMR and pep-tide library screens where set out to derive structure-acti-vity-relationships and to provide data for a theoreticalanalysis of domain specificity. We quantitatively determin-ed the specificity profiles of three representative PDZdomains from the AF6, ERBIN and SNA1 proteins, withrespect to the complete ligand sequence space of C-termi-nal peptides. This quantification was achieved by combi-ning efficiently dissociation constant measurements andstatistical analysis of synthetic peptide libraries, using anAnalysis of Variance (ANOVA) approach. Predicted in vitroaffinities were validated by designing super-binding pep-tides which indeed revealed the lowest Kd values and thehighest PDZ domain specificity. The coverage of the com-

plete ligand sequence space for the three PDZ domainsmade it feasible to compare their ligand selectivity and theoverlap of their recognized ligand sequence subspaces.

NMR structure determination in a structuralgenomics context

As a part of the protein structure factory project, a num-ber of protein structures were generated, including thoseof the subunit B8 of complex I, of the p47 SEP domain, andof a BRCT domain.

The solution structure of the subunit B8 from ubiquinoneoxidoreductase (Complex I) (CI-B8) shows a thioredoxinfold (Fig. 1) with remarkable similarities to thioredoxin-likeFe2S2-ferredoxins. A detailed comparison to these pro-teins show remarkably high similarities of surface proper-ties and possibly catalytically active residues. The redox-potential of the disulfide-bond is comparable to that of thecatalytically active disulfides in thioredoxin-like proteins.This led to the hypothesis that this subunit may be invol-ved in the regulation of complex I.

The BRCT domain occurs in a number of different signaltransduction proteins involved in transcriptional regula-tion, DNA repair, cell cycle progression and cancer sup-pression. The C-terminal region of the breast cancer asso-ciated tumor suppressor protein BRCA1 consists of twosuccessive BRCT domains, separated by a short linker. Wehave, in collaboration with the group of Udo Heinemann atthe MDC, Berlin-Buch, solved the solution structure ofBRCT-c, the C-terminal BRCT domain of BRCA1. Analysisof the structure and comparison with other members of theBRCT superfamily has revealed that only a small number ofconserved residues are necessary for the formation andstabilization of the BRCT motif.

The structure of the SEP domain of the protein p47 wasdetermined and a hypothesis as to its function derived andverified. A particular feature of this fold is the conserva-tion of residues in two loops, and of aromatic residues veryclose to it (Fig. 2). These loops are remarkably rigid, andthe upper part of the structure in Fig. 2 resembles cystei-ne protease inhibitors like cystatins and stefins. An assayshowed a weak inhibition of cathepsin L by the p47 SEPdomain.

Toward structure determination of membraneproteins by NMR

Major forces were concentrated on the further develop-ment of a structure determination concept for proteinsusing magic-angle spinning solid-state NMR. Such amethod would be very welcome for systems which are

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difficult to crystallize and out of range for solution NMR,like membrane proteins, amyloids, and structure formingproteins like actin filaments, for example. Along withvarious membrane proteins, a microcrystalline sample ofthe α-spectrin SH3 domain is studied as a model systemwhich yields suitable NMR spectra for structure deter-mination. At the beginning of the study resonanceassignments for all 13C and 15N resonances were achie-ved, followed by an assignment of non-exchangeable, andlater also of most exchangeable protons. Based on theachieved assignments a first structure of a protein wasdetermined. A key aspect of this study was the generationof biosynthetically site directed labelled samples by usingeither 1,3-13C-glycerol or 2-13C-glycerol as carbon sources.The respective proton driven spin diffusion spectra recei-ved from those samples showed a dramatically increasednumber of long-range restrains that were used for struc-ture calculations. We have now refined the original struc-ture of the α-spectrin SH3 domain using 889 interresiduecarbon-carbon and 6 15N-15N restraints, all obtained bysolid-state MAS NMR. The derived procedure should bewidely applicable to small membrane proteins and amy-loid systems if the proteins can be expressed in bacteria.First steps towards membrane proteins were taken using

the snake toxin neurotoxin II bound to the nicotinic acetylcholine receptor as a test system. The spectra showremarkably sharp lines, and an evaluation of the spectrais under way.

Group members

Dr. Linda Ball Dr. Anne Diehl Dr. Katja Fälber Dr. Jeremy Flinders Dr. Ludwig Krabben Dr. Dirk LabuddeDr. Dietmar LeitnerDr. Ricardo PiresDr. Barth van Rossum Prisca Boisguerin (Doctoral student)Christoph Brockmann (Doctoral student)Federica Castellani (Doctoral student)Michele Fossi (Doctoral student)Matthias Hiller (Doctoral student)Henrik Holtmann (Doctoral student)Mangesh Joshi (Doctoral student)Christian Köhler (Doctoral student)

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FIGURE 1Ribbon diagram of subunit B8 (left) from ubiquinone oxidoreductase (Complex I, right)

Thien-Thi Mac (Doctoral student)Vivien Lange (Doctoral student)Doreen Pahlke (Doctoral student)Ilja Poliakov (Doctoral student)Nikolaj Schröder (Doctoral student)Michael Soukenik (Doctoral student) Carolyn Vargas (Doctoral student)Urs Wiedemann (Doctoral student)Stefan Jehle (Student)Heide Evers (Technical assistance)Lilo Handel (Technical assistance)Martina Leidert (Technical assistance)Kristina Rehbein (Technical assistance)

External funding

Deutsche Forschungsgemeinschaft„Struktur und Mechanismus der 3,4-Dihydroxy-2-butanon-4-phosphat-Synthase“ (OS 106/ 4-2) Hartmut Oschkinat

Deutsche Forschungsgemeinschaft„Analyse von essentiellen WW-Domänen in ß-sheet-Strukturen am Beispiel der WW-Domäne mittes Einbausnicht natürlicher Aminosäuren“ (OS 106/5-1,5-2) Hartmut Oschkinat

Deutsche Forschungsgemeinschaft „Schlüsselreaktionen der biologischen Wasserstoffakti-vierung am Beispiel der [NiFe]-Hydrogenasen“ (TeilprojektC1 im Sonderforschungsbereich 498 “Protein-Kofaktor-Wechselwirkungen in biologischen Prozessen“)Hartmut Oschkinat, Bärbel Friedrich (Humboldt-Universi-tät zu Berlin)

Deutsche Forschungsgemeinschaft„Bestimmung der Raumstrukturen von Rezeptor-gebunde-nen Agonisten und Antagonisten mittels Festkörper-NMR-Spektroskopie“ (Teilprojekt B1 im Sonderforschungsbe-reich 449 „Struktur und Funktion membranständigerRezeptoren“)Hartmut Oschkinat

Deutsche Forschungsgemeinschaft„Theoriegestützte NMR-spektroskopische Analyse vonProtein-Ligand-Wechselwirkungen unter Verwendung vonPeptidbibliotheken“ (Teilprojekt in der Forschergruppe 299„Optimierte molekulare Bibliotheken zum Studium biologi-scher Erkennungsprozesse“)Hartmut Oschkinat, Michael Bienert

FIGURE 2Structural ensemble of the p47 SEP domain. The residues with side chains displayed in black and green are highly conser-ved.

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Deutsche Forschungsgemeinschaft„Analyse von essentiellen Wechselwirkungen in ß-sheet-Strukturen am Beispiel der WW-Domäne mittels Einbaunicht natürlicher Aminosäuren“ (Teilprojekt in der For-schergruppe 475 “Bildung und Stabilität von β-Faltblättern)Hartmut Oschkinat

Bundesministerium für Bildung und Forschung „Strukturanalyse mit hohem Durchsatz für medizinischrelevante Proteine“ (01GG 9812, BMBF-Leitprojekt Protein-strukturfabrik)Hartmut Oschkinat

Bundesministerium für Bildung und Forschung„Aufbau einer Technologieplattform NMR-Messtechnikfür die Proteomforschung“ (0312832)Hartmut Oschkinat

Bundesministerium für Bildung und Forschung “Festkörper-NMR-Spektroskopie“ (0312890G, Teilprojektim Verbund „Proteomweite Analyse membrangebundenerProteine – ProAMP“)Hartmut Oschkinat

Bundesministerium für Bildung und Forschung „Screening von Substanzbibliotheken und Struktur-basier-ten Wirkstoffdesign“ (0312992J, Teilprojekt im Verbundvor-haben „Strukturproteomik“ – Konsortium Hamburg:„Hochdurchsatz-Strukturanalyse von Mycobacterium-tuberculosis-Zielproteinen und ihrer Ligandenkomplexezur Suche nach Wirkstoffen“Hartmut Oschkinat, Jens Peter von Kries

European Community“Exploiting synthetics SH2-scaffolded repertoire librariesto profile cancer cells and to interfere with cancer-relatedphenotypes” (QLK3-2000-00924)Hartmut Oschkinat

European Community„Interactions between growth factors and glycosamino-glycans in arterial disease” (BMH4-98-3289)Hartmut Oschkinat

Selected publications (FMP authors in bold)

Otte L, Wiedemann U, Schlegel B, Pires JR, BeyermannM, Schmieder P, Krause G, Volkmer-Engert R, Schneider-Mergener J, Oschkinat H (2003) WW domain sequenceactivity relationships identified using ligand recognitionpropensities of 42 WW domains. Prot Sci 12, 491-500

Kahmann JD, Wecking DA, Putter V, Lowenhaupt K, KimYG, Schmieder P, Oschkinat H, Rich A, Schade M (2004)The solution structure of the N-terminal domain of E3Lshows a tyrosine conformation that may explain its redu-

ced affinity to Z-DNA in vitro. Proc Natl Acad Sci USA 101,2712-2717

Brockmann C, Diehl A, Rehbein K, Strauss H, SchmiederP, Korn B, Kühne R, Oschkinat H (2004) The oxidized sub-unit B8 from human complex I adopts a thioredoxin fold.Structure 12, 1645-1654

Gaiser OJ, Ball LJ, Schmieder P, Leitner D, Strauss H,Wahl M, Kühne R, Oschkinat H, Heinemann U (2004) Solu-tion Structure, Backbone Dynamics, and AssociationBehavior of the C-Terminal BRCT Domain from the BreastCancer-Associated Protein BRCA1. Biochemistry 43,15983-15995

Collaborations

Adelbert Bacher, GarchingAlan Fersht, FRS, Cambridge, UKPaul Gooley, MelbourneRobert G. Griffin, Cambridge, USA Udo Heinemann, BerlinKlaus-Peter Hofmann, BerlinDieter Oesterhelt, MartinsriedMichael Nilges, ParisJens Schneider Mergener, BerlinRudolf Volkmer-Engert, Berlin

SOLUTION NMRGroup Leader: Dr. Peter Schmieder

APPLICATIONS OF SOLUTION STATE NMRTO ADDRESS BIOLOGICALLY IMPORTANTQUESTIONSPeter Schmieder, Holger Strauss, Christian Appelt, JaninaHahn, Brigitte Schlegel

NMR spectroscopy is an important technique to addressnumerous questions regarding the structure of variouskinds of molecules, including their three-dimensionalstructure. Solution-state NMR is well established as a toolto obtain the three-dimensional structure at atomic reso-lution for soluble biomolecules. Solid-state NMR iscurrently emerging as a technique to obtain structuralinformation, where solution-state NMR is becomingexceedingly difficult, namely in the case of membrane pro-teins.

NMR spectroscopy, preferably in solution, can also provi-de valuable information on the interaction of proteins andligands, either in the case of a particular ligand of interestor in the case of large libraries used in screening proto-cols. Last but not least, NMR is extremely important for thedetermination of the constitution of smaller molecules.

In our group we use the full repertoire of solution-stateNMR techniques in conjunction with a variety of labelingpatterns to address questions of biological and pharma-cological importance. These range from the developmentof new techniques for solution-state NMR to the elucida-tion of the constitution of biologically active peptides andthe determination of the three-dimensional structure ofpeptides and proteins. In addition, NMR spectroscopy isoffered as a tool to researchers and companies throug-hout the Berlin area.

Structure of the chromophore-binding pocket ofthe cyanobacterial phytochrome Cph1

Phytochrome photoreceptors control numerous develop-mental processes in all plants. Light is absorbed by a cova-lently bound tetrapyrrole chromophore, phytochromobilin(PΦB), which is incorporated autocatalytically into a chro-mophore-binding domain in the N-terminal sensory modu-le of the protein. The physiological function of phytochro-me is associated with the photochemical production of thethermodynamically stable Pfr signaling state following lightabsorption by the Pr- ground state (λmax ~660 nm). The Pfr

form itself is photoactive (λmax ~730 nm), quantum absorp-tion leading to the production of Pr. In daylight the photo-cycle establishes a dynamic equilibrium with a constant

level of Pfr. The difference between the Pr and Pfr is attribu-ted to the Z-E-isomerization of a double bond within thechromophore, as suggested from NMR data from isolatedchromopeptides.

With the discovery of Cph1 in the cyanobacterium Syn-echocystis sp. PCC 6803 it was realized that phytochromesare also present in prokaryotes. The native chromophoreof Cph1 in Synechocystis as well as in many other cyano-bacterial phytochromes is phycocyanobilin. Subsequent-ly, phytochromes have also been detected in other cyano-bacteria and even non-photosynthetic bacteria, where thechromophore frequently is biliverdine. Heterologous ex-pression of phytochromes in E. coli yields apoproteins thatare usually capable of self-assembling with the chromo-phore to become red/farred photochromic holoproteins. Adeeper understanding of the photochromic mechanism ofphytochrome proteins requires detailed knowledge of thestructure of the chromophore pocket. No structural infor-mation at atomic resolution, however, is available to date.

We want to use solution state NMR spectroscopy to obtainstructural information on the chromophore binding pockein the two parent states (Pr and Pfr). To separate the signalsof the chromophore from those of the protein, deuterationof the protein is one approach. Since the chromophore isrelatively small compared to the protein, labeling of PCBwith nitrogen and carbon is also necessary. While the pro-duction of proteins in isotopically-labeled form is a well-established procedure and while the same is also possi-ble with DNA and RNA, the production of other types ofmolecules enriched with 13C and/or 15N is usually prohibi-tively expensive. In the case of the PCB chromophorenecessary for the present investigation, the problem issomewhat ameliorated by the fact that PCB is the chromo-phore in the light-harvesting antenna of photosyntheticbacteria and that the phycobilisome which contains thechromophore molecules represents a major fraction of thesoluble proteins in cyanobacteria. We therefore decidedto produce isotopically-labeled PCB using Synechocystissp. PCC 6803 itself (Figure 1).

Using the different labeling pattern, it was possible toobtain NMR spectra of sufficient quality to extract structu-ral information. By recording one-dimensional 15N-spectra,we could solve the much-discussed question whether thechromophore is protonated in the two parent states, whichhas strong implications on the reactions taking place in thephytochrome photocycle. The nitrogen chemical shiftsobtained indicate that both the Pr and the Pfr form are infact protonated. Using carbon-labeled PCB, three-dimen-sional edited NOESY spectra could be obtained that willyield an assignment of the chromophore resonances andwill allow the selection of a most likely structure from

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several structural models. An analysis of those spectra isstill in progress, a next step towards a structure of thechromophore-binding pocket is the introduction of proton-ated amino acids into a deuterated background to detectinteractions between the chromophore and amino acidsresidues lining the binding pocket.

Structure of antimicrobial peptides in a mem-brane-mimicking environment

Antimicrobial peptides are part of the natural immunesystem of many living organisms. A broad range of micro-organisms that involves gram-positive, gram-negativebacteria and fungi is affected by these peptides, which areevolutionary ancient weapons. Usually a cocktail of multi-ple peptides is present, supplementing the pathogen-spe-cific immune response, which occurs relatively slowly.Since bacterial resistance to existing antibiotics is con-stantly increasing, these peptides have gained in interestsince they have the potential to form an entirely new druggeneration.

Despite a growing interest in this type of peptides, theirmechanism of action is largely unknown. Since a replace-ment of L-amino acids with their D-enantiomers does notusually alter the antimicrobial activity of the peptides

unless the overall structure is disrupted, a mechanism viaa specific protein receptor is unlikely. It is rather the bac-terial membrane that is the most likely target of the anti-microbial assault. The outer membrane leaflets of gram-positive as well as gram-negative bacteria are negativelycharged, consisting mainly of phosphatidylglycerol or lipo-polysaccharide, respectively. In contrast, the outer leafletof mammalian cell membranes contains mostly phospha-tidylcholine and is thus charge neutral at physiological pH.It is assumed that the negative charge is responsible forthe selectivity.

In order to gain insight into the mechanism of action ofantimicrobial peptides at the atomic level, we have deter-mined the structure of the cyclic, cationic antimicrobialpeptide cyc-(RRWWRF) free in aqueous solution andbound to detergent micelles using NMR spectroscopy. Thepeptide shows a rather flexible but nevertheless orderedstructure in water, but a distinct structure is formed whenthe peptide is bound to a detergent micelle. The structu-res in the neutral and negatively charged micelles arenearly identical, resembling two β-turns. The orientationof the amino acid side chains creates an amphipathicmolecule with the peptide backbone forming the hydrophi-lic part. The orientation of the peptide in the micelle was

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FIGURE 1Preparation of 15N and 13C, 15N-labeled Phycocyanobilin (PCB) from Synechocystis sp. PCC 6803: The cyanobacteria are grown on the appropria-tely labeled medium (a). After harvesting the cells, the phycobilisome is separated by a sucrose density centrifugation (b). Finally, PCB is extrac-ted using methanol (c).

determined using NOEs and the accessibility of the pepti-de from outside the micelle as probed using paramagneticagents. The peptide is oriented mainly parallel to themicelle surface in both detergents. Substitution of the argi-nine and tryptophan residues is known to influence theantimicrobial activity. Therefore, the structure of the micel-le-bound analogs cyclo(RRYYRF), cyclo(KKWWKF) andcyclo(RRNalNalRF) were determined, having remarkablesimilarities in backbone conformation and side-chain ori-entation. Based on these structures, molecular dynamicssimulations of the peptide in an explicit membrane atvarious peptide-to-lipid ratio were performed. We observethat the NMR structure of the peptide is stable also after100 ns simulation. At a peptide-to-lipid ratio of 2/128, themembrane is only little affected compared to a pure DPPClipid membrane, but at a ratio of 12/128, the water-lipidinterface becomes more fuzzy, the water molecules canreach deeper into the hydrophobic core, and the waterpenetration free energy barrier changes. Moreover, weobserve that the area per lipid decreases and the deute-rium order parameters increase in the presence of thepeptide. We suggest that the changes in the hydrophobiccore together with the changes in the head groups resultin an imbalance of the membrane-stabilizing forces thatwill lead to the disruption of the membrane.

Group members

Holger Strauss (Doctoral student)Christian Appelt (Doctoral student)Janina Hahn (Doctoral student)*Brigitte Schlegel (Technical assistance)

External funding

Deutsche Forschungsgemeinschaft„NMR-spectroscopic investigation of light-induced struc-tural changes in protein-chromophore complexes” (Teil-projekt B6 im Sonderforschungsbereich 498 „Protein-Kofaktor-Wechselwirkungen in biologischen Prozessen“Peter Schmieder

Fonds der Chemischen Industrie„NMR-spektroskopische Strukturuntersuchung von pho-toschaltbaren Peptidliganden für die Numb-PTB-Domänesowie von PTB/Ligandkomplexen“ Kekulé-StipendiumChristian Appelt (Promotionsstipendium)

FIGURE 2Structure of the cyclic peptide cyc-(RRWWRF) bound to DPC micelles. The mole-cule exhibits an amphipatic structure with the peptide backbone as the hydro-philic part (blue) and the aromatic side chains as the lipophilic part (brown). Theorientation of the peptide has been determined to be parallel to the micelle sur-face with the longest axis, with the lipophilic side chains penetrating into themicelle.

* part of period reported

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Strauss H (2003) A device for facilitating the use of theFrench Press. Anal Biochem 321, 276-277

Hupfer M, Rübe B, Schmieder P (2004) Origin and diage-nesis of polyphosphate in lake sediments: A 31P-NMRstudy. Limnol Oceanogr 49, 1-10

Kahmann JD, Wecking DA, Putter V, Lowenhaupt K, KimYG, Schmieder P, Oschkinat H, Rich A, Schade M (2004)The solution structure of the N-terminal domain of E3Lshows a tyrosine conformation that may explain its redu-ced affinity to Z-DNA in vitro. Proc Natl Acad Sci USA 101,2712-2717

Brockmann C, Diehl A, Rehbein K, Strauss H, SchmiederP, Korn B, Kühne R, Oschkinat H (2004) The oxidized sub-unit B8 from human complex I adopts a thioredoxin fold.Structure 12, 1645-1654

Gaiser OJ, Ball LJ, Schmieder P, Leitner D, Strauss H,Wahl M, Kühne R, Oschkinat H, Heinemann U (2004) Solu-tion Structure, Backbone Dynamics, and AssociationBehavior of the C-Terminal BRCT Domain from the BreastCancer-Associated Protein BRCA1. Biochemistry 43,15983-15995

Strauss H, Hughes J, Schmieder P (2005) Heteronuclearsolution state NMR-studies of the chromophore in cyano-bacterial phytochrome Cph1. Biochemistry, 44, 8244-8250

Collaborations

K. Rueck-Braun, TU BerlinP. Hildebrandt, TU BerlinH. von Döhren, TU BerlinT. Lamparter, FU BerlinJ. Hughes, Justus Liebig University Giessen

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STRUCTURAL BIOINFORMATICSGroup Leader: Dr. Gerd Krause

STRUCTURAL BIOINFORMATICS TO UNRAVELMOLECULAR SIGNALING MECHANISMS

The group is focused on genome and protein sequenceanalysis by structural bioinformatics combined with com-parative modeling of protein-protein interaction to studysequence- and structure- function relationships. The mainaim is rational discovery of molecular mechanisms for pro-tein-protein interactions and protein-ligand interaction todeduce ideas for pharmacological intervention. All groupprojects are characterized by close cooperation withexperimental partners to ensure a closed loop betweenthe theoretically derived model and experimental proof asan important prerequisite for a successful application ofbiocomputing methods. The group is concerned with twotopics to elucidate structure - function relationships ofprotein-protein interactions and of G-protein coupledreceptors.

Protein-protein interactionsS. Müller, U. Wiedemann, G. Krause

Protein interaction networks are mediated very often bynon-catalytic protein domains like PDZ- and WW-domains.Narrowing down potential interacting proteins from theprotein interaction network, elucidation of selective pro-tein-protein interaction patterns and subsequent identifi-cation of complementary interaction sites at the levels ofepitopes, residues and atoms are the main aims.

• Interaction sites of junctional proteinsJunctional proteins connect and seal the contact sites inbetween endothelial cells. The exact sites, structures andmolecular mechanisms of interaction between junctionorganizing zona occludence protein 1 (ZO-1) and the tightjunction protein occludin or the adherens junction proteinα-catenin were unknown. Binding studies by surface plas-mon resonance spectroscopy and peptide mapping com-bined with comparative modeling utilizing crystal structu-res led for the first time to a molecular model revealing thebinding of both occludin and α-catenin to the same bind-ing site in ZO-1. Our data support a concept that ZO-1 suc-cessively associates with α-catenin at the adherensjunction and occludin at the tight junction.

Strong spatial evidence indicates that the identified occlu-din C-terminal coiled-coil domain dimerizes and interactsas a helix bundle with the identified structural motifs in ZO-1 (Müller et al. 2005). The selectivity of both protein-

protein interactions is defined by complementary shapesand charges between the participating epitopes. In con-clusion, a common molecular mechanism of forming anintermolecular helical bundle between the hinge region/GuK domain of ZO-1 and α-catenin and occludin is identi-fied as a general molecular principle organizing the asso-ciation of ZO-1 at adherens and tight junctions (Schmidt etal. 2004). The promising results of this joint project in col-laboration with the cell physiology group of I. Blasig, FMP,were primarily due to the fact that the bioinformatic/modeling- and the experimental studies as well have beencarried out by the same person (S.L. Müller). We arecurrently extending the structure-function studies also toother junctional proteins by combining structural infor-mation (bioinformatics/comparative modeling) and bioche-mical-, molecular biological studies.

• Prediction of protein – protein interaction networkpartners

To better understand and to predict the interactions, pre-dictive interaction models (e.g. COMFA and profile hiddenMarkov model recognition activities of WW domains)were derived by U. Wiedemann from experimental scree-ning data. Taking the sequence (Otte et al. 2003) or thestructure (Schleinkofer et al. 2004) of experimentally un-described domains or ligands, these models allowed theprediction of potential binding partners. Suggested opti-mal peptide sequence for binding a subfamily of WW-domains was successfully verified (Schleinkofer et al.2004). Additionally, some models predicted the strength(affinity) of the interaction (www.fmp-berlin.de/nmr/pdz).Using this approach, ligands with higher affinity weredesigned for representative domains of both families. Inparticular, for the hAF6-PDZ, the hERBIN-PDZ and themSNA1-PDZ so-called “super-binding” peptides weredesigned which indeed exhibited the highest affinityachievable by combination of natural amino acids (Wiede-mann et al. 2004).

Molecular signal t ransduction mechanisms ofGPCRG. Kleinau, J. Lättig, G. Krause

• Selective interaction patterns for G-protein subtypesThe molecular interactions at the interface between G-protein-coupled receptors and the G-proteins is of highfunctional importance, since there the decision takesplace about the different specific signaling pathways (viathe G-protein subtypes Gαi, Gαs, and Gαq) into the cell. Forthe thyroid stimulating hormone receptor (cooperationwith the University of Leipzig and NIH Bethesda, MD,USA), we were able to delineate residues of the secondintracellular loop as selective recognition patterns for

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G-protein subtypes by using molecular interaction models.Site-directed mutagenesis confirmed M527 as a residueselectively interacting with Gαq (Neumann et al. 2005).

Our previous studies on peptides and other small molecu-les (lipoamines) which directly interact with G-proteinsrevealed potential interaction sites based on charge pat-terns. Positive charges at ligands interact with negativelycharged residues at the G-protein site. Transferring thesecomplementary cognition patterns to the Endothelin recep-tors led to identification of amino acids that might beresponsible for different signaling pathways of the recep-tor subtypes. To provide support to this hypothesis, sugge-sted site-directed mutagenesis (J Lättig) is currently underinvestigation by the group of Alexander Oksche (FU Berlin).

• Structural determinants of TSH- receptor activation atthe TSHR ectodomain:

Thyroid-stimulating hormone (TSH, thyrotropin) and theTSH-Receptor (TSHR) are key proteins in the control ofthyroid function. TSH plays a critical role in ontogeny. Asa consequence, different mutations of TSHR and TSHresult in pathogenic diseases, for example thyroid toxicadenomas, non-autoimmune and neonatal hyperthyroi-dism, and hypothyroidism. Constitutively active TSH recep-tor mutants (CAMs) are the molecular etiology of > 50% of

hyperthyroidism in Germany. CAMs are thought to mimicto some extent the active conformation of the wild type(wt) receptor and to spontaneously adopt a structure ableto activate G-proteins. A concept for structure-functionrelationships in processes of ligand-dependent and -inde-pendent (constitutive) activity for TSHR would profoundlymodify the understanding of pathophysiology.

TSH belongs to the family of glycoprotein hormones. Themain differences of TSHR and the other glycoprotein hor-mone receptors (GPHR) such as choriogonadotrophic/luteinizing hormone receptor (CG/LHR) and follicle-stimu-lating hormone receptor (FSHR) to other seven transmem-brane-spanning G-protein-coupling receptors (GPCRs) isan even more complex activation mechanism, whichrequires the binding of a large hormone towards a largeN-terminal ectodomain. The intramolecular mechanism ofthe signal transduction to the serpentine domain upon hor-mone binding at the ectodomain is not understood. Theelucidation of the intramolecular mechanisms of TSHreceptor signaling in the large TSHR ectodomain are a pre-requisite for establishing new perspectives for the treat-ment of hyperthyroidism by inverse agonists or for thetreatment of thyroid cancer by small TSH receptor ligands.

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FIGURE 1a: LRR structure of the Nogo-receptor ectodomain (pdbentry 1OZN). A ‘Phe-spine’ (green aromatic rings) inside theLRR is stabilizing the fold instead of the missing helices atthe concave outer face.

b: LRR domain: comparison of the previous model for TSHR(pdb entry 1XUM, grey) based on ribonuclease inhibitor tem-plate (pdb entry 2BNH) and the new generated homologousLRR model for the TSHR (colored) based on the X-ray struc-ture of the hNogo-66-receptor ectodomain. The new tem-plate provides the N-terminal flanking Cys-box1 as an inte-gral structural part of the LRR by contributing with anadditional parallel β-stand (LRR0 in red) to the convex β-sheets of the proposed hormone-binding site.

N-terminalCysteine Box

C-terminaltail

N-terminaltail

Phe-Spine

LRRXN-terminal

Cysteine Box-1

binding site

LRRO

To identify determinants at the GPHR ectodomain that maybe involved in signal transduction, G. Kleinau first addres-sed this issue by searching for homologous structural fea-tures at the ectodomain based on high sequence similarity.

For the LRR motif within the large ectodomain, a new aug-mented structural model based on the Nogo-66-receptorectodomain with 9+2 repeats was generated (Fig. 1a) thatresulted in a strongly enlarged radius at the hormone bin-ding site at the inner convex surface of the LRR-arch. Thestructure is stabilized by an interior Phe-spine instead ofhelices at the concave outer face based on previous tem-plates (Fig. 1b). Moreover, the sequence similarity of thenew template indicated that the N-terminal flankedCysteinbox-1 is also an integral structural part of the LRR-arch by contributing with an additional parallel (LRR0(TSHR37-41)) anti-parallel ‚-strand to the convex ‚-sheet ofthe hormone-binding region.

Furthermore, this model provides for the first time a struc-tural rationale for the previously observed participation ofresidues from Cys-box1 in hormone binding, i.e. for threeresidues at the additional repeat LRR0 of the FSHR29-31 andof TSHR37-41. This LRR model of BHR was later on confir-med by the X-ray structure of the LRR domain for FSHR(Fan & Hendrichson, Nature 2005).

The subsequent structural extracellular component Cys-box2 is attached back-to-back to the 11th ‚-strand of theLRR, very likely via a short turn/loop containing S281. Exten-sive systematic sequence similarity searches of fragmen-ted sequence portions of the ectodomain resulted in ahomologous model based on IL8-IL8RA fragment complex(pdb entry 1ILQ (27) which links Cys-box 2 and Cys-box-3together (Fig. 2).

Subsequently, the residues D403EFNPC408 of the C-b3 arelocated particularly at prominent interface positions of theectodomain, most closely to the transmembrane domain.The hydrophilic residues D403, E404 and N406 are thereforehypothesized to participate very likely in the intramole-cular signal transduction from the ectodomain towards theserpentine domain (Kleinau et al, 2004).

Using our approach, we identified three new mutations(D403A, E404K and N406A) in the ectodomain of the TSHR thatare causing constitutive cAMP activity, and we suggestthat this motif is indeed important for the transduction ofthe signal from the ectodomain to the transmembranedomain. According to the high sequence conservation, theresults are of general relevance for the signal transduc-tion mechanism of other glycoprotein hormone receptors.

FIGURE 2TSHR ectodomain: detail of Cys-box2 (red) and Cys-box-3(yellow) interaction model adopted from IL8 and IL8RA com-plex structure. Aromatic interaction of F286 (C-b2) and F405(C-b3); Disulfide bridge between C408 (C-b3) and C283 (C-b2)based on biochemical data (blue); S281-loop/turn con-formation adopted from Malonyl Coenzyme (cyan); The sub-sequently predicted residues D403, E404, N406 to be involvedin signal transduction at the interface between ectodomainand serpentine domain indeed showed constitutive acti-vities upon mutations.

Cysteine-box-2

F 286Cysteine-box-3

F405

D403 E404

N406

C408

C284

C283

S281turn/loop

TM1

23

Group members

Sebastian L. Müller (Doctoral student)Jens Lättig (Doctoral student)Urs Wiedemann (Doctoral student)Gunnar Kleinau (Doctoral student)

External funding

Deutsche Forschungsgemeinschaft„Wechselwirkungen von Blut-Hirnschranken-Proteinen“(Bl 208/6.2)Ingolf Blasig, Gerd Krause

Deutsche Forschungsgemeinschaft„Identifizierung eines rezeptorinternen stillen Transmittersim TSH-Rezeptor“ (KR 1273)Gerd Krause


Müller SL, Portwich M, Schmidt A, Utepbergenov DI,Huber O, Blasig IE, Krause G (2005) The tight junction pro-tein occludin and the adherens junction protein α-cateninshare a common interaction mechanism with ZO-1. J BiolChem 280, 3747-3756

Schmidt A, Utepbergenov DI, Mueller SL, Beyermann M,Schneider-Mergener J, Krause G, Blasig IE (2004) Occlu-din binds to the SH3-hinge-GuK unit of zonula occludensprotein 1: potential mechanism of tight junction regulation.Cell Mol Life Sci 61, 1354-1365


Schleinkofer K, Wiedemann U, Otte L, Wang T, Krause G,Oschkinat H, Wade RC (2004) Comparative Structural andEnergetic Analysis of WW Domain-peptide Interactions. JMol Biol 344, 865-881

Wiedemann U, Boisguerin P, Leben R, Leitner D, KrauseG, Mölling K, Volkmer-Engert R, Oschkinat H (2004) Quan-tification of PDZ Domain Specificity, Prediction of LigandAffinity and Rational Design of Super-Binding Peptides. JMol Biol 343, 703-718

Neumann S, Krause G, Claus M, Paschke R (2005) Structu-ral Determinants in the Second Intracellular Loop for G-Protein Selectivity of the Thyrotropin Receptor. Endo-crinology 146, 477-485

Kleinau G, Jäschke H, Neumann S, Lättig J, Paschke R,Krause G (2004) Identification of a novel epitope in the TSHreceptor ectodomain acting as intramolecular signallinginterface. J Biol Chem 279, 51590-51600

Wüller S, Wiesner B, Löffler A, Furkert J, Krause G,Hermosilla R, Schaefer M, Schülein R, Rosenthal W,Oksche A (2004) Pharmacochaperones post-translational-ly enhance cell surface expression by increasing confor-mational stability of wild-type and mutant vasopressin V2

receptors. J Biol Chem 279, 47254-47263

Collaborations

R. Paschke, TSH receptor, University of Leipzig activation mechanism

S. Rothemund, IZKF Leipzig Peptide mapping

A. Oksche, FU Berlin V2 receptor

O. Huber, Charite Berlin CBF Interaction of junctionproteins

S. Offermanns, GPR109a receptor/University of Heidelberg ligand interaction

S. Neumann, NIH, NIDDK TSH receptorBethesda, MD, USA

M. Gershengorn, NIH, NIDDK TSH receptorBethesda, MD, USA.

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MOLECULAR MODELLINGGroup Leader: Dr. Ronald Kühne

COMPUTATIONAL TECHNIQUES FORLIGAND DESIGN AND PROTEIN MODELLING

The research of the Molecular Modelling/Ligand DesignGroup is mainly focused on protein modeling, virtualscreening, and lead optimization in close collaborationwith experimental partners within academia as well asindustry. The special expertise of the group includes awide range of molecular modeling technologies, bio-informatics tools, and computational chemistry methods.On top of that, these methods are applied to support theScreening Unit and the medicinal chemistry group withinthe national ChemBioNet initiative. The focus in this fieldis to analyze, design, and purchase a compound databa-se available for academic screening projects within theframework of ChemBioNet. In addition to the alreadyongoing modeling projects, the group has also startedresearch in several new fields during the last two years:structure and function of antimicrobial peptides, highthroughput screening evaluation and virtual screening ofantigen-exchange-inducing agents targeting the majorhistocompatibility complex (MHC) and inhibition of pro-tein-protein interaction in the IL6/IL-6R/gp130 complex. Allof these projects benefit from close collaborations withgroups within the FMP and the MDC as well as with indu-strial partners.

Targeting G-protein-coupled receptorsG-protein-coupled receptor (GPCR) modeling and agonistand antagonist design is one of the key competences ofthe group. The applied computational tools range fromquantum chemically supported ligand design to highthroughput virtual screening of millions of compounds. TheGPCR's are modeled using the bovine rhodopsin x-raystructure and refined using simulated annealing and mole-cular dynamics in implicit as well as explicit lipid environ-ment. These models are exploited in virtual screening andlead optimization using simulated annealing and pharma-cophore mapping.

The foll icle-stimulating hormone receptor (A. Schrey, R. Kühne)

We have constructed a model of the human follicle stimu-lating hormone receptor (FSHR) in a water-vacuum-waterenvironment. The model is consistent with published muta-tional data. A central feature of the model is a sodium ionlocated between Asp408 transmembrane helix 2 (TM2),Asp581 (TM6), and Asn618 and Asn622 (TM7). Activation

of the receptor seems to be associated with the trans-location of this ion and subsequent movements of TM 3, 6,and 7. Further, we recognized a small molecule binding sitewithin the FSHR. Docking simulations supported by scree-ning results served as a basis for lead optimization proces-ses. Refined models of the human and rat FSHR derivedfrom molecular dynamics simulations in explicit lipid envi-ronment have given closer insight into the interactions ofthe residue Histidine615 (TM7), which is specific for thehuman FSH receptor. We found that this residue locatedtowards TM6 is establishing a hydrogen bond to a back-bone carbonyl. In contrast to human FSHR, all known FSHRof other species contain in this position a tyrosine residue.In our simulations the tyrosine establishes hydrogen bondsto residues in TM3. These results are able to explain theknown mutational data and other experimental facts. Thestudies were sponsored by Schering AG.

Gonadotropin-Releasing Hormone Receptor (A. Söderhäll, R. Kühne)

In cooperation with Zentaris AG (Frankfurt am Main), seve-ral models of the human gonadotropin-releasing hormonereceptor (GnRH-R) have been derived. The models form aseries of successively improved generations of the recep-tor, where each generation is based on the latest experi-mental data. The data is based on site-directed mutage-nesis, activity modulation based on mutagenesis of thenative ligand as well as modulation of peptidomimeticligands. The latest generation of the receptor-ligand com-plex was used to derive a pharmacophore pattern, whichwas successfully used in the lead finding and optimization.As a result of this successful cooperation with Zentaris,two patents on new non-peptidic GnRH antagonists arecurrently in process. One of them could be economicallyutilized by the Forschungsverbund Berlin e.V.

Structure and function of antimicrobial peptides (A. Söderhäll, F. Eisenmenger)

Antimicrobial peptides have recently emerged as a pro-mising new generation of antibiotics. This class of pepti-des is generally believed to target the bacterial membra-ne and destroy the chemical gradients over these, eitherby building transmembrane pores or by destabilizing thebilayer structure of the membranes. This mechanism ofaction is believed to make the peptides more stableagainst the evolutionary pressure from bacteria develo-ping antibiotics resistance. By exploiting the in-housecompetence on peptide chemistry in Michael Bienert’sgroup, the antimicrobial peptide cyclo-(RRWWRF) wasselected for NMR structure determination by ChristianAppelt in the group of Peter Schmieder. Using the NMR

25

structure we have simulated realistic membranes at vario-us peptide-to-lipid ratios. From these molecular dynamicssimulations we have concluded that the peptide disturbsthe membrane so that its function as a hydrophobic bar-rier becomes less effective, and may increase the permea-bility of ions as well as water. Interestingly, the perturba-tion of the hydrophobic barrier takes place without theformation of explicit pores, as was often suggested for thiskind of substances. We have elucidated in detail how thiscompound interacts with the membrane lipids and whatimpact this has on the membrane structure /see Fig.1/.

High throughput and virtual screening targetingthe major histocompatibil i ty complex (A. Söderhäll, R. Kühne)

The group of Olaf Rötzschke at MDC has screened theFMP database for antigen-exchange inducing agents tar-geting the MHC. The data from the high throughput screenof the 20,000 compounds has been structured and analy-zed in detail. The analysis was then used in the construc-tion of a prediction model based on the quantitative struc-ture-activity relationships (QSAR) of a hit subfamily fromthe high throughput screening campaign. Based on thisderived model, an extended virtual screening of three

external databases containing a total of more than one mil-lion compounds was carried out. This massive data setwas narrowed down to a list of 5589 ranked compounds.A dozen of these were hand-picked and experimentallytested. Five out of these twelve were found to be active,proving that the hit enrichment of the focused library wasof major help in the second, refined screening round by O.Rötzschkes group. Within the framework of this project,dockings of the lead compounds were carried out in orderto explain the mechanisms behind the antigen exchange.The docking approach was supported by site-directedmutagenesis. Using these dockings together with experi-mental data, we are able to propose a possible mechanismof action of antigen exchange.

NMR structure calculations (C. Brockmann, R. Kühne)

An important part of the scientific activities is related tostructure elucidation of protein domains by NMR. Themain focus in this field is on NMR structure calculationsand methods to refine NMR derived protein structures.

NADH ubiquinone oxidoreductase (complex I) is the lastmajor complex of the respiratory chain for which there isno detailed atomic structure. Since most proteins of this

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FIGURE 1The structure of the antimicrobial peptide cyclo-(RRWWRF)was determined by NMR and exploited in a series of mole-cular dynamics simulations in the presence of explicit lipids.The figure shows the average structure of cyclo-(RRWWRF)from the simulation and a surface representation of themembrane. The amphipatic character of the peptide createsa void in the membrane surface that contributes to theincreased permeability of water and most likely also ionsthrough the membrane.

10

5

0

–5

–10

–10

–5 0 5 10

highly modular complex are rather small, its componentsmake suitable targets for structural studies by NMR. In thisproject the structure of subunit B8 (CI-B8) was investiga-ted by NMR. The solution structure of the oxidized subunitshows a thioredoxin fold with remarkable similarities tothioredoxin-like Fe2S2-ferredoxins (Fig. 2A). A structuralcomparison shows high similarities of surface propertiesand possibly active residues. Since CI-B8 shows somesimilarities to thioredoxins, and since the mammalianhomologues may form a disulfide bridge in a similar posi-tion, we determined the redox potential of oxidized CI-B8to see if it fits into the range covered by active site disulfi-des in thioredoxin-related proteins (Fig. 2B). The redox-potential of the disulfide bond of CI-B8 compares well tothat of the catalytically active disulfides in other thioredo-xin-like proteins. This, in addition to the fact that CI-B8stems from a complex that is involved in a redox-depen-dent reaction, leads to the idea that CI-B8 could assist inredox-associated processes or electron transfer. Thisexample of CI-B8 shows that structural studies of the indi-vidual components of complexes can spark hypothesesabout the function of the component within the complex.

Group members

Dr. Anna SchreyDr. Arvid SöderhällDr. Jörg Wichard*Dr. Frank Eisenmenger (Unix system administration)Christoph Brockmann (Doctoral student)Stefan Hübel (Unix system administration support, data-base management)

External funding

Schering AGTransmembranrezeptoren (Kooperationsvertrag)

Zentaris AG (Kooperationsvertrag)

Conaris AG


Brockmann C, Diehl A, Rehbein K, Strauss H, SchmiederP, Korn B, Kühne R, Oschkinat H (2004) The oxidized sub-unit B8 from human complex I adopts a thioredoxin fold.Structure (Cam) 12, 1645-1654

FIGURE 2Structure and redox-potential determination of CI-B8. A: ribbon-representation of the structure of human CI-B8. Alpha-helices are depicted ingreen, the beta-sheet in blue. The position of the disulfide-bond is indicated in yellow. B: Determination of the redox potential. The change inTrp-fluorescence upon reduction is plotted against the relative concentrations of the redox-buffer system. From a Keq of 0.486 a redox poten-tial of –250 mV was calculated.

1,0

0,8

0,6

0,4

0,2

0,0

1E-4 1E-3 0,01 0,1 1 10 100 1000

[GSH]2 / [GSSG]

R[c

m]

A B


27

Gaiser O, Ball LJ, Schmieder P, Leitner D, Strauss H, WahlM, Kühne R, Oschkinat H, Heinemann U (2004) Solutionstructure, backbone dynamics, and association behaviorof the C-terminal BRCT domain from the breast cancerassociated protein BRCA1. Biochemistry 43, 15983-15995

Pope BJ, Zierler-Gould KM, Kühne R, Weeds AG, Ball LJ(2004) Solution structure of human cofilin: actin binding,pH sensitivity, and relationship to actin-depolymerizingfactor. J Biol Chem 279, 4840-4848

Brockmann C, Leitner D, Labudde D, Diehl A, Sievert V,Bussow K, Kühne R, Oschkinat H (2004) The solution struc-ture of the SODD BAG domain reveals additional electro-static interactions in the HSP70 complexes of SODD sub-family BAG domains. FEBS Lett 558, 101-106

Freund C, Kühne R, Park S, Thiemke K, Reinherz EL, Wag-ner G (2003) Structural investigations of a GYF domaincovalently linked to a proline-rich peptide. J Biomol NMR27, 143-149

Zimmermann J, Kühne R, Volkmer-Engert R, Jarchau T,Walter U, Oschkinat H, Ball LJ (2003) Design of N-substi-tuted peptomer ligands for EVH1 domains. J Biol Chem278, 36810-36818

Karges B, Karges W, Mine M, Ludwig L, Kühne R, MilgromE, de Roux N (2003) Mutation Ala(171)Thr stabilizes thegonadotropin-releasing hormone receptor in its inactiveconformation, causing familial hypogonadotropic hypogo-nadism. J Clin Endocrinol Metab 88, 1873-1879

Collaborations

Dr. W. Karges, University of UlmProf. Schmalz, University of CologneDr. O. Roetzschke, MDC

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SOLID STATE NMRGroup Leader: Prof. Bernd Reif

SOLID-STATE NMR SPECTROSCOPY:PROTEIN AGGREGATION AND MEMBRANEPROTEINS

We use Nuclear Magnetic Resonance (NMR) in order tocharacterize biomolecular systems which are situated atthe interface between solution and solid. In this area,membrane proteins and amyloidogenic peptides and pro-teins are the most interesting “target molecules”. Bynature, structural information of these systems is difficultto obtain by means of X-ray crystallography or standardsolution-state NMR methods. We want to address thesesystems by application of a combination of modern solu-tion state and solid state NMR methods. This requiresdevelopment of especially adapted NMR techniques. Sofar, about 20 proteins are known for which a correlationbetween aggregation and disease is established. Themost prominent examples are Alzheimer's disease, theprion diseases (BSE, CfJ), and Huntington disease. How-ever, little is known about the mechanism which leads toaggregation, as well as on the structure of the amyloidfibrils. We would like to gain more insight into the structu-re of oligomeric intermediate states which are associatedwith protofibril formation. In addition, we are interested incharacterizing dynamic chemical exchange processesbetween the soluble and aggregated state of the respec-tive proteins.

Yeast Prions: Sup35 and Hsp104 Characterization of interactions between the molecularchaperone Hsp104 and the prion protein Sup35 in yeast byNMR spectroscopy Sup35 is a subdomain of the transla-tion-termination complex in S. cerevisiae. Under specificconditions, Sup35 can form protein aggregates that caninduce a conformational change in other Sup35 proteinsand are inherited after cell division. Hsp104 is one of themost important proteins for the thermostability in yeast andis, together with two other proteins, Hsp70/Ssa1 andHsp40/Ydj1, involved in a chaperon complex that can dis-solve protein aggregates that are produced as a heatresponse. Solution-state NMR methods are employed tocharacterize the interactions between the aggregate andthe chaperone. We found that Hsp104 is able to disaggre-gate Sup35-derived peptides in vitro, and that especiallylow oligomeric complexes of Sup35 interact with Hsp104(Narayanan et al., 2003). In this study, we were able to ana-lyze conformational changes of Sup35 induced by Hsp104by NMR in real time.

Structural characterization of l igands targetedagainst beta-amyloid f ibrils (Aß)Alzheimer's disease (AD) is the most common form of age-related neurodegenerative disorder, and is related todeposition of Aβ peptides in the brains of AD patients. Aβ

is formed by processing of APP, the Amyloid PrecursorProtein, a membrane protein of yet unknown function. Weuse the fact that amyloid fibrils orient spontaneously in themagnetic field to determine the structure of peptide inhibi-tors that bind to Aβ aggregates (Chen & Reif, 2004). Thealignment is based on the magnetic anisotropy of the pep-tide bond. A net alignment of amyloid fibrils is induced dueto the arrangement of hydrogen bonds in parallel to thefibril axis. We expect that this approach is useful for thedesign of diagnostic or therapeutic ligands.

Structural characterizat ion of acid denaturedPI3K fibrils Chris Dobson and co-workers demonstrated that not onlyproteins which are related to a disease can form highlystructured aggregates, but almost any other soluble pro-tein under certain solution conditions. One example is theSH3 domain of the phosphatidyl-inositol-3-kinase (PI3K)which forms fibrils under acidic pH. Solid-state NMR expe-riments revealed that His25 of PI3K-SH3 is involved in ter-tiary contacts which stabilize the fibril structure (Venturaet al., 2004). Mutational studies (H25K) confirmed thishypothesis. Most importantly, it could be shown that arti-ficial neurotoxicity is abolished in the mutated proteinusing a MTT reduction assay. We use PI3K-SH3 as a para-digm to study intermediate states as well as the aggrega-ted state and try to obtain a better understanding of themechanism of protein aggregation.

Development of MAS solid-state NMR techniques Solid-state techniques have found great attention in thelast few years since it has turned out to be possible todetermine the structure of uniformly labeled (crystalline)peptides and proteins (Rienstra et al. 2002). However,solid-state NMR experiments are intrinsically insensitivedue to detection of the heteronucleus (carbon or nitrogen).We address the question of sensitivity by employing pro-ton detection in combination with deuteration, which che-mically eliminates the strong proton-proton dipolar coup-lings. This approach allows to detect proteins with highsensitivity (Chevelkov et al. 2003). In addition, long rangeproton-proton interactions are accessible, which areimportant to determine the three-dimensional fold of a pro-tein in the solid state (Reif et al. 2003). Furthermore, weexplore deuterium as a source for dynamic information inthe solid state.

29

Group members

Dr. Alex ChernogolovDr. Maggy Hologne Zhongjing Chen (Doctoral student) Veniamin Chevelkov (Doctoral student)Muralidhar Dasari (Doctoral student)Saravanakumar Narayanan (Doctoral student)Uwe Fink (Technical assistance)

External funding

Deutsche Forschungsgemeinschaft„Entwicklung NMR spektroskopischer Methoden zwi-schen Flüssigkeit und Festkörper. Strukturuntersuchungenan orientierten Biomakromolekülen“ (Re1435/2)Bernd Reif

Deutsche Forschungsgemeinschaft„Strukturelle Charakterisierung des Multidrug-Transpor-ters EmrE mittels MAS Festkörper-NMR-Spektroskopie“(Re1435/3)Bernd Reif

Deutsche Forschungsgemeinschaft„Biochemische und Strukturuntersuchungen an Sup35pim Komplex mit Hsp104, Hsp40 und Hsp70“ Teilprojekt A3im Sonderforschungsbereich 594 „Molekulare Maschinen“Bernd Reif


Chen Z, Reif B (2004) Measurement of residual dipolarcouplings in peptidic inhibitors weakly aligned by transientbinding to peptide amyloid fibrils. J Biomol NMR 29, 525-530

Chevelkov V, Rossum BJv, Castellani F, Rehbein K, DiehlA, Hohwy M, Steuernagel S, Engelke F, Oschkinat H, ReifB (2003) 1H detection in MAS solid state NMR spectro-scopy employing pulsed field gradients for residual solventsuppression. J Am Chem Soc 125, 7788-7789

Narayanan S, Bösl B, Walter S, Reif B (2003) Importance oflow oligomeric weight species for prion propagation in theyeast prion system Sup35/Hsp104. Proc Natl Acad Sci USA100, 9286-9291

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FIGURE 1Hsp104 interacts preferably with low oligomeric Sup35.Interactions between Sup35 and Hsp104 are characterizedby STD NMR experiments. Sup35 oligomeric states aredetermined using DOSY experiments.

Sup35

Hsp104 +Sup35

MonomericSup35

HigherOligomericStates ofSup35

Intermediate 1

Intermediate 2

Reif B, van Rossum BJ, Castellani F, Rehbein K, Diehl A,Oschkinat H (2003) Determination of 1H 1H distances in auniformly 2H,15N labeled SH3 domain by MAS solid stateNMR spectroscopy. J Am Chem Soc 125, 1488-1489

Rienstra CM, Tucker-Kellogg L, Jaroniec CP, Hohwy M,Reif B, McMahon MT, Tidor B, Lozano-Pérez T, Griffin RG(2002) De Novo Determination of Peptide Structure withSolid-State MAS NMR Spectroscopy. Proc Natl Acad SciUSA 99, 10260-10265

Ventura S, Zurdo J, Narayanan S, Parreño M, Mangues R,Reif B, Chiti F, Giannoni E, Dobson CM, Aviles FX, SerranoL (2004) Short amino acid stretches can mediate amyloidformation in globular proteins: Thr Src homology 3 (SH3)case. Proc Natl Acad Sci USA 101, 7258-7263

Collaborations

Dr. W. Boelens, University of Nijmegen, The NetherlandsProf. J. Buchner, TU MunichDr. G. Gast, University of PotsdamProf. Dr. U. Heinemann, MDC Berlin Prof. Dr. W. W. de Jong, University of Nijmegen, TheNetherlandsProf. G. Multhaup, Free University Berlin

Dr. M. Sattler, EMBL Heidelberg Prof. Dr. S. Schuldiner, Hebrew University Jerusalem,IsraelDr. L. Serrano, EMBL HeidelbergDr. S. Ventura, Universitat Autonoma de Barcelona,Spanien. Dr. S. Walter, TU Munich

Fibril AxisMagnetic Field

FIGURE 2Structure of a peptide inhibitor bound to an Aß fibril usingrestrained docking. The spontaneous alignment of fibrils inthe magnetic field induces an ordering of the ligand peptide.Measurement of transfer residual dipolar couplings(trRDCs) yield the relative orientation of the peptide withrespect to the fibril axis.

31

PROTEIN ENGINEERINGGroup Leader: PD Dr. Christian Freund

PROTEIN ADAPTER DOMAINS INVOLVED INT CELL SIGNALING

Our group is interested in the molecular interactions thatgovern the assembly of protein complexes. The focus ison adapter domains that mediate protein-protein interac-tions in immune cells, especially T cells. The intracellularresponse in T cells has to cope with the highly adaptive Tcell receptor engagement process. Subtle changes in thekinetics and affinity of the TCR-MHC-peptide interactionare able to evoke dramatically different immune respon-ses. The fine-tuning of the intracellular processes isdependent on the membrane organization, the molecularpatterning at the inner membrane and cytoskeletal re-arrangements that take place upon stimulation. Cytoplas-mic adapter domains that guide the spatial distribution ofproteins are present in many lymphoid-specific as well asgeneral signaling molecules. They typically recognize pep-tide sequences that are present in the cytoplasmicdomains of transmembrane receptors or within solventexposed regions of intracellular proteins. Deciphering thissequence recognition code and determining the regulato-ry mechanisms of such adapter domain mediated interac-tions is the major topic in our group. Therefore we employNMR spectroscopy, protein biochemistry, fluorescencemicroscopy and screening of biomolecular libraries asresearch tools. Protein Engineering approaches in ourlaboratory implement random and structure-based muta-genesis as strategies to derive biologically relevant infor-mation. Based on the determination of the three-dimensio-nal structure of protein domains, we want to understandrecognition at a molecular level, while biochemical stu-dies are aimed at putting this knowledge in the context ofcellular signaling.

CD2BP2 Michael Kofler, Matthias Heinze, Katharina Thiemke, andChristian Freund

In addition to its binding capacity for the CD2 cytoplasmicdomain, we have identified novel in vitro binding partnersfor the CD2BP2 protein by means of peptide spot analysisand subsequent NMR analysis (Kofler et al. 2004, J. Biol.Chem. 279, 28292-28297). Several spliceosomal proteinscontain recognition sequences that match the require-ments for binding to the GYF domain of CD2BP2. Especial-ly the core splicing protein SmB/B’ contains severalbinding motifs for CD2BP2-GYF, and we have determinedthe epitope for the interaction. Pulldown experiments

confirm a specific interaction between the CD2BP2 andSmB/B’ proteins, and fluorescence microscopy of livecells shows the colocalization of the two proteins in thenucleus of Jurkat and HeLa cells (Fig. 1). Based on our invitro results we are now testing additional proteins fortheir potential to recognize the CD2BP2 protein in vivo.

GYF domainsMichael Kofler, Katrin Motzny, and Christian Freund

The NMR structure of this domain defines a new fold thatis proposed to be present in many eukaryotic proteins. Aconserved set of hydrophobic and aromatic amino acidsdefines the binding site for the proline-rich ligand. Thestructure of the GYF domain in complex with theSHRPPPPGHRV peptide derived from CD2 reveals that theprolines of the ligand form a polyproline type II helix thatcontact the hydrophobic hot spot of the domain (Freund etal. 2002, EMBO J 21, 5985-5995). The glycine residuepromotes a kink in the peptide backbone conformation,thereby allowing the proline helix to be optimally placedwithin the GYF domain binding pocket. Peptide substitutionanalysis shows the importance of the proline-proline-glycine motif for recognition by the GYF domain of CD2BP2.We are currently investigating several other GYF domainsfrom various eukaryotic species by phage display. Thiswill allow us to compare the ligand sequence space ofthese GYF domains, and structural studies are underwaythat complement the peptide binding results. Mapping thesequence space of GYF domains is anticipated to create avaluable tool for the identification of in vivo interactionpartners. It will also allow us to better understand thesequence requirements for individual subclasses of GYFdomains and set the stage for the comparison of sequen-ce recognition between GYF, SH3-, WW-domains.

Cyclophilin Kirill Piotukh and Christian Freund

Cyclophilins contain an intrinsic peptidyl-prolyl-cis-transisomerase activity (Fischer et al. 1989, Nature 337, 476-478), but have also been characterized as binding moduleswithin protein complexes (for a review see Ivery 2000, MedRes Rev 20, 452-484). We are currently investigating thepotential of cyclophilin to act on and bind to proline-richsequences that are also ligands for proline-rich sequencerecognition domains. Conceptually it is believed thatcyclophilins recognize peptide conformation rather thanbeing highly sequence-specific. We will use in vitromethods for the identification of peptide sequences thatcan be recognized by cyclophilins. These peptides arefurther investigated by NMR spectroscopy and other bio-physical methods to gain an understanding of the confor-

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mational restraints imposed on the peptides upon complexformation.

ADAPKatja Heuer, Marc Sylvester, Jürgen Zimmermann,Katharina Thiemke, and Christian Freund

ADAP (adhesion- and degranulation- promoting adapterprotein) was identified as an adapter protein that uponassociation with the TCR complex becomes tyrosine phos-phorylated and thereby creates binding sites for the SH2domains of the Fyn kinase and of the SLP-76 protein (daSilva et al. 1997, Proc Natl Acad Sci 94, 7493-7498; Musciet al. 1997, J Biol Chem 272, 11674-11677). ADAP has alsobeen shown to colocalize with F actin and cytoskeletalproteins such as VASP and WASP in activated Jurkat Tcells. A similar complex has been found enriched in thephagocytic cups of activated macrophages (Coppolino etal. 2001, J Cell Sci 114, 4307-4318; Krause et al. 2000, J CellBiol 149, 181-194). Furthermore, ADAP-deficient mice showa decrease in LFA-1 dependent adhesion of stimulatedperipheral T cells (Griffiths et al. 2001, Science 293, 2260-2263; Peterson et al., 2001, Science 293, 2263-2265) andtherefore ADAP was concluded to be an important regula-tor for inside-out signaling in T cells and other hemato-

poietic cells. From a molecular perspective, ADAP repre-sents a new member of the increasingly large family ofnon-enzymatic, hematopoietic scaffolding proteins. Inaddition to tyrosine-phosphorylation sites, ADAP containsa number of N-terminal proline-rich motifs that mediatethe interaction with the SH3 domain of SKAP55 (Liu et al.1998, Proc Natl Acad Sci 95, 8779-8784; Marie-Cardine etal. 1998, J Biol Chem 273, 25789-25795). ADAP itself con-tains two regions of homology to SH3 domains and wehave recently solved the structure of the ADAP C-terminaldomain by NMR spectroscopy (Heuer et al. 2004, Structu-re 12, 603-610). The structure reveals an extended SH3domain fold, where an N-terminal α-helix contacts the SH3scaffold, thereby creating a composite surface that can-not bind to proline-rich sequences (Figure 2). We are cur-rently investigating the binding properties of this domain,since we believe this will contribute to elucidation ofADAP’s function within immune cell signaling.

FIGURE 1Comparison of the structureof the ADAP hSH3 domainwith a “classical” SH3 do-main. The a-helix of ADAPhSH3 contacts the SH3 foldand several residues that areimportant for binding the pro-line-rich ligand are mutated inADAP hSH3 (compare A andB, in B the peptide ligand isshown in green). These featu-res lead to an altered surfacetopology and the inability ofADAP hSH3 to bind to proline-rich sequences (compare Cand D).

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Group members

Dr. Katja HeuerDr. Kirill PiotukhDr. Jürgen Zimmermann*Michael Kofler (Doctoral student)Matthias Heinze (Doctoral student)Marc Sylvester (Doctoral student)*Katharina Thiemke (Technical assistance)*Ulrike Schneeweiß (Technical assistance)*Uta Ben-Slimane (Technical assistance)** *Katrin Motzny (Technical assistance)**

External funding

Bundesministerium für Bildung und Forschung„Struktur-Funktionsbeziehung wichtiger T-Zell-Proteineund Design von Agonisten und Antagonisten der T-Zellvermittelten Immunantwort“ (Bio-Future 0311879)Sieger im Nachwuchsgruppenwettbewerb Bio-FutureC. Freund

Volkswagen Foundation“Biological Function of Adaptor Domains Controlled by TheActivity of Peptidyl-Prolyl cis/trans Isomerases” (I/77 955)C. Freund

Deutsche Forschungsgemeinschaft„Struktur-Funktionsbeziehung der GYF-Domäne“ (FR1325/2-1)C. Freund

Selected publications (FMP authors in bold)Heuer K, Arbuzova A, Strauss H, Kofler M, Freund C (2005)The helically extended SH3 domain of the T cell adaptorprotein ADAP is a novel lipid interaction domain. J MolBiol, in press

Heuer K, Kofler M, Langdon M, Thiemke K, Freund C (2004)Structure of a helically extended SH3 domain of the T celladapter protein ADAP. Structure 12, 603-610

Kofler M, Heuer K, Zech T, Thiemke K, Freund C (2004)Recognition sequences for the GYF domain reveal a pos-sible spliceosomal function of CD2BP2. J Biol Chem 279,28292-28297

FIGURE 2Binding site of the CD2BP2-GYF domain in complex with aCD2 peptide. Binding site residues of the GYF domain areshown in magenta while the peptide is displayed in yellow.The surface of the domain is rendered transparent.

* part of period reported** part-time

Kofler M, Motzny K, Freund C (2005) GYF domain proteo-mics reveals interaction sites in known and novel targetproteins. Mol Cell Proteomics (in press)

Freund C (2004) The GYF domain. In: Modular ProteinDomains, Wiley-VCH GmbH, Germany

Freund C, Kühne R, Park S, Thiemke K, Reinherz EL, Wag-ner G (2003) Structural investigations of a GYF domaincovalently linked to a proline-rich peptide. J Biomol NMR27, 143-149

Freund C, Kühne R, Yang H, Park S, Reinherz EL, Wagner G(2002) Dynamic interaction of CD2 with the GYF and theSH3 domain of compartmentalized effector molecules.EMBO J 21, 5985-5995

Collaborations

Ellis Reinherz (Dana-Farber Cancer Institute, Boston, MA,USA)Volkhard Helms (University of Saarland, Saarbrücken)Ingo Schmitz (University of Düsseldorf)Richard Kroczek (Robert Koch Institute, Berlin)

CELLULAR SIGNALLING/MOLECULAR GENETICS

INTRODUCTION

SECTION CELLULAR SIGNALLING /MOLECULAR GENETICS

Prof. Ivan HorakDepartment Head: Molecular Genetics(Secretary: Alexandra Kiesling)

Prof. Walter Rosenthal, Department Head: Cellular Signalling(Secretary: Heidemarie Petschick)

Research in the FMP section “Cellular Signalling / Molecu-lar Genetics” covers some of the major aspects of euca-ryotic signal transduction. Increasingly, the developmentof pharmacological strategies to interfere with cellularsignal transduction pathways under study has become amajor point of emphasis.

The Protein Trafficking group is interested in the early sta-ges of the intracellular trafficking of G-protein-coupledreceptors (GPCRs). The intracellular transport of membra-ne proteins starts with their insertion into the membraneof the endoplasmic reticulum (ER). During ER insertion, the

proteins are folded, and correct folding is monitored by aquality control system (QCS). Only correctly folded pro-teins are allowed to enter the vesicular transport via theER/Golgi intermediate compartment (ERGIC) and the Golgiapparatus to the plasma membrane. The study of the earlystages of the intracellular transport of GPCRs is also ofclinical significance. Naturally occuring receptor muta-tions frequently lead to misfolded proteins unable to passthe QCS and consequently to inherited diseases.

The aim of the Anchored Signalling group is to identify furt-her proteins involved in the redistribution of the proteinAQP2 that belongs to the family of water channels. It is cri-tically involved in the Arginine-vasopressin mediatedwater reabsorption in the kidney. Arginine-vasopressinbinds to the vasopressin V2 receptor located on the sur-face of renal principal cells thereby triggering the redistri-bution of AQP2 from intracellular vesicles into the urine-facing plasma membrane. The insertion of AQP2 into theplasma membrane introduces water-selective pores andfacilitates water entry into the cells. The studies in thisgroup may lead to a detailed understanding of the under-

BEREICH SIGNALTRANSDUKTION / MOLEKULARE GENETIK

Prof. Ivan HorakAbteilungsleiter: Molekulare Genetik(Sekretariat: Alexandra Kiesling)

Prof. Walter RosenthalAbteilungsleiter: Signaltransduktion(Sekretariat: Heidemarie Petschick)

Die Forschung im FMP-Bereich „Signaltransduktion/Molekulare Genetik“ befasst sich mit wichtigen Aspektender eukaryotischen Signaltransduktion. Zunehmend liegtdas Augenmerk auf der Entwicklung pharmakologischerStrategien zur Interferenz mit den untersuchten Signal-transduktionswegen.

Die Arbeitsgruppe Protein Trafficking interessiert sich fürdie frühen Stadien des intrazellulären Transports G-Pro-tein-gekoppelter Rezeptoren (GPCRs). Der intrazelluläreTransport von Membranproteinen beginnt mit ihrer Inser-tion in die Membranen des endoplasmatischen Retikulums(ER). Unter der Insertion werden die Proteine gefaltet.Dabei wird die korrekte Faltung durch ein Qualitätskon-trollsystem überwacht. Nur korrekt gefaltete Proteine tre-

ten in den vesikulären Transport über das „ER-cis-Golgiintermediate Compartment“ (ERGIC) und den Golgi-Appa-rat zur Plasmamembran ein. Das Studium der frühen Sta-dien des intrazellulären Transports von GPCRs ist auch vonklinischer Bedeutung. Natürlich auftretende Rezeptor-mutationen führen häufig zu fehlgefalteten Proteinen, diedas Qualitätskontrollsystem nicht passieren können. Aufdiese Weise manifestieren sich Erbkrankheiten.

Das Ziel der Arbeitsgruppe Anchored Signalling ist es, Pro-teine zu identifzieren, die an der Umverteilung des ProteinsAQP2 mitwirken. AQP2 gehört zur Familie der Wasserka-näle und ist an der durch Arginin-Vasopressin-vermittel-ten Wasserreabsorption in der Niere beteiligt. Arginin-Vasopressin bindet an den Vasopressinrezeptor V2, dersich auf der Oberfläche der Prinzipalzellen der Niere befin-det. Auf diese Weise wird die Umverteilung AQP2 ausintrazellulären Vesikeln in die harnseitige Plasmamembranausgelöst. Durch das Eintreten von AQP2 in die Plasma-membran wird diese mit wasserselektiven Poren ausge-stattet, so dass Wasser in die Zellen aufgenommen wer-den kann. Die Untersuchungen in der Arbeitsgruppe sollenzu einem detaillierten Verständnis des molekularenMechanismus der Wasserreabsorption führen und letzt-lich eine Behandlung bestimmter Fälle des Diabetes insi-

lying mechanism, and eventually to a treatment of certaincases of diabetes insipidus. Recently, interference of pro-tein-protein interactions by the means of chemical com-pounds has become a major aspect.

The main field of the Biophysics Group is research in mem-brane transport. Basic research projects aim to explore themolecular mechanisms of water and proton movement.The activities in applied biophysics are devoted to the con-trol of membrane permeability by (bio)polymers and to pho-todynamic reactions of membrane components. Peter Pohlhas been appointed full professor at the University of Linzin Austria and left the FMP at the end of 2004.

The research activity of the Department of MolecularGenetics concerns the molecular mechanisms which con-trol development and function of cells in the blood andimmune system. Elucidation of these processes is an ulti-mate requirement not only for understanding their patho-logical alterations but also for the development of rationaldiagnostic and therapeutic procedures. We have beenusing targeted mutagenesis to analyze genes thought tohave important regulatory functions in the blood and

immune system. This technology is used to generatemouse mutants (“knock-out mice“) that carry an experi-mentally designed gene defect. Thus it is possible to ana-lyze the consequences of a specific gene defect in thecontext of the whole organism. The main interest is focu-sed on cytokine receptor signaling, in particular inter-ferons. These cytokines are used for treatment of humandiseases, despite their unknown functional mechanisms.The highly pleiotropic effects of these cytokines are oftentherapeutically disadvantageous. Therefore, an exactknowledge of signaling pathways and interacting molecu-les which are regulated by these cytokines could lead tothe development of more precisely targeted therapeuticinterventions.

Cytokine receptor signaling involves STATs (Signal trans-ducers and activators of transcription). Frequently, abnor-mal activity of certain STAT family members, particularlyStat3 and Stat5, is associated with a wide variety of humanmalignancies, including haematological, head and neck,breast and prostate cancers. The EMBO research groupof Uwe Vinkemeier is investigating the relationship bet-

pidus möglich machen. Aktuell hat sich insbesondere diemittels chemischer Verbindungen auslösbare Interferenzmit den Protein-Protein-Interaktionen zu einem wesent-lichen Aspekt der Forschungstätigkeit entwickelt.

Der Hauptforschungsgegenstand der Arbeitsgruppe Bio-physik ist der Transport an Membranen. Forschungspro-jekte zielten auf die molekularen Mechanismen der Was-ser- und Protonenbewegung. Die Aktivitäten auf demGebiet der angewandten Biophysik sind darauf gerichtet,die Durchlässigkeit der Membran für (Bio-)Polymere unddie photodynamischen Reaktionen von Membrankompo-nenten zu erforschen. Der Leiter der Gruppe, Peter Pohl,folgte Ende 2004 einem Ruf auf einen Lehrstuhl an der Uni-versität Linz in Österreich.

Die Abteilung Molekulare Genetik befasst sich mit denmolekularen Mechanismen, die die Entwicklung und dieFunktion von Zellen des Blutes und des Immunsystemskontrollieren. Die Erforschung dieser Prozesse ist eineunabdingbare Voraussetzung nicht nur für das Verständnisihrer pathologischen Veränderungen sondern auch für dieEntwicklung rationaler diagnostischer und therapeuti-scher Verfahren. Die Wissenschaftler nutzen zielgerichte-te Mutagenese, um Gene zu analysieren, von denen anzu-nehmen ist, dass sie wichtige regulatorische Funktionen

im Blut und Immunsystem haben. Mit Hilfe dieser Metho-de werden experimentell Mäuse mit einem Gendefekterzeugt (Knockout-Mäuse). Auf diese Weise ist es mög-lich, die Auswirkungen eines spezifischen Gendefekts imKontext des Gesamtorganismus zu analysieren. DasHauptinteresse der Abteilung gilt dem Zytokinrezeptor-Signaling, insbesondere aber den Interferonen. DieseZytokine werden zur Behandlung von Erkrankungen desMenschen eingesetzt, man kennt jedoch ihre Wirkmecha-nismen nicht genau. Der stark pleiotrope Effekt der Inter-ferone ist meist von Nachteil für die Therapie. Deshalbkönnen eine genaue Kenntnis der Signaltransduktionswe-ge und der Molekülinteraktionen, die durch die Zytokinereguliert werden, zur Entwicklung präziserer therapeu-tischer Interventionen führen.

Das Zytokinrezeptor-Signaling verläuft über STATs (Signaltransducers and activators of transcription). Eine abnor-male Aktivität bestimmter Mitglieder der STAT-Familie, ins-besondere von Stat3 und Stat5, steht im Zusammenhangmit einer Vielzahl bösartiger Krebserkrankungen. DieEMBO-Forschungsgruppe von Uwe Vinkemeier untersuchtdie subzelluläre Lokalisation der STATs und die damitzusammenhängenden Konsequenzen für die Gen Induk-tion. Eine der Herausforderungen ist es, herauszufinden,

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ween the subcellular localization of STATs and the resul-ting consequences on gene induction. One of the challen-ges is to determine how target gene access is regulatedand how this might influence the execution of transcrip-tional programs. The goal is a complete description of themechanisms underlying STAT subcellular trafficking byfocusing on the biochemical basis of nuclear accumula-tion as well as phosphorylation-independent nucleocyto-plasmic shuttling. The group will also investigate howthese activities modulate the transcriptional functions ofthe STATs and influence complex phenotypes such asgrowth or antiviral protection.

The Microscopic Technology Group offers a range of con-ventional and advanced light and electron microscopicmethods as well as electrophysiology and microinjectionto all interested research groups in the institute. The groupis available for all types of collaboration and the proces-sing of common research projects. So, e.g. in terms of cha-racterizing cyclic nucleotide-gated cation channels, it haslong standing collaborations with the Synthetic OrganicBiochemistry Group. This group has continued the deve-lopment of photolabile inactive compounds (caged com-

pounds) from which the active biomolecules are liberatedby UV light. These compounds (caged cAMP, cagedcGMP) have proved extremely useful in elucidating intra-cellular signal transduction chains.

The Cellular Physiology Group is concentrating on signaltransduction in the blood brain barrier (BBB), using co-cul-tures of endothelial and glial cells as model systems. Cur-rent emphasis is on the structure and function of tightjunction proteins sealing the interendothelial cleft. Thegroup is also investigating the role of protein kinases in theregulation of the permeability of the BBB. The long-termgoal is to elucidate the molecular basis of such permeabi-lity changes under physiological and pathological condi-tions.

The Biochemical Neurobiology Group is studying bioche-mical and molecular interactions of peptide degradingenzymes and their neuropeptide substrates under patho-logical conditions. The research is e.g. focused on thefunction of angiotensin-converting enzyme (ACE) withalcohol reward in the CNS. This process involves angio-tensin receptors and their signal transduction pathways.

wie der Zugriff auf die jeweiligen Zielgene reguliert ist undwie dies die Tanskriptionsprogramme beeinflusst. Das Zielder Gruppe ist die umfassende Beschreibung der Mecha-nismen, nach denen der subzelluläre Transport der STATsabläuft. Dabei legt sie besonderes Augenmerk auf die bio-chemischen Grundlagen der Kernakkumulation und desphosphorylierungsabhängigen nukleozytoplasmatischenTransports. Zusätzlich wird untersucht, wie diese Aktivitä-ten die Transkriptionsfunktionen der STATs modulieren undkomplexe Phänotypen, wie zum Beispiel Wachstum oderantiviralen Schutz, beeinflussen.

Die Arbeitsgruppe Mikroskopische Techniken bietet alleninteressierten FMP-Gruppen ein breites Spektrum konven-tioneller und spezialisierter licht- und elektronenmikrosko-pischer Methoden sowie Elektrophysiologie und Mikroin-jektion an. Die Gruppe ist offen für Kooperationen undgemeinsame Forschungsprojekte. Sie charakterisiert zumBeispiel seit einigen Jahren zusammen mit der Arbeits-gruppe Synthetische Organische Biochemie erfolgreichdurch zyklische Nukleotide getriebene Kationenkanäle.Dabei kommen in dieser Arbeitsgruppe entwickelte, pho-tolabile chemische Verbindungen (caged compounds) zumEinsatz, aus denen auf Anregung mit ultraviolettem Lichthin aktive Biomoleküle freigesetzt werden. Diese Verbin-

dungen (caged cAMP, caged cGMP) haben sich als außer-ordentlich nützlich für die Untersuchung intrazellulärerSignalketten erwiesen.

Die Arbeitsgruppe Zellphysiologie fokussiert ihre For-schung auf die Signaltransduktion in der Blut-Hirn-Schran-ke (BHS). Dabei verwendet sie gemeinsam kultivierteEndothel- und Gliazellen als Modellsystem. Gegenwärtigstehen insbesondere Struktur und Funktion von Tight-junction-Proteinen, die den interendothelialen Spalt ab-dichten, im Vordergrund des Interesses. Die Gruppeerforscht auch die Rolle von Proteinkinasen bei der Regu-lation der Durchlässigkeit der BHS. Ziel ist es, die moleku-lare Basis der Veränderungen zu erforschen, denen dieDurchlässigkeit der BHS unter verschiedenen physiolo-gischen und pathologischen Bedingungen unterliegt.

Die Arbeitsgruppe Biochemische Neurobiologie unter-sucht die biochemischen und molekularen Interaktionenpeptidabbauender Enzyme und ihrer Neuropeptidsubstra-te unter pathologischen Bedingungen. Die Forschung zieltunter anderem auf die Rolle des Angiotensin-konvertieren-den Enzyms (ACE) und seiner Substrate beim Alkohol-Reward im Belohnungssystem des ZNS. Dieser Prozessverläuft unter Einbeziehung von Angiotensinrezeptorenund deren nachgeschalteten Signalketten.

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PROTEIN TRAFFICKINGGroup Leader: PD Dr. Ralf Schülein

QUALITY CONTROL AND ER INSERTION OFG-PROTEIN-COUPLED RECEPTORS

The group is interested in the early stages of the intracel-lular trafficking of G-protein-coupled receptors (GPCRs).The intracellular transport of membrane proteins startswith their insertion into the membrane of the endoplasmicreticulum (ER). During ER insertion, the proteins are fol-ded, and correct folding is monitored by a quality controlsystem (QCS). Only correctly folded proteins are allowedto enter the vesicular transport via the ER/Golgi interme-diate compartment (ERGIC) and the Golgi apparatus to theplasma membrane.

The study of the early stages of the intracellular transportof GPCRs is of clinical significance. Naturally occuringreceptor mutations frequently lead to misfolded proteinsunable to pass the QCS and consequently to inheriteddiseases. An example is X-linked nephrogenic diabetesinsipidus (NDI), which is caused by mutations in the geneof the vasopressin V2 receptor (V2R). It is noteworthy thatthe QCS may work faultily. Sometimes, non-functionalmutant receptors are transported to the plasma membra-ne, whereas in other cases, still functional proteins areretained.

We use NDI-causing V2R mutants to characterize themechanisms of GPCR quality control in the early secreto-ry pathway and to identify the proteins involved. Our aim isto identify new drug targets for the treatment of diseasescaused by transport-deficient membrane proteins. Inaddition, we study the function of cleavable signal pep-tides of GPCRs.

1. Significance of cleavable signal peptides ofGPCRs

ER insertion of GPCRs is mediated by two different types ofsignal sequences: one group contains signal anchorsequences that are part of the mature receptors. A secondgroup possesses additional signal peptides that are clea-ved off during the insertion process by the signal peptida-ses of the ER. The reason why this second subset requi-res additional signal peptides was not clear for GPCRs andother membrane proteins.

We studied the functional significance of the signal pep-tide of the human endothelin B receptor and showed thatit does not influence receptor expression, but rather isnecessary for the translocation of the receptor’s N tailacross the ER membrane. We have now studied the signal

peptides of the rat corticotropin-releasing factor recep-tors type 1 (CRF1R) and type 2α (CRF2αR). The signal pep-tide of the CRF1R is not necessary for N tail translocationbut strongly promotes receptor expression. The signalpeptide of the CRF2αR also promotes receptor expression.However, it is not cleaved after ER insertion and remains atthe N tail of mature receptor. The CRF2αR thus representsthe first GPCR containing eight hydrophobic helices: seventransmembrane helices and an eighth helix with extracel-lular location. The solubility of the latter helix seems to beensured by N-glycosylation.

2. Analysis of quality control mechanisms ofthe V2R

2.1. Quality control in the ERGICIt was unknown whether quality control of membrane pro-teins is restricted to the ER or whether the ERGIC or theGolgi apparatus are also involved. We have addressed thisquestion using misfolded NDI-causing V2R mutants. Ourresults show that these mutants fall into two classes. ClassA mutants (e.g. L62P, DL62-R64, S167L; Fig. 1) are retainedexclusively in the ER and never leave this compartment.Class B mutants (e.g. R143P, Y205C, R337X, V226E, InsQ292;Fig. 1), in contrast, reach the ERGIC. These mutants arerecognized in this compartment and are most likely rerou-ted to the ER via the retrograde transport system. More-over, we could show that the ability of a mutant to reachthe ERGIC is not determined by its expression level but byits folding state. The ERGIC may represent a second safetynet within the QCS recognizing those proteins that are onlyinefficiently detected at the ER level.

2.2. Compartment-specific rescue of NDI-causing V2Rmutants

Pharmacological strategies to rescue transport-deficientmutant membrane proteins are of clinical significance.Two strategies are being pursued at the moment. The firstinvolves the development of ligands that favor correct fol-ding of the mutant proteins and consequently allow themto pass the QCS (“pharmacological chaperones”). Thesecond strategy is based on the fact that the QCS is some-times overprotective and retains mutant proteins that arestill functional. Here, substances that do not influence fold-ing but prevent the interaction with quality control compo-nents may allow transport and lead to a functional rescue.V2R antagonists mediating the rescue of some NDI-caus-ing V2R mutants as pharmacological chaperones havebeen described. We have now found novel inhibitors of theQCS acting in a compartment-specific way. By using theL62P mutant (ER-retained; class A) and the Y205C mutant(ERGIC-reaching; class B) of the V2R as a model, we showthat the cell-penetrating peptide penetratin rescues the

transport of the Y205C mutant but not that of the L62Pmutant. We have also shown that the peptides exert theirfunction by displacing the BiP chaperone from the mutantreceptor in the ERGIC.

Group members

Dr. Ute Donalies Dr. Claudia Rutz**AnjaThielen (Doctoral student)*Martina Alken (Doctoral student)*Morad Oueslati (Doctoral student)Dagmar Michl (Technical assistance)**

External funding

Deutsche Forschungsgemeinschaft“Struktur und Funktion von Transportsignalen des Vaso-pressin V2-Rezeptors” (Teilprojekt A3 im Sonderfor-schungsbereich 449 “Struktur und Funktion membranstän-diger Rezeptoren”)Ralf Schülein, Walter Rosenthal

Deutsche Forschungsgemeinschaft“ER-Insertion und Qualitätskontrolle von G-Protein-gekop-pelten Rezeptoren” (Teilprojekt A11 im Sonderforschungs-bereich 366 “Zelluläre Signalerkennung und Umsetzung”)Ralf Schülein, Walter Rosenthal


Oueslati M, Hermosilla R, Oorschot V, Donalies U,Schönenberger E, Beyermann M, Oehlke J, Wiesner B,Oksche A, Klumperman J, Rosenthal W, Schülein R (2005)Compartment-specific rescue of nephrogenic diabetesinsipidus-causing vasopressin V2 receptor mutants bycell-penetrating peptides. J Cell Biol, in revision

Thielen A, Oueslati M, Hermosilla R, Krause G, Oksche A,Rosenthal W, Schülein R (2005) The hydrophobic aminoacid residues in the membrane-proximal C tail of the G pro-tein-coupled vasopressin V2 receptor are necessary fortransport-competent receptor folding. FEBS Lett, in press

Alken M, Rutz C, Köchl R, Donalies U, Oueslati M, FurkertJ, Wietfeld D, Hermosilla R, Scholz A, Beyermann M,Rosenthal W, Schülein R (2005) The signal peptide of therat corticotropin-releasing factor receptor 1 promotes

FIGURE 1Two-dimensional model of the V2R. The positions of class A mutants (L62P, DL62-R64, S167L) are indicated by open rectangles, the positions ofclass B mutants (R143P, Y205C, R337X, V226E, InsQ292) are indicated by black rectangles. The following posttranslational modifications of the V2Rare depicted: N-glycosylation at position N22, palmitoylation at positions C341 and C342 and a disulfide bond formed between residues C112 andC192.


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receptor expression but is not essential for establishing afunctional receptor. Biochem J 390, 455-64

Hermosilla R, Oueslati M, Donalies U, Schönenberger E,Krause E, Oksche A, Rosenthal W, Schülein R (2004)Disease-causing V2 vasopressin receptors are retained indifferent compartments of the early secretory pathway.Traffic 5, 993-1005

Wüller S, Wiesner B, Löffler A, Furkert J, Krause G, Her-mosilla R, Schaefer M, Schülein R, Rosenthal W, OkscheA (2004) Pharmacochaperones post-translationally enhan-ce cell surface expression by increasing conformationalstability of wild-type and mutant vasopressin V2 receptors.J Biol Chem 279, 47254-47263

Neuschäfer-Rube F, Rehwald M, Hermosilla R, Schülein R,Rönnstrand L, Püschel G (2004) Requirement of differentSer/Thr residues in the C-terminal domain of the humanEP4 receptor for agonist-induced phosphorylation‚ arre-stin interaction and sequestration. Biochem J 379, 573-585

Engelsberg A, Hermosilla R, Karsten U, Schülein R, Dör-ken B, Rehm A (2003) The Golgi protein RCAS1 controls cellsurface expression of tumor-associated O-linked glycanantigens. J Biol Chem 278, 22998-3007

Collaborations

R.S. Hedge, NIH Bethesda, MD, USAA. Oksche, Charité – University Medicine BerlinR. Hermosilla, Charité – University Medicine BerlinA. Rehm, Max Delbrück Center for Molecular MedicineBerlinG. Püschel, University of Potsdam

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FIGURE 2Influence of penetratin treatment on the subcellu-lar distribution of the GFP-tagged V2R mutantsL62P (class A) and Y205C (class B) in transientlytransfected HEK 293 cells. (A) Laser scanningmicroscopy. Cells were treated with penetratin(Pe, 1 µM) or with vehicle (-). The GFP fluorescen-ce signals of the receptors were recorded (green,left panels), plasma membranes were stained withTrypan blue (red, central panels) and overlay of thesignals was computed (right panels; co-localizati-on is indicated by yellow). Scale bars, 5 µm. (B)Quantitative analysis of the peptide effect. Theratio of cell membrane and intracellular fluores-cence signals (M/I) was determined. Ratios < 1indicate a predominantly intracellular localizationof the receptors and ratios > 1 a predominant loca-lization at the cell membrane. Columns show meanvalues ± SD (n = 30 cells). Note that in (A) and (B)only the transport of the Y205C mutant is rescuedby penetratin treatment.

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ANCHORED SIGNALLINGGroup Leader: PD Dr. Enno Klussmann

WATER REABSORPTION IN THE KIDNEY

A major function of the kidney is the production of urine. Ahuman kidney generates 180 l of primary urine per day,most of which is water. It is obvious that excretion of suchvast amounts of fluid would cause dehydration and death.Therefore, mechanisms have evolved which carefullycontrol body water balance by regulating water reabsorp-tion from primary urine. Most of the water (90%) is reab-sorbed by passive diffusion along an osmotic gradientestablished between the primary urine and the surround-ing tissue. Reabsorption of the remaining 10% of water isregulated by antidiuretic hormone (Arginine-vasopressin,AVP) in particular cells of the kidney, the principal cells.Principal cells line the collecting duct, the terminal part ofthe tubular system transporting urine to the bladder. Lossof responsiveness to the hormone results in a diseaseknown as diabetes insipidus (DI). DI is characterized bya massive loss of water (up to 20 l per day if untreated).

The molecular mechanism underlying AVP-mediatedwater reabsorption in the kidney involves several key pro-teins including the vasopressin V2 receptor, and aquapo-rin-2 (AQP2). The latter belongs to the family of waterchannels whose discovery yielded Peter Agre the NobelPrize in Chemistry in 2003. AVP binds to the vasopressinV2 receptor located on the surface of renal principal cellsthereby triggering the redistribution of AQP2 from intra-cellular vesicles into the apical (urine-facing) plasmamembrane. This process is also known as the AQP2shuttle.

The insertion of AQP2 into the plasma membrane introdu-ces water-selective pores and facilitates water entry intothe cells. Water exits the cells through aquaporins-3 and -4 constitutively located in the basolateral (tissue-facing)plasma membrane. The driving force is again the osmoticgradient established between primary urine and the tis-sue. On the molecular level, AVP stimulates the elevationof the second messenger cAMP followed by activation ofprotein kinase A (PKA). PKA, in turn, transfers a phospha-te group to AQP2. This phosphorylation is a prerequisitefor the redistribution of AQP2. The mechanism is depictedin Figure 1.

The aim of our group is to identify further proteins involvedin the redistribution of AQP2. This will lead to a detailedunderstanding of the underlying mechanism, and mayeventually lead to a treatment of certain cases of DI. Thetranslocation of AQP2 from intracellular vesicles to the

plasma membrane constitutes an exocytosis-like process.A better understanding of this mechanism will also yield abetter understanding of other cAMP-dependent exocyticprocesses such as renin secretion from juxtaglomerularcells, insulin secretion from pancreatic βcells or protonsecretion from gastric parietal cells. Dysregulations ofthese processes cause various diseases including hyper-tension, diabetes mellitus and gastric ulcers.

AKAP18δδ anchors PKA to AQP2-bearing vesi-cles The above mentioned phosphorylation of AQP2 is not thesole prerequisite for its redistribution. We have shown thatthe tethering of PKA to subcellular compartments by socalled A-kinase anchoring proteins (AKAPs) is also essen-tial for the AQP2 shuttle to occur. During the search forAKAP(s) involved in the shuttle, a new splice variant ofAKAP18, AKAP18δ, was identified. Biochemical and cellbiological approaches revealed that AKAP18δ functionsas an AKAP in vitro and in vivo. Immunofluorescencemicroscopy showed that in the kidney, AKAP18δ is mainlyexpressed in principal cells of the terminal section of thecollecting duct, closely resembling the distribution ofAQP2. AKAP18δ was identified on the same intracellularvesicles as AQP2 and PKA. AVP not only recruited AQP2,but also AKAP18δ to the plasma membrane (Fig. 2). AVPcaused the dissociation of AKAP18δ and PKA. The datasuggest that AKAP18δ is involved in the AQP2 shuttle byanchoring PKA in close proximity to AQP2 (Henn et al.2004).

The small GTPase RhoA mediates the diuret iceffects of prostaglandin E2The small GTPases of the Rho family (Rho, Rac, Cdc42)participate in the regulation of the F-actin cytoskeleton. Inpreviously published papers we have demonstrated thatthe small GTPase RhoA, a particular member of the Rhofamily, in its active state (GTP-bound) induces the forma-tion of F-actin-containing stress fibers in primary culturedprincipal cells,and prevents the AQP2 shuttle. We obser-ved that AVP causes a decrease of F-actin-containingstress fibers, and inhibition of RhoA.

Prostaglandin E2 (PGE2) antagonizes AVP-induced waterreabsorption. Using primary cultured rat inner medullarycollecting duct principal (IMCD) cells, we have shown thatstimulation of prostaglandin EP3 receptors induced RhoAactivation and formation of F-actin-containing stress fibersin resting IMCD cells, but did not modify the intracellularlocalization of AQP2. However, the AVP-induced AQP2translocation was strongly inhibited. In addition, stimula-tion of EP3 receptors inhibited the AVP-induced RhoA in-

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activation and the AVP-induced depolymerization of F-actin-containing stress fibers. The inhibitory effect of EP3

receptor stimulation was independent of increases incAMP and cytosolic Ca2+ and is most likely mediated bythe G-proteins G12/13. Further experiments showed thatelevation of cAMP results in the phosphorylation of RhoAby PKA, which is known to inhibit this GTPase. Takentogether, the data suggest that the signaling pathwayunderlying the diuretic effects of PGE2 (and probably thoseof other diuretic agents) include cAMP- and Ca2+-indepen-dent RhoA activation and F-actin formation (Tamma et al.2003a, 2003b).

Group members

Dr. Dorothea Lorenz**Dr. Theresa McSorley Dr. Volker HennDr. Pavel NedvetskyKatja Santamaria (Doctoral student)Viola Weber (Doctoral student)Eduard Stefan (Doctoral student)Christopher Blum (Doctoral student)Christian Hundsrucker (Doctoral student)

Andrea Geelhaar (Technical assistance)Michael Gomoll (Trainee)

External funding

Deutsche Forschungsgemeinschaft„Die Rolle des Zytoskeletts bei der Vasopressin-induzier-ten Translokation von Aquaporin-2 in die Plasmamembranrenaler Hauptzellen“ (Kl1415/1-1)E. Klussmann, W. Rosenthal

Deutsche Forschungsgemeinschaft„Charakterisierung der Proteinkinase A-AnkerproteineHt31 und Rt31 und Untersuchungen zu ihrer biologischenFunktion“ (Ro597/9-1)W. Rosenthal, E. Klussmann

European Community, 5th Frame work programme“Anchored cAMP signalling - implications for treatment ofhuman disease” (QLK3-CT-2002-02149)E. Klussmann, W. Rosenthal

FIGURE 1Schematic representation of the vasopressin-mediated water reabsorption. In resting principal cells (A) aquaporin-2 (AQP2) is located in intra-cellular vesicles. B. The binding of arginine-vasopressin (AVP) to the vasopressin V2 receptor (V2R) activates a cAMP-dependent signalling cas-cade which causes the phosphorylation of AQP2 (phosphorylated AQP2, p-AQP2) and its translocation from intracellular vesicles into the plas-ma membrane facing the lumen of the renal collecting duct. Water from the primary urine enters the cells through AQP2 along an osmotic gradientand exits the cells through aquaporin-3 and aquaporin-4 (AQP3 und AQP4) constitutively present in the basolateral plasma membrane. AC, aden-ylyl cyclase; Gs, stimulatory G-Protein; C and R, catalytic and regulatory subunits of protein kinase A (PKA), respectively.

A B

** part-time


Henn V, Edemir B, Stefan E, Wiesner B, Lorenz D, Theilig F,Schmitt R, Vossebein L, Tamma G, Beyermann M, KrauseE, Herberg FW, Valenti G, Bachmann S, Rosenthal W,Klussmann E (2004) Identification of a novel A-kinaseanchoring protein 18 isoform and evidence for its role inthe vasopressin-induced aquaporin-2 shuttle in renal prin-cipal cells. J Biol Chem 279, 26654-26665

Lorenz D, Krylov A, Hahm D, Hagen V, Rosenthal W, Pohl P, Maric K (2003) Cyclic AMP is sufficient for trigger-ing the exocytic recruitment of aquaporin-2 in renal epi-thelial cells. EMBO Rep 4, 88-93

Tsunoda SP, Wiesner B, Lorenz D, Rosenthal W, Pohl P(2004) Aquaporin-1, nothing but a water channel. J BiolChem 279, 11364-11367

Tamma G, Wiesner B, Furkert J, Oksche A, Schaefer M,Valenti G, Rosenthal W, Klussmann E (2003) The prosta-glandin E2 analogue sulprostone antagonizes vasopres-sin-induced antidiuresis through activation of Rho. J CellSci 116, 3285-3294

Tamma G, Klussmann E, Procino G, Svelto M, RosenthalW, Valenti G (2003) cAMP-induced AQP2 translocation isassociated with RhoA inhibition through RhoA phosphory-lation and interaction with RhoGDI. J Cell Sci 116, 1519-1525

Storm R, Klussmann E, Geelhaar A, Rosenthal W, Maric K(2003) Osmolality and solute composition are strong regu-lators of AQP2 expression in renal principal cells. Am JPhysiol 284 (Renal Section), F189-198

Collaborations

Prof. K. Tasken, University of Oslo, NorwayAKAPs in cardiac myocytes

Dr. M. Zaccolo, University of Padova, ItalyModulation of Ca2+ fluxes in cardiac myocytes

Professor J. D. Scott, Howard Hughes Medical Institute,Vollum Institute, Oregon Health & Sciences University,Portland, Oregon, USAPeptide disruptors of AKAP-PKA interactions

FIGURE 2The effect of AVP on the subcellular distribution ofAQP2 and AKAP18δ in primary cultured rat innermedullary collecting duct principal (IMCD) cells. IMCDcells were left untreated (-AVP) or incubated with AVP(100 nM, 15 min), fixed and permeabilized. AQP2 wasdetected by incubation with goat anti-AQP2 andsecondary Cy3-conjugated antibodies, AKAP18δ withaffinity-purified rabbit anti-AKAP18δ A18δ4 andsecondary Cy5-conjugated antibodies. Immunofluo-rescence signals were detected by laser scanningmicroscopy. The overlay of Cy3 and Cy5 fluorescencesignals is shown in the right panel. Scale bars, 20 µm.

AQP2 AKAP18δ overlay

-AVP

+AVP

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Prof. M. Houslay, University of Glasgow, Scotland, UKRole of phosphodiesterases in renal principal cells

Prof. G. Valenti, University of Bari, ItalyRho-dependent signalling in the AVP-mediated waterreabsorption

Professor F. W. Herberg, University of KasselAnalyses of AKAP-PKA interactions

CELLULAR IMAGINGGroup Leader: Dr. Burkhard Wiesner

ENHANCEMENT OF THE CELL SURFACEEXPRESSION BY INCREASING CONFOR-MATIONAL STABILITY OF VASOPRESSINV2 RECEPTORS

This facility offers a range of conventional and advancedlight and electron microscopic methods as well as electro-physiology to all interested research groups in the insti-tute. The group is available for all types of collaborationincluding advice in preparation techniques, and the pro-cessing of common research projects.

About 50% of nephrogenic diabetes insipidus (NDI)-caus-ing mutations in the vasopressin V2 receptor (V2R) genecode for single amino acid replacements (missense muta-tions). In most cases the encoded mutant V2Rs are misfol-ded and retained in the ER. Improvement of this situationis to be achieved by the application of specific substan-ces (e.g. pharmacochaperones). Such cell permeable ant-agonists should restore cell surface expression. So westudied the effects of SR121463B and SR49059 (V2R andV1R-specific antagonists) on ER-retained V2Rs transientlyexpressed in HEK293 cells. Such investigations can beaccomplished generally by receptor binding studies(radioactive proof of the number of receptors of the cellsurface). If, however, the assigned substance (pharmaco-chaperone) blocks the binding domain for the ligand, thismethod is not applicable. In this situation, the employmentof confocal microscopy is very helpful. So the receptorcan be located with the help of green fluorescent protein(GFP) microscopically. Optical staining of the cell membra-ne by further coloring (trypan blue) makes a simple alloca-tion possible between intracellular and membrane locali-zation of the receptor (see Fig. 1).

In the case of the wild-type murine V2R (mV2R), which ismainly localized in the ER in untreated controls, both ant-agonists promoted a time- and dose- dependent restora-tion of the cell surface expression. SR49059-mediatedrestoration of cell surface expression of the mV2R wasaccompanied by a dramatic increase in [3H]AVP bindingsites.

Figure 2 shows, that with the microscopic investigation therearrangement (from the ER to the cell membrane) is fastrun off. This phenomenon is justified by the optical resolu-tion of light microscopy. The binding studies show thefunctional receptor in the plasma membrane of the cell.

Light-microscopy cannot differentiate whether the recep-tor is located at the intracellular side of the membrane orin the plasma membrane of the cell.

For human NDI-causing mutants (hL62P, h∆LAR 62-64,hH80R, hW164R, hS167T, hS167L, hC319Y, hP322S) andseveral in vitro mutants (hD136A, hD368K/S371X, hF328A)transiently expressed in HEK cells, also predominant ERretention was observed. Interestingly, cell surface expres-sion of h∆LAR 62-64, hD136A, hS167T, hP322S, and hF328Awas only restored by SR121463B. In the case of themutants hL62P, hH80R, hW164R, hS167L, and hD368K/S371X none of the two antagonists restored cell surfaceexpression. Only for the mutant hC319Y, both antagonistspromoted cell surface expression as found for the mV2R.The data show that ER-retained mutant V2Rs differ in theirsensitivity to antagonist-promoted cell surface expression.It is likely that these differences can be attributed to theextent of the folding defect and/or an alteration of the bin-ding pocket. Further studies are required to analyze thegeneral applicability of antagonists in the treatment of NDI.

Further projects involved: A-kinase anchoring proteins and the exocytotic recruit-ment of aquaporin-2 (D. Lorenz, B. Wiesner, M. Ringling, B.Oczko in cooperation with Enno Klussmann, AnchoredSignalling)

Application of novel caged compounds (B. Wiesner, J.Eichhorst, B. Oczko in cooperation with Volker Hagen, Syn-thetic Organic Biochemistry)

Water flux through water channels (D. Lorenz, B. Wiesner,B. Oczko in cooperation with Peter Pohl, Biophysics)

Cellular uptake of peptides (B. Wiesner, B. Oczko in coope-ration with J. Oehlke, Peptide Lipid Interaction/PeptideTransport; and M. Beyermann, Peptide Synthesis)

Constitutive internalization of the human V2 Vasopressinreceptor (A. Schmidt, B. Wiesner in cooperation with R.Hermosilla, Charité group)

DNA binding controls inactivation and nuclear accumula-tion of the transcription factor Stat1 (B. Wiesner, B. Oczkoin cooperation with U. Vinkemeier, Cellular Signal Proces-sing)

Enhancement of the cell surface expression by increasingconformational stability of G protein coupled receptors (B. Wiesner, J. Eichhorst, B. Oczko in cooperation with A. Oksche, Charité group; and S. Wüller, RWTH AachenMedical School).

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FIGURE 1HEK293 cells transiently expressing the mV2R.GFP were treated with the vasopressin receptor antagonist SR49059 for up to 13 h (A: oh; B: 7 h; c: 13 h). Top panel: mV2R.GFP fusion protein. Bottom panel: Plasma membrane stained with trypan blue.

FIGURE 2Antagonist-mediated restoration of cell surface expression analyzed by quantitative laser scanning microscopy and binding analysis. Cells tran-siently expressing the mV2R.GFP were treated for up to 16 h with the specific vasopressin receptor antagonist. The fitted curve representing theincrease in the normalized fluorescence intensities is shown in black. In parallel, membrane preparations were analyzed for specific binding of[3H]AVP. The curve representing the increase in specifically bound [3H]AVP is shown in gray.

A C CTr

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Group members

Dr. Dorothea Lorenz**Antje Schmidt (Student)*Brunhilde Oczko (Technical assistance)Jenny Eichhorst (Technical assistance)*Martina Ringling (Technical assistance)

External funding

Deutsche Forschungsgemeinschaft „Degradations-Mechanismen des humanen Vasopressin-V2-Rezeptors und einiger von Patienten mit X chromoso-maler nephrogener Diabetis insipidus isolierten V2-Rezeptormutanten“ (He 4486/1-1 und 1-2)Ricardo Hermosilla (Charité - University Medicine Berlin),Burkhard Wiesner

Selected publications (FMP authors in bold)Geissler D, Kresse W, Wiesner B, Bendig J, KettenmannH, Hagen V (2003) DMACM-Caged Adenosine Nucleotides:Ultrafast Phototriggers for ATP, ADP, and AMP Activatedby Long-Wavelength Irradiation. ChemBioChem 4, 162-170

Hagen V, Frings S, Wiesner B, Helm S, Kaupp UB, BendigJ (2003) [7 (Dialkylamino)-coumarin 4 yl]methyl-cagedcompounds as ultrafast and effective long-wavelengthphototriggers of 8 Bromo-substituted cyclic nucleotides.ChemBioChem 4, 434-442

Meyer T, Marg A, Lemke P, Wiesner B, Vinkemeier U(2003) DNA binding controls inactivation and nuclearaccumulation of the transcription factor Stat1. Genes Dev17, 1992-2005

Tamma G, Wiesner B, Furkert J, Hahm D, Oksche A,Schaefer M, Valenti G, Rosenthal W, Klussmann E (2003)The prostaglandin E2 analogue sulprostone antagonizesvasopressin-induced antidiuresis through activation ofRho. J Cell Sci 116, 3285-3294

Tsunoda S, Wiesner B, Lorenz D, Rosenthal W, Pohl P(2004) Aquaporin 1, nothing but a water channel. J BiolChem 279, 11364-11367

Henn V, Edemir B, Stefan E, Wiesner B, Lorenz D, Theilig F,Schmitt R, Vossebein L, Tamma G, Beyermann M, KrauseE, Herberg FW, Valenti G, Bachmann S, Rosenthal W,Klussmann E (2004) Identification of a novel A-kinaseanchoring protein 18 isoform and evidence for its role inthe vasopressin-induced aquaporin 2 shuttle in renal prin-cipal cells. J Biol Chem 279, 26654-26665

Wüller S, Wiesner B, Löffler A, Furkert J, Krause G, Her-mosilla R, Schaefer M, Schülein R, Rosenthal W, OkscheA (2004) Pharmacochaperones post-translationally enhan-ce cell surface expression by increasing conformationalstability of wild-type and mutant vasopressin V2 receptors.J Biol Chem 279, 47254-47263

Collaborations

Prof. Ricardo HermosillaPathology of signal transductionCharité-Universitätsmedizin Berlin

PD Dr. Alexander OkscheG protein-coupled receptors: Signal transduction andregulation of cell surface expressionCharité-Universitätsmedizin Berlin


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MOLECULAR CELL PHYSIOLOGYGroup Leader: Dr. Ingolf Blasig

STRUCTURE, FUNCTION, AND REGULATIONOF CELL-CELL CONTACT PROTEINS

This group investigates signal transduction pathwaysincluding cell-cell and protein-protein interactions undernormal and pathological conditions, such as oxidativestress in the brain. The aim of these studies is to exploremolecular aspects of structure, function and regulation ofblood-brain barrier (BBB) proteins for the development ofnew pharmacological approaches to improve treatmentof cerebral diseases, such as stroke, inflammation,epilepsy etc. Another aspect is to develop approaches toopen the BBB for pharmacokinetic application, e. g. impro-ved administration of such antitumor or antiepileptic drugswhich are non-permeable through the BBB.

A main focus was the self-association mechanism oftransmembrane and membrane associated proteins form-ing the tight junctions (TJ). The regulatory and transmem-brane TJ protein occludin was found colocalizing withincell membrane contacts of the same cell and could becoprecipitated with itself (intracellular association).Differently tagged TJ strand-forming claudin-5 also colo-calized in the cell membrane of the same cell and showedfluorescence resonance energy transfer (intracellularassociation). This demonstrates self-association both ofoccludin and of claudin-5 in one cell membrane. For occlu-din, dimerization of the cytosolic C-terminal coiled coil-domain was identified. In claudin-5, we detected that thesecond extracellular loop is a dimer. Thus, occludin mayself-associate via its coiled coil-domain and claudin-5 viaits second extracellular loop. Since the transmembranejunctional adhesion molecule JAM is known to dimerize inTJ, we hypothesize that homodimerization is a structuralfeature of transmembrane TJ proteins. This potential iscorroborated by our finding that the recruiting protein ofthe transmembrane TJ proteins, the membrane-associa-ted ZO-1 also forms a dimer. A general dimerization princi-ple of transmembrane TJ proteins via the ZO-1 has beenworked out. This principle may serve as a common featureof TJ assembly (Figure). This work was supported bygroups of the FMP (G. Krause, Structural Bioinformatics;E. Krause, Mass Spectrometry) and MDC (K. Gast; M.Kolbe).

The occludin-ZO-1 interaction was studied in more detail.Binding studies by SPR and peptide mapping combinedwith CD studies and molecular modelling indicated that

occludin’s coiled-coil domain interacts as a three-helixbundle with three helices on the SH3-hinge-GuK unit ofZO-1. A similar association was found between ZO-1 andthe adherens junction protein α-catenin. Ion bindings bet-ween the basic helices of ZO-1 and the acidic ones inoccludin were also identified. In conclusion, a commonmolecular mechanism of forming intermolecular helicalbundles between ZO-1 and α-catenin and occludin wasidentified as a general molecular principle organizing theassociation of ZO-1 at adherens and tight junctions. Colla-boration within the FMP (G. Krause, Structural Bioinfor-matics; M. Beyermann, Peptide Chemistry), and with theFree University (O. Huber) and the Humboldt University (J. Schneider-Mergener) Berlin.

Further studies were accomplished on the effects of hypo-xia on cultures of brain capillary endothelial cells (BCEC).In addition to identifying proteins that are up- or down-regulated after hypoxia, activity data were acquired forselected proteins. Novel proteomic techniques have beenintroduced which allow the elucidation of signalling path-ways by analyzing post-translational modifications (e. g.,protein phosphorylation). Moreover, investigations wereaimed at improving the conditions for targeting low abun-dant proteins using the Rotofor© technology (collaborationE. Krause, Dept. Peptide Chemistry; D. Stanimirovic/Otta-wa, Canada) as well as for membrane proteins. Earlierresults suggested that cytokines are involved in theresponse to oxidative stress. Continuative investigationsshowed that exposure to interleukin-1ß results in prolife-ration of the cells, nerve growth factor (NGF) release andexpression of NGF receptors. This is a new pathway inBCEC which, hence, can modulate BBB functions (colla-boration C. Humpel, Innsbruck/Austria).

Clinical studies were continued to support our experimen-tal findings that oxidative stress may injure the BBB andthat these disturbances can be treated. Thus, mood dis-orders with depression were studied and indications werefound for opening of the BBB. BBB disturbances and schi-zophrenic events were reduced by pharmacologicalapproaches targeted, among others, to astrocytes main-taining BBB properties in the barrier forming brain endo-thelium (collaboration M. Schroeter, Max Planck Institute,Leipzig).

Group members

Dr. Reiner F. HaseloffDr. Jörg PiontekDr. Christine Rückert*Dr. Darkhan Utepbergenov*Anna Y. Andreeva (Doctoral student)*


Manjot Singh Bal (Doctoral student)Dörte Lorberg (Doctoral student)*Kerstin Mikoteit (Doctoral student)*Sebastian L. Müller (Doctoral student)Juliane Walter (Doctoral student)*Lars Winkler (Doctoral student)Inga Roswadowski* (Student)Jenny Kirsch (Student)*Markus Heine (Student)*Birgit Lassowski (Student)*Christian Niehage (Trainee)*Katrin Schulz (Student)*Ariane Schuster (Student)*Constanze Wolf (Student)*Nikolaj Zuleger (Student)*Barbara Eilemann (Technical assistance)Gislinde Hartmann (Technical assistance)

External funding

Deutsche Forschungsgemeinschaft„Extrazelluläre Loops von Blut-Hirnschranken-Proteinen“(BL 308/7-1)Ingolf Blasig

Deutsche Forschungsgemeinschaft„Wechselwirkungen von Blut-Hirnschranken-Proteinen“(BL 308/6-1, 6-2, 6-3)Ingolf Blasig, Gerd Krause

Deutsche Forschungsgemeinschaft „Lokalisation und Phophorylierungsmuster von Occludin“(GK 238/3, Teilprojekt im Graduiertenkolleg „Schadenme-chanismus im Nervensystem – Einsatz von bildgebendenVerfahren“ Ingolf Blasig

Bundesministerium für Bildung und Forschung„Molecular Fingerprinting of the Blood-Brain Barrier inHypoxia – Targeting Brain Vessels to Treat Stroke“, NRCReiner Haseloff

Deutscher Akademischer Austauschdienst„Molecular pharmacology of the HISS-resistin system”(PPP-Ungarn 324/ssch)Ingolf Blasig

Deutscher Akademischer Austauschdienst„Natural polyphenols in the cardiovascular system”(323/bis Slowakei)Ingolf Blasig

FIGURE 1Scheme of the dimerization concept showing that transmembrane TJ proteins, as well as ZO-1 may dimerize. Claudin-5 dimerizes via its secondextracellular loop (2.ECL; solid double arrow) and occludin via its coiled coil-domain (CC; interacting cylinders). JAMs are known to dimerize viaits extracellular domain (asterisk). ZO-1 dimerizes via its SH3-GuK unit. The positively charged (+) helices of ZO-1 (H1, H2 in GuK; CC1 betweenSH3 and GuK) bind to negatively charged (-) CC-domain of occludin (two dotted arrows). PDZ-domain 3 of ZO-1 can associate to JAMs, PDZ-1to claudins. N, N-terminus; yellow circles, disulfide bridge in claudin-5 first and occludin last loop.

Claudin-5 dimer JAM dimer Occludin dimer

PDZ domains

GuK domain

extra cellular

cell membrane

SH3 domain

ZO-1 dimer


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European CommunityMarie Curie Grant (HPMT-CT-2001-00399)G. Schreibelt

Schering-Stiftung„Phosphorylierung von Occludin” (Stipendium)Anna Andreeva


Müller SL, Portwich M, Schmidt A, Utepbergenov DI,Huber O, Blasig IE, Krause G (2005) The tight junction pro-tein occludin and the adherens junction protein β-cateninshare a common interaction mechanism with ZO-1. J BiolChem, in press

Zassler B, Blasig IE, Humpel C (2005) Protein delivery ofcaspase-3 induces cell death in malignant C6 glioma andbrain capillary endothelial cells. J Neuro-Oncology, inpress

Haseloff RF, Blasig IE, Bauer H-C, Bauer H (2005). Insearch of the astrocytic factor(s) modulating blood-brainbarrier functions in brain capillary endothelial cells in vitro.Mol Cell Neurobiol 25, 25-39

Schmidt A, Utepbergenov DI, Mueller SL, Beyermann M,Schneider-Mergener J, Krause G, Blasig IE (2004) Occlu-din binds to the SH3-hinge-GuK unit of zonula occludensprotein 1 - potential mechanism of tight junction regula-tion. Cell Mol Life Sci 61, 1354-1365

Moser KV, Reindl M, Blasig IE, Humpel C (2004) Braincapillary endothelial cells proliferate in response to NGF,express NGF receptors and secrete NGF after inflamma-tion. Brain Res 1017, 53-60

Schroeter ML, Abdul-Khaliq H, Frühauf S, Höhne R, SchickG, Diefenbacher A, Blasig IE (2003) Serum S100B is increa-sed during early treatment with antipsychotics and in defi-cit schizophrenia. Schizophrenia Res 62, 231-236

Collaborations

Hartwig Wolburg, Universität TübingenWolf-Hagen Schunck, Max-Delbrück-Center für Moleku-lare Medizin Berlin-Buch, GermanyMarina Bigl, Universität Leipzig, GermanyHannelore Haase, Max-Delbrück-Center für MolekulareMedizin Berlin-Buch, GermanyDanica Stanimirovic, Institute of Biological Sciences, NRC,Ottawa, CanadaHans-Christian Bauer, Universität Salzburg, AustriaMaria Balda, University College London, UKChristian Humpel, Universität Innsbruck, AustriaStephan Christen, Universität Bern, Switzerland

BIOCHEMICAL NEUROBIOLOGYGroup Leader: Dr. Wolf-Eberhard Siems

UNEXPECTED FUNCTIONS OF PEPTIDOLY-TIC ENZYMES

The Biochemical Neurobiology Group deals with the bio-chemical, pharmaceutical and molecular aspects of pep-tidases. The current research is focused on angiotensin-converting enzyme (ACE), neutral endopeptidases (NEP)and some related enzymes. We evaluate their biochemi-cal and functional relations to various disorders inhumans, like alcohol addiction, obesity, neuronal disor-ders, problems in male fertility and heart diseases.

Peptidases play a frequently underestimated role in main-taining and controlling essential functions in the body.Anthony Turner, one of the Nestors of research on NEP,wrote: “Peptidases and peptidolysis play vital roles in cel-lular processes from fertilization – to death”. A look at theACE confirms this sentence: This enzyme plays a key rolenot only for regulation of blood pressure, but also at thebeginning and at the end of life: it is essential for mamma-lian fertilization (male ACE-knock out mice are infertile inspite of normal sperm and libido) as well as for apoptosis,in which angiotensin II (Ang II, the main product of ACE) isessentially involved.In recent years, the use of molecular techniques and ofgenetically modified animals in peptidase research resul-ted in tremendous progress and innovative perspectives.In 2003 A. Turner commented the newly found functions ofACE and NEP as follows: “... (they) provide new avenuesfor the treatment of some of the major human diseases ofthe aging population of the Western world: cardiovascu-lar disease, cancer and dementia”.

ACE, NEP and related enzymes are transmembranalmetallopeptidases, which are prevalent in many tissuesand cleave a broad spectrum of endogenous substrates.They consist of a short cytoplasmatic domain, a singletransmembranal part and a very big extracellular part,which includes the catalytic domain(s), each of them con-taining one zinc ion.

ACE and NEP play an important role in circulation, andinhibitors of the two enzymes, especially ACE inhibitors,are widely used for treatment of hypertension and forcardioprotection. Because of their broad spectrum ofother substrates, these enzymes are also significantlyinvolved in many other pathophysiological processes.

In recent years, a lot of novel, in part surprising resultswere published on ACE and NEP. One example concerns

ACE2, an enzyme closely related to ACE: in addition, to itspeptidolytic activity, ACE2 was identified as the essentialreceptor of the SARS virus, and represents the entry por-tal of the body for the virus.

Kohlstedt et al. (2002) described receptor-like functionsalso for ACE. They proved e.g. that the reaction of ACE withsome, but not all substrates or inhibitors resulted in spe-cific phosphorylation at its own cytoplasmatic site.Another unexpected finding: the two enzymes, especiallythe NEP, play an important role in degrading the Alzhei-mer’s disease (AD) peptide ß-amyloid (Aß). This knowl-edge is expected to lead to new basic approaches forunderstanding and treatment of this disease.

In the Biochemical Neurobiology Group additional func-tions of these peptidases have been discovered during thelast years.

ACE and voluntary alcohol consumptionOur experiments of the last years had demonstrated thatAng II, the main product of ACE, directly influences volun-tary consumption of alcohol. Open questions concern therole of central and peripheral Ang II, the involved recep-tor(s) and the downstream signaling.

We characterized the role of central Ang II in alcohol in-take by using transgenic rats [TGR(ASrAOGEN)680], whichexpress an antisense RNA against angiotensinogen andconsequently have sharply reduced Ang II levels exclu-sively in the central nervous system. These rats consumedmarkedly less alcohol in comparison to their wild-typecontrols. Moreover, Spirapril, an ACE inhibitor, which pas-ses the blood-brain barrier, did not alter voluntary alcoholconsumption in the TGR(ASrAOGEN)680, but it significant-ly reduced alcohol intake in wild-type rats. Studies in differ-ent types of knockout mice have proved that the effect ofAng II on alcohol consumption is mediated by the angio-tensin receptor AT1, whereas the AT2 receptor and thebradykinin B2 receptor are not involved. With reference tosignal transduction we found that the TGR(ASrAOGEN)680with the very low central Ang II showed a markedly redu-ced dopamine concentration in their ventral tegmentalarea (VTA), confirming a role of dopaminergic transmissionin Ang II-controlled alcohol preference. Our results indi-cate that a distinct drug-mediated control of the centralrenin-angiotensin system (RAS) could be a new principlefor therapy of alcohol disease.

NEP-deficient mice – a model of human obesityIn long-lasting experiments on the functions of NEP, weobserved that elderly NEP-deficient mice (NEP-/-) had aconsiderably higher body weight than wild-types. In con-

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trast to other animal models of obesity, but in accordancewith typical human obesity, the differences in body weightbecame most apparent in the second half of life. NMR-stu-dies showed that the higher body weight in NEP-/- mice isexclusively due to an accumulation of fat. The molecularbasis of NEP-related obesity in mice and the pathophysio-logical consequences are presently being investigated.NEP is known to hydrolyze a great number of hormones,among them orexigenes (peptides stimulating food intake),like NPY, MCH and some of the opioids. Furthermore, seve-ral other peptides with effects on food ingestion also con-tain amino acid sequences that should be hydrolyzed byNEP (e.g. galanin, ghrelin, orexin). The catabolic effect ofNEP on these peptides and a more detailed biochemicalanalysis of old NEP-/- mice with regard to a new obesitymodel are still being investigated at present.

In contrast to other genetically modified animals expres-sing obese phenotypes, the alterations in NEP-/- mice con-cern a relatively great number of substrates and receptorsystems. Therefore, these mice may be a better animalmodel of the typical, polyfactorial human obesity. Theseresults and derived diagnostic and therapeutic applica-tions are summarized in a joint patent application of FMP,Charité und Free University of Berlin.

NEP deficiency in mice: learning experimentsNEP belongs to the enzymes which are able to catabolizethe amyloid ß-peptide (Aß). This important function of NEPwas recently confirmed by several in vitro as well as invivo studies and is now being intensively discussed as toits impact in the pathogenesis and therapy of AD.

But in even very old NEP-/- animals immunostaining exper-iments with brain slices and monoclonal antibodiesagainst murine Aß-peptide (cooperation with Multhaup,Heidelberg/Berlin) did not reveal any Aß depositions.

Therefore, further experiments focused on differences inthe NEP-induced degradation of human and murine Aß orAß-partial sequences by using purified enzyme as well asmembrane preparations of NEP-/- and NEP+/+ mice. Aßpeptides of humans and rodents had indeed differentdegradation rates (mAß>hAß). However, the differencesin Aß degradation by NEP cannot be the only reason forthe general lack of AD-like processes in rodent brains. Ourdegradation studies indicated that further membraneenzymes are involved in Aß catabolism. Surprisingly, alsoACE degraded Aß by means of an endopeptidolytic attack.

However, we found in behavioral experiments that the veryold homozygous NEP-knockout mice (>2 years) displayed

FIGURE 1Catabolism of murine ANP and BNP by lung membranes of wild-type and NEP-knockout mice: NEP is involved in the cleavage of ANP only.A large percentage of the ANP-degradation and the total BNP-degrading activity (green arrows) cannot be ascribed to NEP (black arrow). Aninteresting result is the upregulation of the non-NEP activity (yellow arrows) in NEP-deficient membranes, so that the degradation is faster thanin wild-type membranes.

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an even better performance in learning tests, e.g. in a Mor-ris water maze, compared to their wild-type littermates.This result was confirmed in long-term potentiation (LTP)experiments in two brain regions (hippocampus and amyg-dala). In contrast, there were no differences between thetwo genotypes in younger animals (~9 months). The mole-cular background of this effect is still unclear. Theseexperiments demonstrate the Janus head-like action ofNEP in brain function: it improves the brain function bydegradation of the plaque-forming Aß-peptides, and itapparently degrades other peptides which improve learn-ing processes. Involved peptides are still unknown. Gluca-gon-like Peptide-1 (GLP-1) could play a role. This peptidestrengthens cognitive functions, and we found a greaterstability of this peptide in membrane preparations of NEP-/-- mice.

Domain-selective forms of ACETo analyze the newly discovered ACE-functions and theunexpected interactions with substrates and inhibitors inmore detail, we transfected CHO cells with the followingmurine constructs: a) wild-type ACE (both domains areintact), b) C-terminal ACE, c) N-terminal ACE, d) inactivat-ed ACE (no intact domain). The specificity of these ACE-

forms was characterized by Western blots and enzymaticstudies with domain selective substrates (Hip-His-Leu, Z-Phe-His-Leu, Ac-SDKP), and the clones each with thehighest enzyme activities were selected.

These different domain-selective ACE forms are now usedto investigate

(i) the receptor-like function of cell-bound ACE,(ii) the unusual endopeptidolytic activity of ACE on Aß and

Aß partial sequences,(iii) the role of angiotensin (1-7) as substrate and inhibitor.

Peptidolytic degradation of natriuretic peptidesNatriuretic peptides (NP), like atrial- (ANP), B-type- (BNP)and C-type natriuretic peptide (CNP), are cyclic peptidehormones with relevance to cardiovascular, endocrineand renal homeostasis. All three peptides contain an intact17 amino acid disulfide-linked loop which is essential fortheir biological activity. Two different mechanisms are dis-cussed as being responsible for the inactivation of the NP:

(i) binding to specific receptors (C-type-receptor) withsubsequent internalization and degradation,

(ii) degradation by extracellular peptidases.

FIGURE 2A: Three-dimensional structure of the catalytic centre of

NEP. B: Hypothesis of the NEP – NP interaction.

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two large tails:spatial clashes

• NP moves into the cavity of NEP• Complementary recognition sites are supporting the orien-

tation of NP within the cavity• Large N - and C - terminal tails (simultaneously) hinder

the orientation towards the catalytic site

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The NEP is generally accepted to be the main enzymewhich initially catabolizes NP by cleavage within the loopat the Cys-Phe bond.

In HPLC-monitored degradation studies we analyzed thecatabolism of mouse ANP and BNP by membranes of dif-ferent organs from NEP-deficient and wild-type mice. Weobserved an unexpected result: Both NP peptides wererapidly degraded by mouse lung and kidney membranes,but in contrast to the generally published opinion, NEP wasonly involved in the cleavage of ANP. A large percentageof the ANP-degrading and the total BNP-degrading can-not be ascribed to NEP. An interesting side result was theup-regulation of the catabolic activity in NEP-deficientmembranes resulting in an unequivocally faster degrada-tion than in wild-type membranes. (Figure 1)

The next aim of the project will be the characterization ofthis/these still unknown NP-degrading enzyme(s) and itsmode of action. A specific inhibition of these peptidaseswould protect the NP. This would potentiate the cardiopro-tective action of NPs and possibly enable new therapeu-tic treatments.

In a second part of this project we want to find out themolecular basis of the different degradation of NP by NEP.In a first step, we proved the influence of the chemicalstructure of NP on their catabolism by comparing NP ofdifferent species with various structure modifications.These degradation studies were performed with recombi-nant NEP. The observed degradation rate was dependenton the size of the N- and C-terminal tails of the NP. Onlyhuman BNP was totally resistant in our test. These resultsenabled a first model for the molecular interactions bet-ween the catalytic center of NEP and NP (Figure 2).

To prove this model, we plan studies with NEP and NPderivates from human BNP with (a) shorter terminal tails or(b) replaced amino acids in the highly conserved inner-loop regions. This should help to detect why human BNPis not a substrate for NEP. Moreover, modified peptidase-resistant NP-analogues are interesting for potential thera-peutic use.

Group members

Dr. Winfried Krause**Matthias Becker (Doctoral student)* Xiaoou Sun (Doctoral student) Kristin Pankow (Doctoral student)*Bettina Kahlich (Technical assistance)

External funding

Deutsche Forschungsgemeinschaft„Neuropeptidasen und Alkoholkonsum – Untersuchungenan transgenen und knockout-Tieren“ (SI 483/3-1/2) Wolf-Eberhard Siems

ASTA MedicaWolf-Eberhard Siems

Strathmann AGWolf-Eberhard Siems


Gembardt F, Sterner-Kock A, Imboden H, Spalteholz M,Reibitz F, Schultheiss H-P, Siems WE, Walther T (2005)Organ specific distribution of ACE2 mRNA and correlatingpeptidase activity in rodents. Peptides 26, 1270-1277

Heringer-Walther S, Moreira MC, Wessel N, Saliba JL,Silvia-Barra J, Pena JL, Becker M, Siems WE, SchultheissHP, Walther T (2005) Brain natriuretic peptide predicts sur-vival in Chagas disease more effectively than atrial natriu-retic peptide. Heart 91, 385-387

Maul B, Walther T, Krause W, Pankow K, Becker M,Gembardt F, Alenina N, Bader M, Siems WE (2005) CentralAngiotensin II Controls Alcohol Consumption via its AT1Receptor. FASEB J 19:1474-1481

Walther T, Stepan H, Pankow K, Becker M, Schultheiss HP,Siems WE (2004) Biochemical analysis of neutral endopep-tidase activity reveals independent catabolism of atrialand brain natriuretic peptide. Biol Chem 385, 179-184

Walther T, Stepan H, Pankow K, Gembardt F, Faber R,Schultheiss HP, Siems WE (2004) Relation of ANP and BNPto their N-terminal fragments in fetal circulation: evidencefor enhanced neutral endopeptidase activity and resist-ance of BNP to neutral endopeptidase in the fetus. BJOG111, 452-455

Siems WE, Maul B, Wiesner B, Becker M, Walther T,Rothe L, Winkler A (2003) Effects of kinins on mammalianspermatozoa and the impact of peptidolytic enzymes.Andrologia 35, 44-54

Collaborations

Thomas Walther, EMC Rotterdam, The NetherlandsMichael Bader, MDC BerlinDoris Albrecht, Humboldt-Universität zu BerlinGisela Grecksch, University of MagdeburgSergej Danilov, University of Chicago, USA


BIOPHYSICSGroup Leader: PD Dr. Peter Pohl

COMBINED TRANSPORT OF WATER ANDIONS THROUGH MEMBRANE CHANNELS

Life in all its diversity became possible due to the separa-tion of living entities from the lifeless and hostile environ-ment by means of membranes. It requires preservation ofselective transmembrane material and informationexchange. During evolution cells developed, the functionof which depends on the well-controlled interplay andmaterial exchange between different compartments. Thedifferent transport functions are performed by a sophisti-cated apparatus of membrane proteins, each of them isfulfilling a very distinct function. Investigation of the lattercomprises the main interest of the biophysics group. Weaim to explore the molecular mechanisms of water, pro-ton and oxygen movement. Additional activities in the areaof applied biophysics are devoted to selected modifiers ofmembrane permeability (e.g. ribosome inactivating pro-teins and block copolymers). In this short report, we willfocus on water transport. The investigations are carriedout on different levels of organization: starting from pro-teins reconstituted into planar and vesicular membranes,followed by proteins overexpressed in different cell linesand completed by proteins identified in primarily culturedcell monolayers.

Water transport by aquaporinsWater transport is essential to all forms of life. Never-theless, the pathways taken by water across a membranebarrier and the mechanism of solute-solvent coupling arestill not entirely resolved. Commonly, it is considered thatwater may pass the lipid bilayer by diffusion. The extent towhich the lipid part of membranes contributes to watertransport varies considerably between different cells. Epi-thelial cells, for example, have to maintain large chemicaland osmotic gradients. Consequently, the membranematrix has to be effectively impermeable to ions and mostother small molecules. Tightening of the lipid barrier requi-res proteinaceous transport routes of water. Proteins spe-cialized on water transport are called aquaporins. Wehave investigated the selectivity of several members of theaquaporin family both in artificial model membranes andin their cellular environment.

For example, in cooperation with the department “Mole-cular Medicine” (Burkhard Wiesner, Dorothea Lorenz,Walter Rosenthal) Dr. Tsunoda investigated the transportmediated by aquaporin-1 (AQP1). It is a membrane chan-nel which allows rapid water movement driven by a trans-

membrane osmotic gradient. The protein was claimed tohave a secondary function as a cyclic-nucleotide gatedion channel. However, upon reconstitution into planarbilayers, the ion channel exhibited a tenfold lower singlechannel conductance than in Xenopus oocytes and a hun-dredfold lower open probability (<10-6) of doubtful physio-logical significance (Saparov et al., 2001, J.Biol.Chem.276:31515). Investigating AQP1 expressed in humanembryonic kidney cells, we now have shown that thediscrepancy is not due to alterations of AQP1 propertiesupon reconstitution into bilayers but rather due to regula-tory processes of the oocyte expression system that mayhave been misinterpreted as AQP1 ion channel activity. Asconfirmed by laser scanning reflection microscopy, from0.8 to 1.4 x 106 AQP1 copies per cell contributed to osmo-tic cell swelling. The proper plasma membrane localiza-tion was confirmed by observing the fluorescence of theN-terminal yellow fluorescent protein tag. Whole cellpatch-clamp experiments of wild-type or tagged AQP1expressing cells revealed that neither cGMP nor cAMPmediated ion channel activity. The lack of significant CNGion channel activity rules out a secondary role of AQP1water channels in cellular signal transduction.

Anomalously high water mobili ty in membraneion channelsAlong with aquaporins, a variety of other membrane pro-teins is likely to contribute to transmembrane water move-ment as well. Dr. Saparov carried out simultaneous meas-urement of the electrical and hydraulic conductivities of abacterial potassium channel. He folded the artificial mem-branes from two protein containing lipid monolayers in theaperture of a teflon film. Water flow was measured byimposing an osmotic gradient across these bilayers anddetecting the resulting small changes in solute concentra-tion close to the membrane surface with scanning ionselective microelectrodes. Dr. Saparov made the surpris-ing observation that the amount of transported watermolecules exceeds the number of transported ions byseveral orders of magnitude. Water molecules are con-strained to move with K+ ions through the narrow part ofthe KcsA channel because of the single file nature oftransport. In the presence of an osmotic gradient, a watermolecule requires less than 10 picoseconds to cross thepurified protein reconstituted into planar bilayers. RinsingK+ out of the channel, water may be 1000 times faster thanthe fastest experimentally observed K+ ion and 20 timesfaster than the one dimensional bulk diffusion of water(Fig. 1). Both the anomalously high water mobility and itsinhibition observed at high K+ concentrations are consist-ent with the view that liquid-vapor oscillations occur dueto geometrical confinements of water in the selectivity

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filter. These oscillations, where the chain of moleculesimbedded in the channel (the “liquid”) cooperatively exitsthe channel leaving behind a near vacuum (the “vapor”),so far have only been discovered in hydrophobic nanopo-res by molecular dynamics simulations (Hummer, G.,Rasaiah, J.C. & Noworyta, J.P. (2001) Nature 414, 188-190;Beckstein, O. & Sansom, M.S.P. (2003) Proc. Natl. Acad.Sci. USA. 100, 7063-7068).

Our results clearly show that in single file transport, watermolecules are more than just spacer molecules betweenthe ions. They are not only required for electrostatic rea-sons but add important features to the overall transportprocess. Whether the fast transport mode occurs in Na+,Ca2+, other K+ channels or even in members of the aquapo-rin family, which all realize single file transport, remains tobe elucidated, as well as the physiological importance ofthis phenomenon. With respect to the high density of K+

channels in the nodes of Ranvier, for example, a contribu-tion to water homeostasis in neurons is likely.

Confinement of water into a very narrow geometry led toprofound changes of its physico-chemical properties. As aresult the mobility of water molecules inside the channeland in the bulk differed by more than an order of magni-tude. Further investigation of the ultrafast diffusion will be

conducted in the frame of the priority program of the Deut-sche Forschungsgemeinschaft “Micro- and Nanofluidics”.

Group members

Dr. Sapar M. SaparovDr. Satoshi Tsunoda Dr. Oxana O. Krylova Dr. Valentina Margania**Steffen Serowy (Doctoral student )*Rustam Mollajew Matthias Prigge (Student)*

Prof. Dr. Yuri Antonenko (Guest scientist)*Dr. Valerij Sokolov (Guest scientist)*Dr. Artem Ayuyan (Guest scientist)*Christina de Souza (Guest scientist)*Alexander Lentz (Guest scientist)*Elena Sokolenko (Guest scientist)*

External funding

Deutsche ForschungsgemeinschaftHeisenbergstipendium (Po533/7-1)Peter Pohl

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FIGURE 1Fast osmotic water flow through the bacterial potassiumchannel (KcsA) occurs only when all ions are rinsed out ofthe filter. This situation is illustrated in the figure where allbinding sites are occupied by water molecules (red). Thepotassium ion (yellow) has been placed into the cavitywhich in contrast to the selectivity filter is wide enough toallow different molecules to pass each other (Fig.3 from Biol.Chem. 2004, 385: 921–926).


Deutsche Forschungsgemeinschaft“Single file water transport across peptidic nanopores”(Po533/11-1)Peter Pohl

Deutsche Forschungsgemeinschaft„Molekulare Mechanismen des Wassertransports durchEpithelzellmonoschichten“ (Po533/8-1, 8-2)Peter Pohl

Deutsche Forschungsgemeinschaft„Wechselwirkung zwischen ebener Bilipidmembran undPhotosensibilisator“ (Po533/4-3)Peter Pohl

Deutsche Forschungsgemeinschaft„Migration von Protonen an der Oberfläche ebener Bilipid-membranen“ (Po533/5-2, 5-3)Peter Pohl

Deutsche ForschungsgemeinschaftKooperation mit der Lomonossov-Universität Moskau (436RUS113/551)Peter Pohl

Deutsche ForschungsgemeinschaftKooperation mit dem Frumkin-Institut für ElektrochemieMoskau (436 RUS113/634)Peter Pohl

Volkswagenstiftung ”Control of membrane permeability with novel types ofamphiphilic macromolecules” (I/77743)Peter Pohl

Volkswagenstiftung ”Control of membrane permeability with novel types ofamphiphilic macromolecules” (I/80074)Peter Pohl


Pohl P (2004) Combined transport of water and ionsthrough membrane channels. Biol Chem 385, 921-926

Saparov SM, Pohl P (2004) Beyond the diffusion limit:Water flow through the empty bacterial potassium chan-nel. Proc Natl Acad Sci U. S. A 101, 4805-4809

Krylova OO, Pohl P (2004) Ionophoric activity of pluronicblock copolymers. Biochemistry 43, 3696-3703

Sun J, Pohl EE, Krylova OO, Krause E, Agapov II, Tonevit-sky AG, Pohl P (2004) Membrane destabilization by ricin.Eur Biophys J 33, 572-579

Tsunoda SP, Wiesner B, Lorenz D, Rosenthal W, Pohl P(2004) Aquaporin-1, Nothing but a Water Channel. J BiolChem 279, 11364-11367

Lorenz D, Krylov A, Hahm D, Hagen V, Rosenthal W, PohlP, Maric K (2003) Cyclic AMP is sufficient for triggering theexocytic recruitment of aquaporin-2 in renal epithelialcells. EMBO Rep 4, 88-93

Serowy S, Saparov SM, Antonenko YN, Kozlovsky W,Hagen V, Pohl P (2003) Structural Proton Diffusion alongLipid Bilayers. Biophys J 84, 1031-1037

Collaborations

- Proton migration (DFG Po 533/5-1, 2, 3; 436 RUS113/551):Prof. Yuri N. Antonenko, Lomonossov University Mos-cow, Belozersky Laboratory

- Photosensibilisator (DFG Po 533/4-3; 436 RUS113/634):Dr. Valerij Sokolov, Russian Academy of Science, Frum-kin Institute of Electrochemistry

- Blockcopolymers and multidrug resistance (VW): Prof.J. Kressler, Martin-Luther-Universität Halle; Prof. Dr.Frey, University of Mainz

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CYTOKINE SIGNALINGGroup Leader: Dr. Klaus-Peter Knobeloch

FUNCTIONAL ANALYSIS OF POSTTRANSLA-TIONAL PROTEIN MODIFICATION BY UBI-QUITIN AND UBIQUITIN LIKE MOLECULESUSING GENE TARGETING IN THE MOUSE

Covalent attachment of Ubiquitin and Ubiquitin like (UBL)molecules serves as an important regulatory mechanismcontrolling a pleiotropy of biological processes includingembryogenesis, cell cycle, growth control, and immuneresponse. Beside serving as a marker that targets proteinsfor proteasomal degradation Ubiquitin conjugation hasbeen demonstrated to be involved in the regulation of awide range of molecular mechanisms like protein localiza-tion, receptor degradation, transcriptional control andinteraction of proteins.

Thus distinct components of the Ub and UBL conjugationand deconjugation pathway might serve as novel targetsfor drug intervention.

In our group targeted mutagenesis in the mouse is usedto address the biological relevance and function directlyin the context of the whole organism. Current research isfocused on ISG15, an interferon stimulated ubiquitin likemolecule and UBPy, a deubiquitinating enzyme.

ISG15 is one of the most strongly induced genes uponinterferon treatment and like SUMO or NEDD8 belongs tothe family of ubiquitin-like modifiers. ISG15 can be conju-gated to distinct proteins and has been implicated in avariety of biological activities which encompass antiviraldefence, immune responses, and pregnancy. Mice lackinUBP43 (USP18), the ISG15 deconjugating enzyme whichbelongs to the family of deubiquitinating enzymes, deve-lop a severe phenotype with brain injuries and lethalhypersensitivity to Poly(I:C). It was reported that an aug-mented conjugation of ISG15 in the absence of UBP43induces prolonged STAT1 phosphorylation and that theISG15 conjugation plays an important role in the regulati-on of JAK/STAT and interferon signaling (Malakhova OA etal. 2003, Genes & Development 17, 455-460). It was alsopublished that UBP43-/-mice are more resistant againstinfection with lymphocytic choriomeningitis virus (LCMV)and vesicular stomatitis virus (VSV).

To directly assess the functional role of ISG15, we gene-rated mice lacking ISG15 via gene targeting in embryonicstem cells. ISG15-/- mice display no obvious abnormalities,and lack of ISG15 did not affect the development and com-position of the main cellular compartments of the immune

system. In contrast to UBP43-/- mice, the interferon-indu-ced antiviral state and immune responses directed againstVSV and LCMV were not significantly altered in the absen-ce of ISG15. Furthermore, interferon or endotoxin inducedSTAT1 tyrosine-phosphorylation, as well as expression oftypical STAT1 target genes remained unaffected by thelack of ISG15. Thus the role of ISG15 in the pathology of thephenotype of UBP43-/- and the specificity of UBP43 needsto be reassessed. Therefore, we generated mice deficientfor both molecules in an attempt to rescue phenotypicalterations of UBP43-/- mice, providing they are caused byan enhanced ISG15 conjugation. This approach is alsosuitable to test whether UBP43 possesses additionalfunctions beside acting as an ISG15 deconjugating enzy-me. These double knockout mice are currently under ana-lysis.

We were also able to clone a new mouse gene, which wenamed UBL14, that encodes a protein with 95% amino acidhomology to ISG15. To elucidate the role of this moleculein vivo, we also used a loss of function approach andgenerated mice deficient for UBL14, as well as for both,ISG15 and UBL14.

Conditional inactivation of the ubiquitin-speci-fic protease UBPy/USP8 Ubiquitination of proteins is now recognized to target pro-teins for degradation by the proteasome and for internali-zation into the lysosomal system, as well as to modifyfunctions of some target proteins. Although much progresshas been made in characterizing enzymes that link ubiqui-tin to proteins, the understanding of deubiquitinating enzy-mes is just beginning to evolve. The importance of deubi-quitinating enzymes has been demonstrated by theobservation that cellular key molecules like p53 or NF-kappa B are controlled by ubiquitin isopeptidases (Li et al2002, Trompouki et al 2003). However, so far there are noreports challenging the function of deubiquitinating pro-teins in the context of the whole organism.

UBPy (USP8) is an ubiquitin isopeptidase that is upregula-ted upon serum induction and was shown to bind to SH3domains of certain target proteins via an unusual recogni-tion motif. From patients with a myeloproliferative disordera fusion product of the p85beta subunit of phosphatidyli-nositol-3-kinase and UBPy could be isolated. In an initialapproach we have generated mice lacking UBPy. As thesemice die early in embryogenesis we generated mice thatallow conditional inactivation of the gene using the cre-loxP system. By mating the mice to Mx-cre we have in-activated the gene in adult mice and are analyzing thephenotypic alterations, and molecular mechanisms thatare affected by the lack of UBPy. In parallel, we genera-

ted mice lacking UBPy in specific hematopoietic cell linea-ges like T-cells and granulocytes/macrophages in order todetermine the functional role of UBPy in these cell linea-ges.

Group members

Sandra Niendorf (Doctoral student)Agnes Kisser (Doctoral student)Markus Wietstruck (Technical assistance)Anna Osiak (Doctoral student)*Liane Boldt (Technical assistance)

External funding

Deutsche Forschungsgemeinschaft „Herstellung und Haltung genetisch veränderter Mäuse“(TP Z3 im Sonderforschungsbereich 366 „Zelluläre Signal-erkennung und Umsetzung“)Ivan Horak

Deutsche Forschungsgemeinschaft„Untersuchungen zur Funktion des Interferon-stimuliertenGenes 15 (ISG15).“ (KN 590/1-1)Klaus-Peter Knobeloch


Osiak A, Utermohlen O, Niendorf S, Horak I, Knobeloch KP.(2005) ISG15, an interferon-stimulated ubiquitin-like pro-tein, is not essential for STAT1 signaling and responsesagainst vesicular stomatitis and lymphocytic choriomenin-gitis virus.Mol Cell Biol., 6338-6345

Van Spriel AB, Puls KL, Sofi M, Pouniotis D, Hochrein H,Orinska Z, Knobeloch K-P, Plebanski M, Wright MD (2004)A regulatory role for CD37 in T-Cell Proliferation. J Immu-nol 172, 2953-2961

Rosenbauer F, Wagner K, Zhang P, Knobeloch KP, IwamaA, Tenen DG (2004) pDP4, a novel glycoprotein secreted bymature granulocytes, is regulated by transcription factorPU.1. Blood 103, 4294-4301

Schuh K, Cartwright EJ, Jankevics E, Bundschu K, Lieber-mann J, Williams JC, Armesilla AL, Emerson M, OceandyD, Knobeloch KP, Neyses L (2004) Plasma membrane Ca2+ATPase 4 is required for sperm motility and male fertility. JBiol Chem 279, 28220-28226

FIGURE 1ISG 15 conjugation cycle

FIGURE 2Editing functions of deubiquitinating enzymes. Deubiquitinating enzy-mes may negatively regulate proteolysis or other signaling functionsof ubiquitination such as internalization or altered protein function byremoving the ubiquitin chain from the target proteins.


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Huebner A, Kaindl AM, Knobeloch KP, Petzold H, Mann P,Koehler K (2004) The triple A syndrome is due to mutationsin ALADIN, a novel member of the nuclear pore complex.Endorcr Res 30, 891-899

Collaborations

InternationalHerbert W. Virgin IV, Department of Pathology Washington University School of Medicine

Prof. Dr. Robert Krug, Institute for Cellular and MolecularBiology University of Texas at Austin

NationalProf. Dr. med. Oliver Liesenfeld, Institut für Mikrobiologieund HygieneCharité – Universitätsmedizin Berlin

PD Dr. Marco Prinz, Abt. NeuropathologieGeorg-August-Universität Göttingen

Dr. Olaf Utermöhlen, Institut für Medizin. Mikrobiologie,Immunologie und Hygiene Universität zu Köln

MOLECULAR MYELOPOIESISGroup Leader: Dr. Dirk Carstanjen

ICSBP – A CRITICAL TRANSCRIPTIONFACTOR FOR MYELOPOIESIS

The research aim of our group is to elucidate the role ofthe transcription factor ICSBP (interferon consensussequence binding protein) within hematopoietic cell deve-lopment. This hematopoietic specific transcription factoris a major element regulating transcriptional control ofmyelopoiesis. Mice deficient in this transcription factordevelop a proliferative syndrome characterized by theexpansion and accumulation of immature myeloid proge-nitor cells within the bone marrow and spleen. Further-more, it has been shown by our group that ICSBP influen-ces lineage choice of granulocyte-monocyte progenitorcells (GMP) leading to an imbalance in development andmaturation of progenitor cells in favor of granulocytes.

Genetic profile of myeloid developmentThis asymmetric lineage choice is at least partially due toa reduced response to a macrophage specific cytokine(macrophage colony stimulating factor: M-CSF). Our grouphas recently shown that M-CSF signal transduction inmacrophages is altered due to altered protease expressi-on in these cells and enhanced proteasomal degradationleading to shortened signaling. In contrast, the reason foraccumulation of immature progenitor cells and preferen-tial production of granulocytes is further obscure. In anapproach to decipher the function of ICSBP in immatureprogenitor cells as well as to define the expression profi-le of granulocyte versus erthrocyte precursors, we sortedGMP and erythrocyte progenitor (EP) cells by flow cyto-metry in collaboration with H.R. Rodewald, Ulm. Globalgene expression profiling was performed in these highlypurified cells and analyzed using Affymetrix technology.The data sets of this analysis have been published via theGEO database. These data now allow for the first time tostudy systematically the transcriptional profile of thesefunctional distinct cell populations. As a first approach, weidentified the global pattern of transcription factors speci-fically expressed in either lineage. While this analysis con-firmed the exclusive or highly preferential expression ofseveral transcription factors with pivotal importance forerythroid development (e.g. erythroid Krüppel-like factor,GATA-1, Friend of GATA-1 (FOG1)), we also identified seve-ral transcription factors specifically expressed in myeloidprogenitors. Several of those are known: Cebpα, Cebpβ,Gfi1, Fli1, PU.1, and Meis1 to name but a few, others havenot yet been implicated in myeloid development, e.g.Mef2c, KLF4, and SAT1b. Importantly, ICSBP has been

shown to be specifically expressed by GMP, and the RNAexpression level was within the group of the five transcrip-tion factors the most prominently expressed transcriptionfactors in these cells, in concert with prominent proteinsas PU.1, and Cebpα. These important findings further con-firms the pivotal role of ICSBP for myeloid developmentand will now allow to study systematically the analysis oftranscription factors regulating myeloid and erythroid celldevelopment using genetic approaches e.g., mouse dele-tion-mutants, RNA interference or retroviral overexpres-sion. These studies are currently being undertaken.

Dissecting the transcriptional network orche-strated by ICSBPWe further analyzed the global gene expression profile inthe GMP of the ICSBP mouse deletion mutant. This analy-sis revealed insight into the developmental program indu-ced by the loss of ICSBP within myeloid progenitor cells.We found several major myeloid transcription factorsdownregulated in the GMP of ICSBP-deleted mice. We arecurrently studying the contribution of these transcriptionfactors to the myeloproliferative syndrome of these miceusing mouse deletion mutants and retroviral overexpres-sion in bone marrow derived stem and progenitor cells.Furthermore, we are studying potential direct transcriptio-nal regulation of ICSBP on genomic target sequences. Wetherefore cloned several murine genomic sequences withknown or potential regulatory functions for the genes wefound differentially expressed in GMPs of ICSBP-deletedmice. Luciferase promoter studies are currently beingundertaken to identify the impact on ICSBP in direct orcombinatorial gene expression regulation with other pro-minent myeloid transcription factors. Furthermore, manyof the genes overexpressed in ICSBP-deleted mice aregenes preferentially expressed in mature granulocytic andmonocytic cells. Interestingly, at the GMP stage, there wasno clear preferential expression pattern of granulocyticgenes implicating that branching between monocytic andgranulocytic development occurs beyond the GMP stageand additional extrinsic factors, e.g. cytokine signaling, areresponsible for the preferential generation of granulocy-tes versus macrophages in the ICSBP deleted mouse, ahypothesis consistent with our previous findings showingabnormal M-CSF signaling in macrophages derived fromthe ICSBP deleted mouse.

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Group members

Maja Djurica (Doctoral student)*Jessica Königsmann (Doctoral student)*Axel Kallies (Doctoral student)*Joanna Selfe (Doctoral student)*Melanie Benedict (Technical assistance)

External funding

Deutsche Forschungsgemeinschaft„Myeloproliferatives Syndrom bei ICSBP-defizienten Mäu-sen: Identifizierung neuer potentieller onkotherapeuti-scher Targetstrukturen“ (TP G1 im Sonderforschungsbe-reich 506 „Onkotherapeutische Nukleinsäuren“)I. Horak

Deutsche Forschungsgemeinschaft„Gene in Entwicklung und Funktion von myeloiden Zellen“HO 493/12-1 und 12-2Ivan Horak

Deutsche Forschungsgemeinschaft„Die Rolle des Adaptorproteins Disabled-2 bei Zytokin-signalvermittlung in hämatopoetischen Zellen“ (CA 306/1-1)Dirk Carstanjen


Zatóvicova M, Tarabkova K, Svastova E, Gibadulinova A,Mucha V, Jakubickova L, Biesova Z, Rafajova M, Ortova GutM, Parkkila S, Parkkila AK, Waheed A, Sly WS, Horak I,Pastorek J, Pastorekova S (2003) Monoclonal antibodiesgenerated in carbonic anhydrase IX-deficient mice recog-nize different domains of tumour associated hypoxia-indu-ced carbonic anhydrase IX. J Immunol Methods 282, 117-134

Barmeyer C, Harren M, Schmitz H, Heinzel-Pleines U,Mankertz J, Seidler U, Horak I, Wiedenmann B, Fromm M,Schulzke JD (2004) Mechanisms of diarrhea in the inter-leukin-2-deficient mouse model of colonic inflammation.Am J Physiol Gastrointest Liver Physiol 286, G244-G252

Terszowski G, Waskow C, Conradt P, Lenze D, Koenigs-mann J, Carstanjen D, Horak I, Rodewald HR (2004) Pro-spective isolation and global gene expression analysis ofthe colony-forming unit-erythrocyte (CFU-E). Blood 105,1937-1945

FIGURE 1Bone marrow cells from ICSBP wt (upper) and deleted(lower part) were depleted of mature cells expressing mar-ker for granulocytes, monocytes, T-, B-cells and erythro-cytes. Those so-called lineage negative cells were furtherstained with antibodies against c-kit (stem cell factor recep-tor) as well as interleukin-3 receptor. c-kit high, Il-3 recep-tor positive cells contain so called granulocyte-monocyteprogenitor cells (GMP), megakaryocyte progenitor cells(MP), and common myeloid progenitor cells (CMP). Thosepopulations can be identified through the use of antibodiesagainst FcγR I and CD41. The GMP population contains onlycells that give rise to monocytes/macrophages and granu-locytes. This population was sorted to purity, RNA isolated,and global gene expression profiling was performed usingAffymetrix™ technology.

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MOUSE MODELSGroup Leader: Dr. Rüdiger Pankow

BCL-2 ASSOCIATED TRANSCRIPTIONFACTOR AND FMS INTERACTING PROTEIN

Btf (Bcl-2 associated transcription factor) and FMIP (fmsinteracting protein) are two genes which have been impli-cated in cellular regulatory processes. Both are intracel-lular proteins with subcellular distributions in the cyto-plasm and the nucleus. We have raised antibodies againstBtf and found its expression in a broad spectrum of tissu-es and organs including those of the hematopoieticsystem. Its highest expression, however, is found in brainregions of young developing mice. This expression de-clines with age, with Btf being detectable in only a fewneural cells in adulthood.

Due to its association with Bcl-2, Btf was thought to be atranscriptional repressor, with an apoptotic function. Sinceno rigorous evidence for a role of Btf in apoptosis wasfound, its biological function remains unknown. To gene-rate mice deficient for Btf we inserted a marker cassettecontaining the β-galactosidase gene into the btf gene. Theinserted β-galactosidase gene is under the control of theendogenous btf promoter which allows the detection ofbtf gene expression. Mice, homozygous for this mutation,lack all wild type btf transcripts and are deficient inBtf protein. These mice display a severe phenotype resul-ting in premature death. Molecular mechanisms behindthe phenotypic changes are being analyzed. To assess therole of Btf in the hematopoietic system we reconstitutedlethally irradiated wild type mice with fetal liver or bonemarrow cells from Btf deficient mice. These mice reconsti-tuted a Btf-/- hematopoietic system including all resultingcell lineages but do not develop any of the phenotypicsigns characteristic of Btf deficient mice. We could there-fore rule out the influences of the hematopoietic systemand conclude that other factors provide the major causefor the phenotype.

For FMIP, an interacting partner of the activated M-CSFreceptor, a possible role in myeloid differentiation wassuggested. By means of targeted gene disruption, wegenerated a mouse model with a null mutation of the fmipgene. Our studies revealed, however, that embryos lackingFmip die early during embryogenesis. Thus, to allow stu-dies of Fmip in physiological processes in the mouse, wehave generated FMIP conditional knockout mice using thecre/loxP system. By intercrossing these mice with Cre-deleter mouse strains we have obtained Fmip floxed micethat allow organ or cell lineage specific deletion of Fmip

and have used these models to study the function of FMIPand in particular, its relevance in myeloid development.

Group members

Dr. Rosel BlasigKerstin Bohne (Technical assistance)Janet Klemm (Technical assistance)Mina Thakur (Technical assistance)*


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CELLULAR SIGNAL PROCESSINGGroup Leader: Dr. Uwe Vinkemeier

KINETIC CONTROL OF GENE TRANSCRIPTION

We are studying the effects on gene transcription of agroup of extracellular signaling molecules termed cyto-kines, more than a hundred of which are presently known.Cytokines are secreted small proteins that fulfill crucialroles in cell differentiation and innate immunity. Bestknown are the interleukins and erythropoietine (Epo) fortheir role in immune cell differentiation, inflammation andlactation. Other cytokines such as LIF and Oncostatin Mcontrol stem cell development and cell growth, while theinterferons are known to cause growth arrest and protectcells against viral and microbial attack. Not surprisingly,cytokines are highly relevant for clinical applications.Several cell-activating cytokines are being tested in clini-cal trials, and interleukin-2 was the first drug approved forthe treatment of tumors. Today, treatment with interferonsis the standard therapy for several severe viral infections(e.g. hepatitis) as well as for some tumors (e.g. skin can-cer). However, overexpression of cytokines may alsocause diseases such as rheumatoid athritis. In thosesituations, inhibition of cytokine action is a therapeuticgoal. As the alteration of gene transcription is the basis ofcytokine action, we need to explore the intracellularevents that are triggered by cytokines in order to under-stand their effects in the normal and diseased state.

The Janus kinase (JAK) / signal transducer and activatorof transcription (STAT) pathways, first identified in theinterferon systems, are responsive to a wide range of cyto-kines and growth factors. The STAT proteins receive cyto-kine signals at intracellular receptor chains in the cyto-plasm and carry them into the nucleus, where they thenact as transcription factors. Thus, these proteins need tocross the nuclear envelope to functionally link the cellmembrane with the promoters of cytokine-responsivegenes. Movement of STATs in either compartment is diffu-sion controlled and not directed along permanent structu-res. However, passage through the nuclear gateways,named nuclear pore complexes (NPC), provides a formi-dable diffusion barrier to proteins the size of STATs (> 85kD for the monomer), as only ions and small molecules notexceeding 40 kD can freely enter the cell nucleus. Theanalysis of their nucleocytoplasmic translocation has toconsider that the STAT proteins exist in two different statesin terms of signaling: before the stimulation of cells withcytokines the STAT molecule exists in a non-tyrosine phos-phorylated state. Stimulation with cytokines increases theactivity of receptor-associated JAK kinases, leading to the

formation of tyrosine-phosphorylated STATs, whichinstantly assemble into homo- or heterodimers via cano-nical phosphotyrosine-SH2 domain interactions. Tyrosinephosphorylation is often described as STAT activationsince only the dimer is a high-affinity DNA-binding proteinrequired for the induction of cytokine-responsive genes.This is achieved by directly targeting cognate recognitionelements named gamma activated sites (GAS).

Carrier-dependent as well as carrier-independent modesof translocation are known to exist for the passage of pro-teins through the nuclear pore. The nuclear pore complexis a multi-protein structure that creates an aqueous chan-nel spanning the double membrane of the nuclear enve-lope. This structure with an estimated molecular mass of125 MDa in mammalian cells is composed of severalcopies of about 30 different proteins collectively callednucleoporins (Nups), many of which contain multiple phe-nylalanine-glycine (FG) repeats that are interspersed withpolar residues of varying number. Although the moleculardetails that allow passage through the pore remain to beelucidated, it is widely accepted that permeating molecu-les need to overcome the hydrophobic repulsion that isexerted by the FG repeat-rich interior of the nuclear pore.Biochemically carrier-free and carrier-dependent nucleo-cytoplasmic translocation are related. Docking to thenuclear pore is diffusion controlled for the two pathways,and in both cases the actual translocation process appe-ars to occur independent of metabolic energy via identi-cal interactions with channel components. Proteins thatcontain regions that enable them to directly engage in pro-ductive interactions with the nucleoporins are capable ofcarrier-independent nucleocytoplasmic transport, where-as the remaining proteins (also called cargo proteins)need to associate with transport factors, which act aschaperones during passage through the pore. The vastmajority of transport factors belongs to the karyopherinsuperfamily of proteins, which mediate either import orexport from the nucleus. Recognition of cargo proteins bythe transport factors requires the presence of loosely con-served stretches of amino acids on the cargo surface, ter-med nuclear localization -NLS- or nuclear export -NES-signals, respectively. The stability of cargo/karyopherincomplexes is determined by the small GTPase Ran, asRanGTP disrupts importin/cargo complexes but stabilizesthe formation of exportin/cargo-complexes. Thus, theRanGDP/RanGTP gradient that forms through the asymme-tric distribution of nucleotide exchange factors across thenuclear membrane discriminates cytosol from nucleo-plasm and hence confers directionality to the transportprocess.

Our lab discovered that the STATs make use of both ofthese translocation mechanisms. Due to their constantcarrier-independent and -dependent nucleocytoplasmiccycling the STATs are distributed throughout the cell, allthe time. Deviations from an even pancellular distributionresult from modulated nuclear export. This is caused eit-her by enhancing the translocation rate or by nuclearretention due to a co-factor-independent mechanism. Therecognition that the dephosphorylation reaction is underkinetic control of DNA binding provided a strikingly simpleexample of a self-controlling mechanism that integratescentral elements of cytokine-dependent gene regulation,namely receptor monitoring, promoter occupancy, andtranscription factor activity. It is important to point out thatnuclear accumulation is not a mechanism sui generis, butmerely reflects the formation of a conformation that is resi-stant to dephosphorylation. For this reason nuclear accu-mulation is not suited as a diagnostic marker of transcrip-tional activity. In contrast, the translocation rate of STATtranscription factors critically determines the outcome ofcytokine signaling and hence constitutes a potential spe-cificity determinant. The molecular mechanisms that giverise to flux modulation, either physiologically or in patho-logical situations such as microbial infections, and howthis affects both duration and strength of cytokine signals,as well as the overall sensitivity and latency of the systemwill be the task of future research. A further important areaof our research is post-translational modifications, withfocus on arginine methylation and sumoylation.

In the last years our lab has made a number of exciting andunexpected discoveries, which revealed that the STATsare an intriguing model system for studying the intracellu-lar dynamics of a signal transducer. In addition, it is increa-singly being appreciated that STAT nucleocytoplasmiccycling also constitutes an important area for microbialintervention. Currently, we are generating mice thatexpress STAT mutants. This will allow us to study theeffects of defective nucleocytoplasmic shuttling on deve-lopment and disease. These approaches are complemen-ted by chemical and structural biology to explore thepossibilities of rational intervention.

Group members

Prof. Dr. mult. Thomas Meyer Dr. Andreas BegittDr. Regis Cartier Dr. Ying Shan**Dr. Andreas Marg*Torsten Meissner (Doctoral student)*Inga Lödige (Doctoral student)Nicola Venta (Doctoral student)*

Mandy Kummerow (Technical assistance)Stephanie Meyer (Technical assistance)

External funding

Deutsche Forschungsgemeinschaft„Konstitutiver nucleocytoplanmatischer Transport vonSTAT1“ (VI 218/2) Uwe Vinkemeier Deutsche Forschungsgemeinschaft

Deutsche Forschungsgemeinschaft„Einfluss der Tyrosin-Dephosphorylierung auf die Zielgen-findung von STAT1“ (VI 218/3) Uwe Vinkemeier

Deutsche Forschungsgemeinschaft„Analyse einer konservativen Protein-Interaktionsdomä-ne“ (VI 218/4) Uwe Vinkemeier

Bundesministerium für Bildung und Forschung„Molekulare Grundlagen der zellulären Signalverarbei-tung“ (Sieger im Nachwuchsgruppenwettbewerb „Bio-Future“)Uwe Vinkemeier

European Molecular Biology Organization“Regulation of Transcription” (EMBO-Young-InvestigatorAward 2002)Uwe Vinkemeier

Selected publications (FMP authors in bold)Chen X, Bhandari R, Vinkemeier U, Van Den Akker F, Dar-nell JE Jr, Kuriyan J (2003) A reinterpretation of the dime-rization interface of the N-terminal domains of STATs. Pro-tein Sci 142, 361-365

Meyer T, Vinkemeier U, Meyer U (2003) MedizinischeImplikationen pharmakogenomischer Behandlungsstrate-gien. Ethik in der Medizin 12, 207-209

Meyer T, Vinkemeier U, Meyer U (2003) Evidence-basedmedicine – Was geht verloren? Ethik in der Medizin 14, 3-10



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Marg A, Shan Y, Meyer T, Meissner T, Brandenburg M,Vinkemeier U (2004) Nucleocytoplasmic shuttling bynucleoporins Nup153 and Nup214 and CRM1-dependentnuclear export control the subcellular distribution of latentStat1. J Cell Biol 165, 823-833

Meissner T, Krause E, Vinkemeier U (2004) Ratjadone andleptomycin B block CRM1-dependent nuclear export byidentical mechanisms. FEBS Lett 576, 27-30

Meissner T, Krause E, Lödige I, Vinkemeier U (2004)Arginine Methylation of STAT1: A Reassessment. Cell 119,587-589

Meyer T, Hendry L, Begitt A, John S, Vinkemeier U (2004)A single residue modulates tyrosine dephosphorylation,oligomerization, and nuclear accumulation of stat trans-cription factors. J Biol Chem 279, 18998-19007

Meyer T, Vinkemeier U (2004) Nucleocytoplasmic shuttlingof STAT transcription factors. Eur J Biochem 271, 4606-4612

Vinkemeier U (2004) Getting the message across STAT!Design principles of a molecular signaling circuit. J CellBiol 167, 197-201

Collaborations

Susan John, Kings’s Kollege London, Department of Immu-nobiology

Ilian Jelesarov and H. R. Bosshard, Universität Zürich,Institut für Biochemie

Gino Cingolani, Stony Brook University, USA

Ulrich Kubischeck, Friedrich-Wilhelm-Universität Bonn,Institut für Physikalische und Theoretische Chemie

Markus Czub, Canadian Science Center for Human andAnimal Health, Winnipeg, Canada

Thomas Höfer and Reinhard Heinrich, Institut für Theore-tische Biophysik, Humboldt-Universität zu Berlin

CHEMICAL BIOLOGY

INTRODUCTION

SECTION CHEMICAL BIOLOGY

Prof. Dr. Michael Bienert, Department Head: Peptide Chemistry (Secretary: Marianne Dreissigacker)

Hartmut BergerMichael BeyermannMargitta DatheVolker HagenEberhard KrauseJens-Peter von KriesJohannes OehlkeJörg Rademann

Although in the last decade our knowledge about genomesequences (genomics) and the occurrence of cellular pro-teins (proteomics) has increased dramatically, this pro-gress is often not sufficient to understand the function andthe significance of biomacromolecules in cellular systemsor in organs of different organisms. All proteins evoke theirfunction by specific interactions with other proteins orconstituents of membranes and the cytoskeleton, and dee-

per insights at a molecular and atomic level are requiredin order to unravel the subtle modes of protein-proteincommunication. The analysis of interactions between che-mical moieties of proteins, and the identification of proteinsubstructures, affected by drug-like compounds, as wellas the design and the synthesis of compounds with modi-fied recognition characteristics, demand chemical thin-king and chemical methodologies. This makes clear thatchemistry has become a more central science in ourefforts to understand the molecular basis of life. This dra-matic trend has led to the creation of the term “ChemicalBiology”, defining all kinds of chemical research focusedon the unravelling of biological phenomena. Naturally,there is an overlap with other areas of chemistry, such as“Medicinal Chemistry” and “Bioorganic Chemistry”. Theinteraction with a powerful Structural Biology and theinvolvement of biochemical, biophysical and analyticalmethodologies are necessities for the further successfuldevelopment of the novel science “Chemical Biology”.

In the context of Chemical Biology, chemical and biologi-cal research in the FMP are traditionally excellently inter-connected. Research efforts of the Department of Peptide

BEREICH CHEMISCHE BIOLOGIE

Prof. Dr. Michael Bienert, Abteilungsleiter: Peptidchemie(Sekretariat: Marianne Dreissigacker)

Hartmut BergerMichael BeyermannMargitta DatheVolker HagenEberhard KrauseJens-Peter von KriesJohannes OehlkeJörg Rademann

Obwohl unser Wissen über Gensequenzen (Genomics)und über das Auftreten zellulärer Proteine (Proteomics)dramatisch angewachsen ist, reicht der Fortschritt oftnoch nicht aus, um die Funktion und Bedeutung von biolo-gischen Makromolekülen in zellulären Systemen oder inden Organen der verschiedenen Organismen zu verste-hen. Alle Proteine funktionieren, indem sie spezifischeInteraktionen mit anderen Proteinen oder mit Bestandtei-len der Membranen und des Zytoskeletts eingehen, undtiefergehende Einsichten auf molekularer und atomarer

Ebene sind nötig, um die Feinstruktur der Protein-Protein-Kommunikation zu erfassen. Sowohl die Analyse derWechselwirkungen zwischen chemischen Resten an Pro-teinen und Identifizierung von Proteinsubstrukturen, diedurch Wirkstoffe beeinflussbar sind, als auch das Designund die Synthese von Verbindungen mit veränderterErkennungscharakteristik erfordern chemisches Denkenund chemische Methodologie. Dies macht klar, dass dieChemie für unseren Bemühungen, die molekulare Basisdes Lebens zu verstehen, zu einer zentralen Wissenschaftgeworden ist. Dieser dramatische Trend hat zur Ent-stehung des Begriffs „Chemische Biologie“ geführt, deralle Arten chemischer Forschung beschreibt, die auf dieUntersuchung biologischer Phänomene gerichtet sind.Natürlich gibt es Überlappungen mit anderen Gebieten derChemie, wie zum Beispiel der Medizinischen und der Bio-organischen Chemie. Datenaustausch mit einer starkenStrukturbiologie und die Einbeziehung biochemischer, bio-physikalischer und analytischer Methoden sind eine Not-wendigkeit für eine weitere erfolgreiche Entwicklung derjungen Wissenschaft Chemische Biologie.

Im Kontext der Chemischen Biologie ist die chemische undbiologische Forschung am FMP traditionell exzellent ver-netzt. Forschungsarbeiten der Abteilung Peptidchemie

Chemistry and Biochemistry are focused on the molecularmechanisms of peptide-protein (Peptide Synthesis Group,Peptide Biochemistry Group) and peptide-lipid interactions(Peptide-Lipid Interaction Group), leading to a betterunderstanding of the action of biologically active peptideson their respective targets. The study of protein-proteininteractions in the cell requires the improvement of thecellular uptake of peptides and other low-molecular com-pounds (Peptide-Lipid Interaction Group). The function ofproteins is often regulated by distinct chemical, post-translational modification, which can be analyzed bynewly developed mass spectrometric procedures (MassSpectrometric Group). In addition, the department is in-volved in methodological studies in order to improve thechemical synthesis of small-sized proteins, as well as tosynthesize fluorescently labeled peptide analogues forlocalization and interaction studies.

Very useful tools for the study of intracellular biologicalprocesses, “caged compounds”, are synthesized andcharacterized in the Synthetic Organic BiochemistryGroup. Caged compounds are photolabile, inactive deriva-tives of biologically active substances, from which the

active biomolecule, e. g. a transmitter, is rapidly liberatedby UV light, thus allowing the analysis of fast biologicalprocesses in cells.

In 2004, Chemical Biology at the FMP was considerablystrengthened by the installation of the MedicinalChemistry Group. A central aim of this group consists inthe creation and development of synthetic methods for thecombinatorial organic chemistry and solid phase organicsynthesis. Focused libraries of potential ligands are gene-rated in order to find small organic molecules which bindspecifically to selected target proteins, thus assisting inthe analysis of functions and interactions of the proteins.Naturally, such small molecules could also serve as leadstructures for the design of drug candidates. For analyzingthe biological activity of small-molecule libraries, aScreening Unit is now established, developing conceptsfor the handling of libraries and providing the technicalequipment for testing hundreds and thousands of chemi-cal compounds.

und Biochemie zielen auf die molekularen Mechanismenvon Peptide-Protein- (Arbeitsgruppe Peptidsynthese;Peptidbiochemie) und Peptid-Lipid-Interaktionen (Arbeits-gruppe Peptid-Lipid Interaktion) und verhelfen zu einembesseren Verständnis der Wirkung biologisch aktiver Pep-tide auf ihre jeweiligen Zielstrukturen. Die Untersuchungvon Protein-Protein Interaktionen in der Zelle erforderteine verbesserte Aufnahme der Peptide und anderer klei-ner Moleküle (Arbeitsgruppe Peptid-Lipid Interaktion). DieFunktion von Proteinen ist oftmals durch bestimmte che-mische posttranslationale Modifikationen reguliert, diemittels neuartiger massenspektrometrischer Verfahrenanalysiert werden können (Arbeitsgruppe Massenspek-trometrie). Darüber hinaus ist die Abteilung an methodo-logischen Studien beteiligt, die die chemische Synthesekleiner Proteinmoleküle verbessern und fluoreszenz-markierter Peptidanaloga für Lokalisierungs- und Inter-aktionsstudien gewährleisten sollen.

Äußerst nützliche Werkzeuge für die Untersuchung intra-zellulärer biologischer Prozesse, „caged compounds”,werden in der Arbeitsgruppe Synthetische OrganischeBiochemie synthetisiert und charakterisiert. Caged com-pounds sind photolabile, inaktive Derivate biologischaktiver Substanzen, die aktive Biomoleküle, z. B. einen

Transmitter, auf Einwirkung von ultraviolettem Licht hinfreisetzen und so die Analyse schneller biologischer Pro-zesse in Zellen ermöglichen.

Im Jahr 2004 wurde die Chemische Biologie am FMP durchdie Einrichtung der Arbeitsgruppe Medizinische Chemiebeträchtlich gestärkt. Ein zentrales Ziel der Gruppe ist dieEntwicklung und Etablierung von Methoden für die kombi-natorische organische Chemie und Festphasensynthese.Fokussierte Bibliotheken potentieller Liganden werdenaufgestellt, um kleine organische Moleküle zu suchen, diespezifisch an ausgewählte Zielproteine binden. So wird dieAnalyse der Funktionen und Interaktionen von Proteinenunterstützt. Natürlicherweise können solche kleinen Mole-küle auch als Leitstrukturen für das Design von Wirkstof-fen dienen. Um die biologische Aktivität von Bibliothekenkleiner Moleküle untersuchen zu können, hat das FMPeine Screening Unit etabliert, die Konzepte für das Hand-ling von Substanzbibliotheken erarbeitet und technischeVoraussetzungen für die Testung Hunderter und Tausen-der chemischer Verbindungen schafft.

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PEPTIDE SYNTHESISGroup Leader: Dr. Michael Beyermann

DEVELOPMENT OF A MODEL FOR LIGAND-RECEPTOR INTERACTION

Insights into the molecular basis of interaction betweenpeptide ligands and their membrane-embedded receptors,in particular G-protein-coupled receptors class B, willopen a way for development of new drugs for treatmentof a number of diseases, since those receptors are veryimportant targets of peptides regulating many essentialbiological functions. Those receptors are functional onlywhen embedded in the membrane, which is why it is dif-ficult to obtain direct structural information by spectros-copic methods such as X-ray crystallography or NMRmethods. Indirect methods, such as structure-activityrelationship studies, modifying the ligand or/and receptorstructure, e.g. by point substitutions, and looking at corre-sponding effects on ligand-receptor binding/activation,may give information about interaction modes betweenligand and receptor. The incorporation of light-directedcrosslinkers into ligands gives the possibility to crosslinkthe ligand bound to receptors. By this approach, contactpoints between ligand and receptor can be determined.Furthermore, many evidence points to a crucial contribu-tion of extracellular receptor domains to ligand binding.Therefore, we are investigating ligand binding to extracel-lular receptor domains. From all these studies, which alsorequire development of efficient chemical and biotechno-logical methods for preparation of receptor domains andconstructs as artificial receptors for appropriate structu-re analysis in complex with ligands, we will stepwisededuce an appropriate interaction model, also taking intoconsideration whether receptors interact with ligands asmono- or oligomers.

CRF receptor antagonists showing di f ferentefficiency (1)Corticotropin-releasing factor (CRF), urocortin I (Ucn I),sauvagine (Svg), and urotensin (Uts) are non-selective,endogenous agonists of the G-protein-coupled receptorsCRF1 and CRF2. Determinants of subtype receptor selecti-vity, particularly for such long-chain (~40 aa) peptides, arewidely unknown. First subtype (CRF2) selective agonistshave only been discovered on basis of genomic DNA infor-mation and subsequent cDNA cloning. A strikingly diffe-rent amino acid motif (VPIG) at the N-terminus of theselective agonist Ucn II led to CRF2 selective agonists,when incorporated into non-selective Ucn I and Svg butnot Utn. Receptor antagonists generated by N-terminaltruncation of CRF2 selective VPIG-Ucn I and Ucn II exhibi-

ted significant CRF2/CRF1 selectivity in terms of affinity (19 and 260 fold, respectively). Replacing the apparentselectivity motif VPIG in truncated Ucn II by TFH, residuesat corresponding positions of non-selective Ucn I gave themost selective antagonist for CRF2, exhibiting an about1500-fold higher affinity for CRF2 compared to CRF1. Mostremarkably, the CRF2/CRF1 selectivity in terms of antago-nistic potencies and receptor affinity of those antagonistsstrongly diverge, although no significant intrinsic activitywas found. The quotient of the relative selectivity in termsof affinity and potency of a certain antagonist may provi-de a basis to evaluate its antagonistic efficiency. The an-tagonistic efficiency varied between 2.35 and 33.2 for thepeptides investigated, showing that looking at receptoraffinity only in screenings for antagonists as a global meas-ure may be insufficient to reflect their inhibitory potency.

Dimerization of cort icotropin-releasing factorreceptor type 1 is not coupled to ligand binding(2)As reported, receptor dimerization of G-Protein-coupledreceptors may influence signaling, trafficking and regula-tion in vivo. Up to now, most studies aiming at the possiblerole of receptor dimerization in receptor activation andsignal transduction are focused on class 1 GPCRs. Wehave investigated dimerization behavior of CRF1 receptorstagged with fluorescence labels (CFP/YFP) transientlyexpressed in HEK293 cells. We measured FluorescenceResonance Energy Transfer and found that CRF1 receptorsform mainly dimers. Upon addition of CRF-related agonistsor antagonists, no significant change of the FRET signalwas observed, indicating no change in the dimer-mono-mer ratio by ligand binding.

Photoaff ini ty cross-l inking of the cort icotro-pin-releasing factor receptor type 1 using pho-toreactive urocortin analogues (3)Interaction of peptide ligands to G-protein-coupled recep-tors of class B, such as glucagon, secretin and corticotro-pin-releasing factor (CRF), is characterized by a commonorientation of two binding domains, in that ligand C-termi-nus binds to extracellular receptor N-terminus (high affini-ty binding) and receptor juxta-membrane domain bindsligand N-terminus. N-terminal truncation, particularly ofresidues 6-8 in the case of CRF, leads to antagonists, sug-gesting that those residues constitute the receptor acti-vating sequence. Here, we identified by photoaffinitycross-linking using Bpa-analogues (Bpa: p-benzoyl-L-phe-nylalanine) of urocortin (Ucn) interaction domains of thereceptor (CRF1) with individual amino acids. Specificdigestion patterns of ligand-receptor complexes, obtainedusing different cleavage methods and SDS-PAGE for frag-

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ment separation, have shown that 125I-Y-1-Bpa0-Ucn and125I-Y0-Bpa12-Ucn bind to the second, whilst 125I-Y0-Bpa17-Ucn and 125I-Y0-Bpa22-Ucn bind at the first extracellularreceptor loop. These results show that the suggested acti-vating sequence, which might penetrate into the trans-membrane segment, is fixed to juxta-membrane domain byspecific interaction of amino acid residues 12, 17 and 22.Our findings are in conflict with recently published modelsin that peptide antagonists bind to receptor N-terminusenclosing those residues.

Hexa-histidine tag position influences disulfi-de pattern (4)The oxidative folding, particularly the arrangement of dis-ulfide bonds of recombinant extracellular N-terminaldomains of the corticotropin-releasing factor receptortype 2a bearing 5 cysteines (C2 to C6) was investigated.Depending on the position of a His-tag, two types of disul-fide patterns were found. In the case of a N-terminal His-tag, the disulfide bonds C2-C3 and C4-C6 were found, leav-ing C5 free, whereas the C-terminal position of the His-tagled to the disulfide pattern C2-C5 and C4-C6, and leavingC3 free. The latter pattern is consistent with the disulfidearrangement of the extracellular N-terminal domain of the

CRF receptor type 1, which has six cysteines (C1 to C6) andin which C1 is paired with C3. However, binding data of thetwo differently disulfide-bridged domains show no signifi-cant differences in binding affinities to selected ligands,indicating the importance of the C-terminal portion of theN-terminal receptor domains, particularly the disulfidebond C4-C6 for ligand binding.

An unexpected side reaction gets a beneficialtool : N↔O-acyl shifts (5) N-to-O acyl migrations at serine or threonine residues inpeptides or proteins have previously been observed upontreatment with strong acids. We observed such shifts, sur-prisingly, for the moderately strong acid trifluoroaceticacid (TFA) used in Fmoc chemistry for the final removal ofall protecting groups. N,O-acyl shifts have not been recog-nized as leading to serious side reactions in Fmoc-basedpeptide synthesis and, thus, are not even mentioned inrecent textbooks. The extent of undesired N→O shiftsdepends on the peptide sequence and even in the case ofTFA may give rise to large amounts of depsipeptide by-pro-ducts. Moreover, we found that syntheses of sequencesin which the ester unit appears at appropriate positionswere far more efficient than the syntheses of those with-

FIGURE 1MALDI-MS spectra of H-(Val-Thr)10-NH2

(crude products) synthesized using stan-dard Fmoc-chemistry (a) and as the all-depsi isomer (b) (M[H]+calc: 2019.18)

900,0 1162,8 1425,6 1688,4 1951,2 2214,0

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out the ester unit. A final ammonium hydroxide-inducedO→N-shift yielded the authentic peptides in much betteryields. This new method opens a way for efficient synthe-sis of difficult sequences, thus facilitating investigationson the role of individual amino acid residues on structuralstability of the WW domain FBP-28. Compared with othermethods, such as pseudoproline methodology, an impor-tant advantage of the depsipeptide method results fromthe fact that the conformation-disrupting modificationintroduced by the depsipeptide unit is still present duringchromatographic purification of the deblocked peptide.The suppression of conformation-induced associationleads to an increased solubility and narrower peaks fordepsi isomers, thus allowing a much more efficient purifi-cation.

Group members

Dr. Jana Klose (4)DC Angelika Ehrlich (SPOT library synthesis)*Doreen Wietfeld (Doctoral student) (1)*Sandra Tremmel (Doctoral student) (5)Oliver Krätke (Doctoral student) (2,3)Stephan Pritz (Doctoral student)*Irene Coin (Doctoral student) (5)*Annerose Klose (Technical assistance peptide chemistry)Dagmar Krause (Technical assistance peptide purification,analysis)Barbara Pisarz (Technical assistance Biacore)**Bernhard Schmikale (Technical assistance peptide syn-thesis)**

External funding

Deutsche ForschungsgemeinschaftProjekt im Schwerpunktprogramm „Molekulare Sinnes-physiologie“ (SPP 1025)Michael Beyermann

Deutsche Forschungsgemeinschaft„Untersuchungen von CRF- und CRF-Rezeptor-Mutantenzur Entwicklung eines Modells für die Ligandenerkennungvon G-Protein-gekoppelten Rezeptoren der Familie 2“ (TPA6 im Sonderforschungsbereich 449 „Struktur und Funk-tion membranständiger Rezeptoren“)Michael Bienert, Michael Beyermann, Walter Rosenthal

Deutsche Forschungsgemeinschaft„Organisation und Funktion eines Transduktionskomple-xes in Sehstäbchen“ (BE 1434/3-3)Michael Beyermann

Deutsche Forschungsgemeinschaft„Struktur, Stabilität und Spezifikationen von nichtkataly-tischen Proteindomänen und deren Verwendung alsWerkzeuge für das Design einer stabilen minimalen ß-Falt-blattstruktur und das Verständnis von pathologischen Pro-zessen“ (TP 2/2-1 in der Forschergruppe 299 „Optimiertemolekulare Bibliotheken zum Studium biologischer Erken-nungsprozesse“)Hartmut Oschkinat, Michael Bienert


Klose J, Fechner K, Beyermann M, Krause E, Wendt N,Bienert M, Rudolph R, Rothemund S (2005) Impact of N-terminal domains for corticotropin-releasing factor(CRF) receptor-ligand interactions. Biochem, in press

Klose J, Wendt N, Kubald S, Krause E, Fechner K, Beyer-mann M, Bienert M, Rudolph R, Rothemund S (2004) Hexa-histidin tag position influences disulfide structure but notbinding behavior of in vitro folded N-terminal domain of ratcorticotropin-releasing factor receptor type 2a. Protein Sci13, 2470-2475

Carpino LA, Krause E, Sferdean CD, Schümann M, FabianH, Bienert M, Beyermann M (2004) Synthesis of ”Difficult”Peptide Sequences: Application of a Depsipeptide Tech-nique to the Jung-Redemann 10- and 26-mers and theAmyloid Peptide A‚(1-42). Tetrahedron Lett 45, 7519-7523

Eggelkraut-Gottanka R, Klose A, Beck-Sickinger AG, Bey-ermann M (2003) Peptide (alpha)thioester formation usingstandard Fmoc-chemistry. Tetrahedron Lett 44, 3551-3554

Kaupp UB, Solzin J, Hildebrand E, Brown JE, Helbig A,Hagen V, Beyermann M, Pampaloni F, Weyand I (2003) Thesignal flow and motor response controlling chemotaxis ofsea urchin sperm. Nature Cell Biol 5, 109-117

Wietfeld D, Fechner K, Heinrich N, Seifert R, Bienert M,Beyermann M (2003) Development of CRF receptor-2selective peptide ligands. Biopolymers 71, 376

Collaborations

In a number of projects we have collaborated either withgroups within and/or outside the FMP, particularly withL.A. Carpino (University of Massachusetts/USA) on devel-opment of a new method for synthesis of difficult peptides,with A.G. Beck-Sickinger (University of Leipzig) on prepa-ration and use of peptide thiol esters for peptide and pro-tein synthesis, and with U.B. Kaupp (FZ Jülich)/V. Hagen(FMP) on studies of chemotaxis using appropriately cagedpeptides. Of particular impact is the joint work with thePeptide Biochemistry Group.


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PEPTIDE LIPID INTERACTION / PEPTIDETRANSPORTGroup Leaders: Dr. Margitta Dathe and PD Dr. JohannesOehlke

PROPERTIES OF CELLULAR UPTAKE-MEDIATING PEPTIDES

The application of many potential, biotechnologically avail-able drugs of e.g. proteinaceous or nucleic acid origin islimited by their inefficient cellular uptake caused by thelow permeability of the cell membrane. Commonly useduptake-promoting carrier systems based on cationic lipidsor virus components are hampered by low efficiency, poorspecificity, poor bioavailability, toxicity and immunologi-cal risks. Thus, promising new drugs will not be introdu-ced in diagnostics and therapy if we are not able to deve-lop safe, efficient application forms. As an alternative,during the last decade small peptide carriers designatedas cell-penetrating peptides (CPP) or protein transducti-on domains (PTDs) have been introduced as uptake-faci-litating compounds. Covalent linkage or complexation ofsuch peptides with bioactive polymers up to the size ofproteins and nucleic acids led to highly efficient deliveryinto and across a variety of cells, thereby avoiding thedrawbacks of the common delivery systems. Peptidecoupling to potential drug containers such as liposomeswhich can be loaded with hydrophobic as well as hydro-philic compounds provide the additional advantage oftransport of large amounts of drug molecules with unfavo-rable properties such as poor solubility and low stability.Since many of the peptides promote membrane-reorien-tation processes involving temporary membrane destabi-lization and energy-independent uptake, it has been sug-gested that the lipid bilayer of cell membranes is targeted.Peptides which contain recognition motifs suitable foraddressing well-defined receptor structures at the cellmembrane provide an opportunity for site-specific deli-very.

The main goal of our group is the elucidation of the struc-tural requirements for peptides to deliver covalently orcomplex-bound drugs across cell membranes by nonen-docytotic as well as endocytotic mechanisms. Theresearch efforts in this context are focused on studyinginteractions of CPPs with lipid membranes to get mecha-nistic insights into transmembrane transport routes and onthe development of peptide-liposome complexes for drugdelivery to the brain (M. Dathe). As a further topic, thestructural requirements for the delivery ability of CPPs arebeing studied, using covalently bound peptide nucleicacids (PNAs) as cargo molecules by pursuing the uptake

into different cell types in comparison with the biologicaleffects of the PNAs in various systems (J. Oehlke). Additio-nally, we are interested in elucidating the structural prin-ciples and the mechanism of action of peptides whichselectively kill bacteria by destroying their cell membrane.These studies aim at contributing to the development of anew class of peptidic antibiotics (M. Dathe).

Cellular uptake of PNA after conjugation withposi t ively and negatively charged ∝∝ -hel icalamphipathic, β-sheet forming and unstructuredpeptidesYvonne Wolf, Angelika Ehrlich, Burkhard Wiesner, MichaelBienert, and Johannes Oehlke

In order to contribute to an elucidation of the structuralrequirements for the shuttling ability of cell-penetratingpeptides, we investigated the cellular uptake of a 12-merPNA directed against the mRNA of the nociceptin/orpha-nin FQ receptor after disulfide bridging with various pepti-des showing different structure forming properties, char-ge and size (Table). As the lead we used an amide-boundconjugate of the PNA with the cell-penetrating α-helicalamphipathic model peptide KLALK LALKA LKAAL KLA-NH2

(KLA). We have shown previously that KLA exhibits up totenfold higher cellular uptake and biological activity thanthe naked PNA. The cellular uptake was studied by meansof capillary electrophoresis combined with laser-inducedfluorescence detection (CE-LIF). Using this approach aseparate quantification of the conjugate and the nakedPNA generated by cleavage of the disulfide bond in thereducing environment of the cell interior was possible,thus avoiding bias of the results by surface adsorption.

Assessment of the cellular uptake into CHO cells andcardiomyocytes by CE-LIF revealed enrichments within thecell interior of about two and tenfold for the naked PNAand its amide bound KLA conjugate, respectively (relatedto the external concentration and an experimentally deter-mined ratio of about 10 µl cell volume/mg protein). Confo-cal laser scanning microscopy (CLSM) and fluorescenceactivated cell sorting (FACS) supported these results. Anextensive cellular uptake was found also for the analogousdisulfide bridged KLA-PNA-conjugate. But, for reasonswhich still remain unclear, the intracellular PNA concen-tration in this case only approached that provided exter-nally. Even strong structural alterations in the peptide partof the disulfide bridged conjugate did not seriously influ-ence the extent of uptake into HEK- and CHO cells (Fig. 1),contradicting speculations about specific structural requi-rements for the shuttling ability of peptides. Surprisingly,after energy depletion an enhanced uptake into CHO cellsas well as into HEK cells was found for conjugates bearing

an acidic helical amphipathic and a ß-sheet forming pep-tide, respectively (Fig. 1). It can be inferred from the latterfindings that the uptake under normal conditions can becounteracted differently by active transport systems independence on both the peptide and the cell type, thusproviding a potential basis for achieving cell selectivecargo delivery.

Peptide transport across l ipid bilayersS. Keller, E. Bárány-Wallje1, S. Serovy2, M. Bienert, M.Dathe

Conflicting reports on the translocation ability of the cell-penetrating peptides through pure lipid bilayers havestimulated biophysical studies on the interaction of pene-tratin, a peptide capable of transporting large hydrophiliccargos into the cell with model lipid membranes to com-pare its bilayer behavior with the ability to enter cells. Theresults, which provided no evidence for peptide trans-location through pure lipid bilayers, support suggestionsthat the highly cationic peptide enters living cells by anendocytotic pathway rather then by disturbing the integ-

rity of the lipid matrix and formation of transient and locallipid structures.

Brain target ing by apolipoprotein E-peptide-liposome complexesI. Sauer, O. Liesenfeld3, H. Scharnagel4 , K. Weisgraber5,M. Bienert, M. Dathe

The treatment of central nervous system diseases is a par-ticular challenge. Different from blood vessels of otherorgans which are equipped with small pores, the tightlyconnected brain capillary endothelial cells forming theblood-brain barrier (BBB) prevent paracellular transport.Furthermore, most potential drugs and modern moleculartools are neither able to diffuse across the cell layer norare accessible to the specialized receptor-mediated trans-port systems. An approach to overcome unfavorable prop-erties provides for drug incorporation into carriers. Lipo-somes have emerged as a very potential drug container.

1) Dept. of Biochemistry and Biophysics, Stockholm University2) Junior Research Group Biophysics

3) Institute for Infectious Medicine, Charité Campus Benjamin Frank-lin, Berlin

4) Dept. of Clinical Chemistry, University Hospital Freiburg im Breisgau5) Gladstone Institute of Cardiovascular Disease, San Francisco6) NMR-Spectroscopy Group, FMP

Compound Sequence Structural properties

Peptide nucleic acid Fluos-GGA GCA GGA AAG-Cys

KLA Dansyl-G-C-KLALK LALKA LKAAL KLA-NH2 α-helical, amphipathic

KGL Dansyl-G-C-KGLKL KGGLG LLGKL KLG- NH2 unstructured

ELA Dansyl-G-C-ELALE LALEA LEAAL ELA-NH2 α-helical, amphipathic

RLA Dansyl-G-C-RLALR LALRA LRAAL RLA-NH2 α-helical, amphipathic

Penetratin Dansyl-G-C-RQIKI WFQNR RMKWK K-NH2 poor α-helical amphipathic

VT5 Dansyl-G-C-DPKGDPKGVTVTVTVTVTGKGDPKPG- NH2 β-sheet, amphipathic

TABLE 1Components of disulfide-bridged PNA-peptide conjugates.

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The solvent-containing interior of the nanoscopic particlesself-assembled from lipids can be loaded with polar drugsand the hydrophobic shell with non-polar compounds.Equipped with an appropriate transport-mediating com-pound that recognizes membrane constituents of braincapillary endothelial cells, thousands of incorporated drugmolecules could be transported.

It was our objective (M. Dathe and coworkers) to developpeptidic vectors which recognize the low density lipopro-tein receptor (LDL receptor) on brain capillary endothelialcells, and to develop strategies of their efficient couplingto liposomal carriers. The receptor was found to be highlyexpressed on capillary endothelial cells of different spe-cies. A peptide encompassing the highly cationic tandemdimer peptide (141-150)2 derived from the LDL receptor-binding domain of apolipoprotein E (apoE) was used asvector sequence. A cost-effective approach to coat lipo-somes with an apoE peptide and to induce the structurethat enables binding to the LDL receptor relies on theadsorption to the liposomal surface. Amphipathic andtransmembrane helices and fatty acid chains served asanchoring domains. Since coating vesicle surfaces withpeptides could promote the destabilization of liposomalpreparations rendering vesicles leaky and peptides

released in circulation could cause perturbation of cellmembranes, their stable anchorage at the liposomal sur-face, the potential to assume a receptor-recognizing heli-cal conformation, their ability to conserve the integrity ofthe vesicles, and their toxic effect were evaluated.Complexes of liposomes with a dipalmitoyl-modified apoEpeptide proved to be the most promising to combine thepurposes of a stable drug container and targeting to braincapillary endothelial cells. Furthermore, covalent couplingof the cationic apoE peptide onto sterically stabilized lipo-somes provide stable complexes.

The peptide-coated liposomes were efficiently internalizedinto endothelial cells of brain microvessels by an energy-dependent endocytotic process (Figure 1). Low peptideaffinity to the LDL receptor and internalization into cellswith up- and down-regulated receptor level pointed to thedominating role of an LDL receptor-independent transportroute. Studies to influence unspecific charge interactionof the cationized liposomes with anionic membrane consti-tuents suggested that the ubiquitously expressed cellmatrix component heparan sulfate proteoglycan (HSPG)played a decisive role in the uptake process.

FIGURE 1Quantities of PNA-Cys in the extracts of HEK- and CHO-cells exposed before to 0.2 µM PNA alone or disulfide bridgedwith various peptides (table) for 60 min at 37 °C without (normal) and with energy depletion (after 60 min preincubati-on with 2 deoxy-glucose/Na-azide 25/10 mM). Each bar represents the mean of three samples + SEM.

The efficient uptake via HSPG-involving endocytosis mayprovide a promising strategy for facilitated drug deliveryacross the blood brain barrier. Furthermore, the observedinternalization emphasized the potential of cationic pepti-des to ferry complex drug carriers into cells, and theadsorptive coupling strategy offers great adaptability to abroad spectrum of ligands destined to diverse biologicaltargets.

Group members

Ines Sauer (Doctoral student)Sandro Keller (Doctoral student)*Yvonne Wolf (Doctoral student)* Axel Wessolowki (Doctoral student)*Heike Nikolenko (Technical assistance)Gabriela Vogelreiter (Technical assistance)

External funding

Deutsche Forschungsgemeinschaft “Linear and cyclic hexapeptides: interaction with membra-nes and modulation of selective cell-lytic processes” (DA324/4-1, 4-2)Margitta Dathe

Deutsche Forschungsgemeinschaft“Passage of the blood-brain barrier using surface-modi-fied nanosuspensions and apolipoprotein-E peptide-covered carrier systems” (DA 324/5-1)Margitta Dathe

Deutsche Forschungsgemeinschaft“Hirntargeting mittels oberflächenmodifizierter Nano-suspensionen und Apolipoprotein E-Peptid beladenerTrägersysteme“ (Teilprojekt 7B der Forschergruppe 463“Innovative Arzneistoffe und Trägersysteme – IntegrativeOptimierung zur Behandlung entzündlicher und hyperpro-liferativer Erkrankungen“) Margitta Dathe

European Community“Investigation of structural prerequisites for cell-selectivedrug uptake mediated by peptide vectors” (Subproject ofQLK3-CT-2002-01989 “Target specific delivery systems forgene therapy based on cell penetrating peptides”)Johannes Oehlke, M. Bienert

European Community“Interaction of cell-penetrating with artificial lipid mem-branes and peptide translocation through lipid layers”(Subproject of QLK3-CT-2002-01989 “Target specific deli-

FIGURE 2Confocal laser scanning microscopic picture of mousebrain capillary endothelial cells exposed to fluores-cence-labeled and apoE peptide-covered liposomes atT = 37 °C for t = 30 min. Uptake is documented by thespotted fluorescence within the cytoplasm.


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very systems for gene therapy based on cell penetratingpeptides”)Margitta Dathe, Michael Bienert

Deutscher Akademischer Austauschdienst“Untersuchungen zu strukturellen Voraussetzungen füreinen zellselektiven Wirkstofftransport durch Peptidvek-toren“(DAAD-Stipendium – 3-monatiger Studienaufenthalt ander Universität Montpellier, Frankreich)Yvonne Wolf

Deutscher Akademischer Austauschdienst“Interaction of amphipathic ApoE-peptides with membra-nes: modulation of a receptor-mediated drug transport intothe brain”(DAAD-Stipendium: 4-monatiger Studienaufenthalt am J. David Gladstone Institute, San Francisco, USAInes Sauer

Deutscher Akademischer Austauschdienst“Lineare und cyclische Hexapeptide: Interaktion mit Mem-branen und Modulation selektiver zelllytischer Prozesse“(DAAD-Stipendium: 2-monatiger Studienaufenthalt amKarolinska Institut, Stockholm, Schweden)Axel Wessolowski

Organizing committee of the Congress “Cellular TransportStrategies for Targeting of Epitopes, Drugs and ReporterMolecules“, (Budapest 2003), Conference grantInes Sauer


Dathe M, Nikolenko H, Klose J, Bienert M (2004) Cycliza-tion increases the antimicrobial activity and selectivity ofarginine- and tryptophan-containing hexapeptides. Bio-chemistry 43, 9140-9150

Wessolowski A, Bienert M, Dathe M (2004) Antimicrobialactivity of arginine- and tryptophan-rich hexapeptides: theeffect of aromatic clusters, D-amino acid substitution andcyclization. J Pept Res 64, 159-169

Oehlke J, Wallukat G, Wolf Y, Ehrlich A, Wiesner B, Ber-ger H, Bienert M (2004)Enhancement of intracellular concentration and biologicalactivity of PNA after conjugation with a cell-penetratingsynthetic model peptide. Eur J Biochem 271, 3043-3049

Hällbrink M, Oehlke J, Papsdorf G, Bienert M (2004)Uptake of cell-penetrating peptides is dependent on thepeptide-to-cell-ratio rather than on peptide concentration.Biochim Biophys Acta 1667, 222-228

Sauer I, Dunay IR, Weisgraber K, Bienert M, Dathe M(2005) An apolipoprotein E-derived peptide mediatesuptake of sterically stabilized liposomes into brain capilla-ry endothelial cells. Biochemistry 44, 2021-2029

Collaborations

Prof. Oliver Liesenfeld, Dept. of Medical Microbiology andImmunology of Infection, Charité Campus BenhaminFranklin, Berlin Dr. Hubert Scharnagel, Dept. of Clinical Chemistry, Univer-sity Hospital Freiburg im BreisgauProf. Karl Weisgraber, Gladstone Institute of Cardiovascu-lar Disease, San Francisco

PEPTIDE BIOCHEMISTRYGroup Leader: Dr. Hartmut Berger

CORTICOTROPIN-RELEASING FACTORRECEPTOR TYPE 1 (CRFR1): REGULATIONOF THE COUPLING TO DIFFERENT G-PRO-TEINS AND THE TWO-DOMAIN RECEPTORMODEL

Corticotropin-releasing factor (CRF) receptor type 1(CRFR1) is a G-protein-coupled receptor (GPCR) of theclass B, secretin receptor family, which is activated byseveral peptide ligands and which is thought to be theprincipal physiological mediator of stress responses. Forthe activation of the receptor by peptide ligands, a two-domain model has been established by several authors:the N-terminal domain of the receptor (N-domain) is re-quired for ligand binding, whereas the juxtamembraneregion, consisting of transmembrane regions and inter-vening loops (J-domain), is involved in the activation of thesignaling steps by the ligand, bitethered between N and J.Furthermore, whereas peptide antagonists bind predomi-nantly to the N-domain with high affinities, non-peptideantagonists bind only to the J-domain. We have providedevidence that the CRFR1 expressed in HEK (HEK-CRFR1)cells couples to Gs- and Gi-proteins with different charac-teristics. The two-domain binding model and our model onG-protein coupling regard different aspects of the activa-tion of the CRFR1. Therefore, it is important to investigatewhether they can be fitted into one combined model.

For this reason, we studied the regulation of G-proteincoupling of the CRFR1 in HEK-CRFR1 cell membranes andthe influence of peptide and non-peptide antagonists onthe coupling of the receptor, using the GTPγ35S bindingassay (Hartmut Berger, Nadja Heinrich, Monika Georgi,Doreen Wietfeld*, Jens Furkert**). Corresponding tobiphasic binding of the ligand to the CRFR1, Gs- and Gi-pro-tein coupling of the receptor were seen in the biphasicligand-evoked stimulation of GTPγ35S binding to HEK-CRFR1 membranes (Fig. 1). Gs-activation could be separa-ted by inactivation of Gi with pertussis toxin, Gi activationby pre-stimulation of the cells with a ligand, which onlydesensitized Gs activation (Fig. 1). The specificity of Gs andGi activation was confirmed by immunoprecipitation ofGTPγ35S-bound Gα subunits. By this means and a substan-tial accumulation of inositol phosphates (IP3) in cells, itwas found that the receptor also activates Gq-proteins.

Studying the characteristics of the coupling of CRFR1 toGs, Gi, and Gq in more detail, we developed the followingmodel for the activation of the signaling by CRFR1 in HEKcells. A peptide agonist ligand binds to a high-affinity, low-capacity receptor binding site activating Gs-protein andadenylate cyclase with high ligand potency. At higher con-centration the ligand additionally binds to one or more low-affinity, high-capacity sites of which the main part remainsnon-coupled, however a small number couples to Gi-pro-teins, attenuating the ligand-stimulated production ofcAMP. The activation of the G-proteins is accomplished byincreasing the affinity of the nucleotide-binding site in theGα chain for GTP, the affinity of GTPγS to Gi being strongerincreased as compared to Gs. Very high ligand concentra-tions also stimulate IP3 accumulation through activationof Gq and, in addition, through a pertussis toxin-dependentway, obviously by activation of phospholipase C by the Gβγ

chain released from Gi when Gi is activated. Stimulation ofthe receptor by ligands desensitizes Gs and Gq activation,but not Gi, so that the inositol phosphate pathway is abouthalf-desensitized.

From our findings it seems clear that the CRFR1 can beadded to the growing list of GPCRs that simultaneouslycouple to unrelated G-proteins, possibly through differentactive receptor states and/or different affinities of the G-proteins to one or more such states. To get more insightinto these possibilities, we studied the influence of pepti-de and non-peptide antagonist on the G-protein coupling,on the basis of the two-domain model for CRFR1. The pep-tide antagonist α-helical CRF(9-41), assumed to bind pre-dominantly to the N-domain, antagonized Gs as well as Gi

coupling competitively with equal potencies, suggestingthat similar ligand binding to N is responsible for Gs as wellas Gi activation. However, the non-peptide antalarmin,assumed to bind exclusively to the J-domain, antagonizedGs activation competitively but the Gi response non-com-petitively (Fig. 2). This should mean that nonpeptide ant-agonists antagonize Gs and Gi coupling of CRFR1 by com-petitive and allosteric mechanisms, respectively, and thatdifferent conformation ensembles of two possibly over-lapping J domains of the receptor are responsible for thecoupling to Gs and Gi.

Taken together, it is concluded that CRFR1 signals throughGs-, Gi-, and Gq-proteins. The concentrations of the stimu-lating ligand and GTP and, furthermore, selective desensi-tization may be part of a regulatory mechanism determin-ing the actual ratio of the coupling of CRFR1 to differentG-proteins. Gs and Gi coupling of the receptor can be de-scribed within the framework of the two-domain receptor

* Peptide Synthesis Group, **Cellular Biology/Molecular Medicine Group

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FIGURE 1Gs- and Gi-protein coupling of CRFreceptor type 1 as measured by sauva-gine-stimulated binding of GTPγ35S tomembranes obtained from HEK cellsexpressing the receptor.

FIGURE 2Competitive and noncompetitive antago-nism by the non-peptide antalarmin of,respectively, Gs and Gi coupling of theCRF receptor type 1 expressed in HEKcells. G-protein coupling was measuredas sauvagine-stimulated binding ofGTPγ35S to membranes obtained fromthe cells after inactivation of Gi by per-tussis toxin (Gs activity left, A) or desen-sitization of Gs coupling by sauvagine (Gi activity left, B).

model in that they are accomplished by different activeconformations of the J-domain.

Our group was also engaged in receptor assays for study-ing structure-activity relationships of CRF peptides (KlausFechner, Gabriela Vogelreiter, results are given by the Pep-tide Synthesis Group).

Group members

Dr. Nadja Heinrich**Dr. Klaus Fechner*Monika Georgi (Technical assistance)Gabriela Vogelreiter (Technical assistance)**

Selected Publications (FMP authors in bold)

Wietfeld D, Heinrich N, Furkert J, Fechner K, BeyermannM, Bienert M, Berger H (2004) Regulation of the couplingto different G proteins of rat corticotropin-releasing factorreceptor type 1 in human embryonic kidney 293 cells. JBiol Chem 279, 38386-38394

Oehlke J, Wallukat G, Wolf Y, Ehrlich A, Wiesner B, Ber-ger H, Bienert M (2004) Enhancement of intracellular con-centration and biological activity of PNA after conjugationwith a cell-penetrating synthetic model peptide. Eur J Bio-chem 271, 3043-3049


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MASS SPECTROMETRYGroup Leader: Dr. Eberhard Krause

MASS SPECTROMETRY-BASED PROTEINANALYSIS

Recent results emphasize the role of mass spectrometryas an essential tool for proteom studies in the field of cel-lular biology. The identification and quantitation of verycomplex protein mixtures as well as of low abundancesignaling proteins require highly sensitive analytical tech-niques. Based on the methodological advances in peptideand protein ionization (awarded with the Nobel Prize in2002) and new powerful fragmentation techniques(MS/MS), state of the art mass spectrometry is able to pro-vide very accurate and sensitive mass measurements aswell as sequence information of peptides and high mole-cular weight proteins. The MS group contributed to seve-ral proteom projects which include the identification ofspecific proteins of molecular networks in cells followingfunctional stimulation and the elucidation of functionallyimportant post-translational modifications of proteins. Inorder to gain insight into the ionization behavior of peptidesand proteins, the group is performing methodological stu-dies. Using chemical derivatization strategies and stableisotope-labeling, improved methods are being developedfor the MS-based analysis of phosphoproteins and for thehigh sensitive identification and quantitation of low-expres-sed signaling proteins, respectively. Both the proteomicswork and the methodological studies are being carried outin collaboration with groups from both within and outsidethe FMP.

Examination of erythropoietin receptor functi-ons using dif ferent proteomic strategiesThe interaction between erythropoietin (EPO) and itsreceptor, which is expressed on late erythroid progenitorcells, initiates multiple signaling pathways by the recruit-ment of cytosolic src homology 2 (SH2) domain-containingproteins to the phosphorylated receptor. The phosphory-lation of receptor tyrosine residues is mainly due to theactivity of the Janus family kinase 2 (JAK2), a receptor-associated enzyme which is undergoing transphosphory-lation after receptor dimerization. Studies using mutatedforms of the EPO receptor (EPOR) have identified singletyrosine residues that are critical for the recruitment ofdistinct signaling proteins. However, it is still difficult torelate the function of single tyrosine residues/receptorsubdomains or distinct signaling molecules/pathways tothe induction of proliferative, antiapoptotic or differentia-tive cellular responses initiated after the binding of EPO toits receptor. Proteomic strategies seem to have an enor-

mous potential in the analysis of changes in the abundan-ce of proteins, but also in their post-translational modifi-cation. A major challenge in creating phosphoproteomemaps, however, remains the fact that phosphorylatedregulatory molecules are expressed at rather low levels.

We applied different proteomic strategies to the investiga-tion of structure-function relationships in the EPOR signa-ling complex. Using a set of EPOR mutants expressed in anidentical cellular background (Fig. 1), we focused on rapidchanges in the tyrosine phosphorylation state of proteins.However, the analysis using the classical approach com-bining two-dimensional gel electrophoresis (2-DE) andidentification by MALDI-MS revealed that low expressedsignaling proteins cannot be detected by this technique. Analternative strategy using 1-D gel separation of phospho-proteins and LC-tandem mass spectrometry (MS/MS)allowed us to identify proteins which are involved in intra-cellular signaling. Besides proteins of already establishedpathways, proteins could be identified which have not beenlinked to EPO-induced signalling so far. Thus, the phospho-rylation of SUMO-1 has been suggested to be part of stra-tegies repressing cytokine induced signals in analogy tothe covalent ubiquitin attachment. We identified severalGTP binding proteins as well as proteins involved in themodification/regulation of G-proteins which have not beenreported to be involved in EPOR induced regulatory events.

In addition, a stable isotope labeling method (Fig. 2) wasused for quantitative analysis of gel-separated proteins ofthe EPOR signaling. The results show that identificationand relative quantification of more than 200 EPOR-depen-dent proteins could be achieved. We can conclude fromour experiments that a proteomic strategy based on acombination of one-dimensional gel separation, in-gel18O-labeling, and LC-MS/MS provides the sensitivityrequired for the detection of low expressed signalingmolecules and offers the potential for a more comprehen-sive analysis of complex cellular responses (cooperationwith T. Bittorf and S. Körbel, Institute for Medical Bio-chemistry and Molecular Biology, University of Rostock).

Chemical derivatization strategies for analysisof post-translational modifications of proteins Esterification of the hydroxyl functions of threonine, seri-ne, and tyrosine with a phosphate group is the ubiquitousmodification of proteins which is involved in many signaltransduction processes, for example, cell differentiation,proliferation, energy storing, and apoptosis. Althoughmass spectrometry has become a preferred method toanalyze phosphorylated proteins, the determination of thesite of phosphorylation, especially for high molecularweight proteins, remains far from routine. The low abun-

FIGURE 1Schematic representation of wild-typeand mutated EGF/EPO receptors. Allreceptors consist of the human EGFRligand-binding domain and the trans-membrane and cytoplasmatic domainsof the murine EPOR.

FIGURE 2Scheme for the identification and rela-tive quantitation of EPOR-dependentphosphoproteins by enzymatic isotopelabeling.

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dance of phosphoproteins, the difficulty of obtaining fullsequence coverage by specific proteolysis, and the lowionization efficiency of phosphopeptides compared withtheir non-phosphorylated analogs may prevent the detec-tion of specific phosphopeptides. In our group, the beta-elimination/Michael addition reaction was used to replacethe phosphate moiety of phosphoserine or phosphothreo-nine peptides by a group which should give rise to remar-kably enhanced signal intensities in MALDI mass spectro-metry. Several N- and S-nucleophiles were studied withregard to enhanced ionization efficiency, and optimizedreaction conditions for beta-elimination/addition procedu-res were developed. Based on in-gel derivatization with 2 phenylethanethiol (PET) we demonstrate that enhancedionization efficiencies can be used for sensitive MS ana-lysis of protein phosphorylation. This method was appliedto determine the phosphorylation of Stat1. Stat proteins(signal transducers and activators of transcription) arecytokine-responsive transcription factors which play animportant role in the regulation of gene expression. Seve-ral post-translational modifications are required for acti-vation, whereas serine phosphorylation at position 727 isnecessary for transcriptional activity. The derivatizationallowed direct MS detection of the peptide sequencecovering the phosphoserine position 727 of Stat1, which isnormally suppressed in complex peptide mixtures.

In addition, mass spectrometric analysis was able to pro-vide evidence contradicting recent results on the role ofArg31 methylation in the DNA binding of Stat1. MS andtandem MS measurements clearly revealed that argininein position 31 of Stat1 is not methylated to a significantextent. In conclusion, alternative explanations to methyla-tion have to be explored to understand the molecularmechanism of reduced interferon sensitivity of tumor cellswhich accumulate high levels of the methyltransferaseinhibitor methylthioadenosine (in cooperation with T.Meissner and U. Vinkemeier, FMP).

Group members

Dr. Michael SchümannClementine Klemm (Doctoral student)Heidi Lerch (Technical assistance)Kareen Tenz (Technical assistance)*Stephanie Lamer (Technical assistance)*

External funding

Deutsche Forschungsgemeinschaft„Simultane Untersuchung Erythropoietinrezeptor-abhän-giger Signale durch funktionelle Proteomanalyse“ (BI 599/2)Thomas Bittorf (University of Rostock), E. Krause

Bundesministerium für Bildung und Forschung„Identifizierung hepatozellulärer Biomarker“ (0312618/UAMassenspektrometrie) Eberhard Krause


Kleuss C, Krause E (2003) G alpha s is palmitoylated at theN-terminal glycine residue. EMBO J 22, 826-832

Czupalla C, Culo M, Müller EC, Brock C, Reusch HP,Spicher K, Krause E, Nürnberg B (2003) Identification andcharacterization of the autophosphorylation sites of phos-phoinositide 3-kinase isoforms beta and gamma. J BiolChem 278, 11536-11545

Kraus M, Bienert M, Krause E (2003) Hydrogen exchangestudies on Alzheimer’s amyloid-beta peptides by massspectrometry using matrix-assisted laser desorption/ioni-zation and electrospray ionization. Rapid Commun MassSpectrom 17, 222-228

Carpino LA, Krause E, Sferdean CD, Schümann M, FabianH, Bienert M, Beyermann M (2004) Synthesis of “difficult“peptide sequences: application of a depsipeptide techni-que to the Jung-Redemann 10- and 26-mers and the amy-loid peptide Abeta(1-42). Tetrahedron Lett 45, 7519-7523

Baumgart S, Lindner Y, Kühne R, Oberemm A, WenschuhH, Krause E (2004) The contributions of specific amino acidside chains to signal intensities of peptides in matrix-assi-sted laser desorption/ionization mass spectrometry. RapidCommun Mass Spectrom 18, 863-868

Klemm C, Schröder S, Glückmann M, Beyermann M,Krause E (2004) Derivatization of phosphorylated peptideswith S- and N-nucleophiles for enhanced ionization efficien-cy in matrix-assisted laser desorption/ionization mass spec-trometry. Rapid Commun Mass Spectrom 18, 2697-2705

Meissner T, Krause E, Lödige I, Vinkemeier U (2004) Argini-ne methylation of STAT1: a reassessment. Cell 119, 587-589

Collaborations

Thomas Bittorf, University of RostockPhosphoproteomics of EPO receptor signaling

Ursula Gundert-Remy, Axel Oberemm, BfR BerlinProteom analysis for risk assessment of chemicals

Christiane Kleuss, Institute for Pharmacology, Charité –University Medicine BerlinModifications of G-proteins

Louis A. Carpino, University of Massachusetts, Amherst,MA, USASide reaction in peptide synthesis


SYNTHETIC ORGANIC BIOCHEMISTRYGroup Leader: Dr. Volker Hagen

CAGED COMPOUNDS

Controlled temporal and spatial release of biomoleculesfrom photolabile precursors, called caged compounds,have become increasingly interesting in the chemical andbiological communities. When caged, a biomolecule isrendered biologically inactive by derivatization with aphotolabile protecting or caging group. As a result, it canbe applied to cells under steady state conditions withoutevoking biological responses. Flash photolysis using UVlight cleaves the modifying group and rapidly generatesthe biologically active molecule. Because photolysis ofcaged compounds generates effectors in situ, much fasterand more spatially uniform, concentration jumps can betriggered than with other techniques. There are no diffu-sion problems and no spatial inhomogeneity problemswith addition of substrates. Desensitization is minimized.Caging and uncaging of biomolecules are widely used forstudies of mechanisms and the kinetics of cellular proces-ses. Other applications of photoremovable protectinggroups which are currently being explored are time resol-ved X-ray studies, protein folding, gene expression con-trol, and also combinatorial chemistry and photolitho-graphy.

Caged compounds must meet specific requirements. Theyshould undergo fast and highly efficient photochemicalreactions and display high molar absorptivities at wave-lengths >300 nm, or better >350 nm. Furthermore, theyshould be sufficiently soluble in aqueous solutions, stabletowards solvolysis, and biologically inert. Sometimesmembrane-permeability is required. Commonly usedcaged compounds do not meet these requirements, the-refore the design of novel caging groups and the develop-ment of novel caged biomolecules are necessary. Further-more, a comparatively small number of caged compoundsexists at present and the variety of applications is suchthat it is desirable to have a range of compounds with dif-ferent characteristics to choose from.

Our group deals with the development of novel caginggroups and the synthesis, characterization and applicationof caged compounds. In the past few years, we developed

[5,7-bis(carboxymethoxy)coumarin-4-yl]methyl (5,7-BCMCM), [7,8-bis(carboxymethoxy)coumarin-4-yl]methyl (7,8-BCMCM),

{7-[bis(carboxymethyl)amino]coumarin-4-yl }methyl(BCMACM), [6,7-bis(ethoxycarbonylmethoxy)coumarin-4-yl]methyl(BECMCM), [7-(diethylamino)-α-methylcoumarin-4-yl]methyl(DEAMCM), α-carboxy-4,5-dimethoxy-2-nitrobenzyl (CDMNB), and 4-carboxymethoxy-2-hydroxycinnamoyl (CMHC)

moieties as novel caging groups. Nearly all these caginggroups bear anionic substituents which confer highaqueous solubility. Furthermore, the coumarinylmethylcaging groups absorb at wavelengths >350 nm and there-fore allow uncaging at long-wavelength irradiation.

Using the novel coumarinylmethyl protecting groups andour earlier introduced caging groups, we prepared impro-ved caged versions of cyclic nucleoside monophosphates(cNMPs). Their usefulness was demonstrated in physiolo-gical studies of different types of cyclic nucleotide-gatedion channels of olfactory sensory neurons (B. Wiesner,FMP; A. Menini, Trieste; K. Benndorf, Jena) and of sperm(U. B. Kaupp, Jülich). The most useful caged cNMPs arethe BCMACM, BCMCM and BECMCM-caged derivatives.We could show that photocleavage of these coumarinyl-methyl-caged compounds was possible with two-photonexcitation as well (collaboration with B. Wiesner, FMP)and that the liberation of the cNMPs occur in the nanose-cond time scale. A fluorescence spectroscopic method forthe calculation of the rate constants of the steps of thephotolysis pathways was developed (collaboration with J.Bendig, Berlin and R. Schmidt, Frankfurt/Main). The highsoluble BCMACM-caged compounds allow unpreceden-ted large instantaneous steps of the cNMP concentration.Using membrane-permeable BECMCM-caged cGMP andcAMP, it was demonstrated for the first time that cGMPpromotes the influx of Ca2+ into sperm of sea urchin or star-fish and it succeeds in combination with caged resact theidentification of the motor response in free swimmingsperm (Kaupp, Jülich).

The CDMNB group was used for caging and uncaging ofthe vanilloid receptor agonist capsaicin (collaboration withS. Frings, Heidelberg). Caged capsaicin is a novel tool forkinetic examinations of TRPV1 channels in somatosenso-ry neurons. First applications of CDMNB-caged capsaicinto noniceptors from rat dorsal root ganglia confirmed thatit can be used as a phototrigger for the activation of noni-ceptors in physiological studies.

The group was also concerned with the development ofcaged protons. Protons interact with all proteins, whichmodulate their structure and their catalytic properties.Therefore protons trigger events in protein folding/unfold-

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ing and play a crucial role in cellular signal transduction.Kinetic studies of all these processes may be aided byphotoactivatable proton precursors for the generation ofrapid pH jumps. A number of caged protons have beendescribed, but they have a lot of disadvantages. We syn-thesized and characterized caged protons based onvariants of our newly introduced 7-(dimethylamino)couma-rinylmethyl (DMACM) caging group. The novel caged pro-ton sodium DMACM sulfate exhibits a photorelease rateconstant of at least 5x108 s-1, long-wavelength absorptionproperties, a high extinction coefficient and a high quan-tum yield. Furthermore, DMACM sulfate is very easily solu-ble in water. The combination of high photosensitivity andhigh solubility allows to induce large pH jumps. We foundalso sensitivity of the compound to two-photon photolysis(collaboration with B. Wiesner, FMP) that should allow truethree-dimensional resolution. The utility of the caged pro-tons was demonstrated in studies of the H+ migration alonglipid bilayers (P. Pohl, S. Keller, FMP).

Group members

Daniel Geißler (Doctoral student)*Ralf Lechler (Doctoral student)Nico Kotzur (Student)*Brigitte Dekowski (Technical assistance)

External funding

Deutsche Forschungsgemeinschaft„Neue ‚caged’ cyclische Nucleotide – Synthese, Photo-chemie und biologische Anwendungen“ (HA 2694/1-2)Volker Hagen

European Community„Cyclic nucleotides for the study of cellular events“ (Bio4-CT98-0034)Volker Hagen, U. Benjamin Kaupp (Research Center Jülich)


Kaupp UB, Solzin J, Hildebrand E, Brown JE, Helbig A,Hagen V, Beyermann M, Pampaloni F, Weyand I (2003) Thesignal flow and motor response controling chemotaxis ofsea urchin sperm. Nature Cell Biol 5, 109-117

FIGURE 1Large jumps in pH can be achieved uponflash photolysis of phototriggers for H+

(F = pH-sensitive fluorescence indica-tor).


Geißler D, Kresse W, Wiesner B, Bendig J, Kettenmann H,Hagen V (2003) DMACM-caged adenosine nucleotides:Ultrafast phototriggers for ATP, ADP and AMP activated bylong-wavelength irradiation. ChemBioChem 4,162-170

Serowy S, Saparov SM, Antonenko YN, Kozlovsky W,Hagen V, Pohl P (2003) Structural proton diffusion alonglipid bilayers. Biophys J 84, 1031-1037

Lorenz D, Krylov A, Hahm D, Hagen V, Rosenthal W, PohlP, Maric K (2003) Cyclic AMP is sufficient for triggering theexocytic recruitment of aquaporin-2 in renal epithelialcells. EMBO Rep 4, 88-93

Hagen V, Frings S, Wiesner B, Helm S, Kaupp UB, BendigJ (2003) (7-Dialkylaminocoumarin-4-yl)methyl-caged com-pounds as ultrafast and effective long wavelength photot-riggers of 8-bromo-substituted cyclic nucleotides. Chem-BioChem 4, 434-442

Matsumoto M, Solzin J, Helbig A, Hagen V, Ueno S,Kawase O, Maruyama Y, Ogiso M, Godde M, Minakata H,Kaupp UB, Hoshi M, Weyand I (2003) A sperm-activatingpeptide controls a cGMP-signaling pathway in starfishsperm. Dev Biol 260, 314-324

Collaborations

Photochemistry of caged compoundsJ. Bendig and P. Wessig, Institute of Chemistry, HumboldtUniversity Berlin

Time-resolved fluorescence spectroscopyR. Schmidt, Institute for Physical and Theoretical Chemi-stry, Johann Wolfgang GoetheUniversity, Frankfurt / Main

Studies of cellular signaling using caged compounds andcaged chemoattractantsU. B. Kaupp, Institute for Biological Information Process-ing, Research Center Jülich

Caged vanilloid receptor agonists as tools for kineticexaminations of TRPV1 channelsS. Frings, Department of Molecular Physiology, Universityof Heidelberg

Analysis of receptor systems in CNS using caged com-poundsH. Kettenmann, Max Delbrück Center for Molecular Medi-cine, Berlin-Buch

Controling of gene expression by using caged compoundsS. Cambridge, Max Planck Institute of Neurobiology,Munich

Caged cNMPs as tools for studies of gating kinetics ofCNG channels K. Benndorf, Friedrich Schiller University Jena

Studies of the function of CNG channels on isolated olfac-tory sensory neurons using caged cNMPs A. Menini, International School for Advanced Studies,Trieste, Italy

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MEDICINAL CHEMISTRYGroup Leader: Prof. Jörg Rademann

CHEMICAL BIOLOGY WITH SMALLMOLECULES

Small molecules are powerful biological tools that can beemployed as bioactive protein ligands. They are used toelucidate and modulate the functions of proteins; more-over, they have been efficient in the discovery of novelproteins or the assignment of an observed functions tospecific proteins. In addition, protein ligands are thepotential starting point for drug development efforts.

Small molecule ligands are generally accessible by chemi-cal synthesis. They can be targeted by classical organicsynthesis methods. However, for the identification of newbiological activities and the subsequent optimization of theinitial compounds, rationally designed collections of mole-cules, denominated as chemical libraries, have to be pro-vided. Library generation poses novel challenges to synthe-tic methods including product isolation and analysis.

Therefore, the starting point of our work is the creation ofsynthetic and analytical methodology to furnish designedchemical libraries. For this purpose combinatorial organ-ic chemistry and solid phase synthesis are applied anddeveloped. The syntheses in most cases are operated inparallel with medium throughput.

On the basis of the developed synthetic methods, focusedscreening libraries are designed and constructed. Focu-sed libraries are composed of potential ligands targetingone protein or a group of target proteins. They are prepa-red for subsequent bioassaying in external collaborationsor in-house screens.

Beyond our own synthetic efforts directed at focusedligand libraries, we are engaged in the set-up of a desi-gned generic (base) screen library. This compound collec-tion is created to target proteins without having a focusedlibrary available or with little structural information known.In order to limit this library to a manageable size, the focuswill be on the detection of fragment-based interactionswith low or medium affinity. The primary hits identified inan initial screen can be taken as the starting point for asubsequent chemical refinement.

For the planning of both synthetic and generic screenlibraries, the input of computational chemistry and mole-cular modelling tools is essential. Within the FMP we haveorganized a medicinal chemistry panel together with themodelling group and the Screening Unit for the coherentplanning of library composition and hit evaluation.

To strengthen FMP’s activities in the area of chemical bio-logy, we co-initiated the national collaboration network“ChemBioNet” and represent the FMP on the board of thisnetwork. The FMP has offered to set up a central screen-ing library that is to consist of focused libraries, a genericscreen library of limited size, and specialized compoundcollections in which synthetic chemists primarily from out-side the FMP can deposit their compounds to make themaccessible for screening. The library concept will beaccompanied by a database to communicate the contentsof libraries together with screening results to externalusers.

In the specific projects, the central part of our work overthe last two years was the development of novel polymercarriers and polymer reagents for focused library produc-tion. Based on polyethyleneimines, a multi-ton industrialproduct, very high loaded resins (“ultraresins”) weredeveloped and used in the synthesis of peptides, hetero-cycles and polymer reagents. The new carriers are veryeconomical in solid phase synthesis as they allow a muchhigher yield of products per gram of resin compared tocommercially available carriers. The ultraresin concepthas been extended towards the synthesis and delivery ofdendritic, multivalent peptides to cell targets. Via a rever-sible cross-linking of branched polyethyleneimine, thefacile preparation of high molecular weight dendrimers (upto 60 kDa) was feasible, carrying a controlled number ofcopies of small molecules, e.g. peptide chains or peptido-mimetics. These complex constructs were shown to effi-ciently deliver peptide ligands across cell membranes viaan endocytotic pathway. Currently the specific biologicalactivity of such dendritic constructs is under investigati-on. The multivalent and dendritic structure of these artifi-cial macromolecules is expected to render the peptideligands less prone to proteolysis and increase their bio-logical activity through multivalent binding.

For the preparation of glycolipids we have set up a novelsynthetic strategy for library production that was denomi-nated as “hydrophobically assisted switching phase(HASP) - synthesis”. This concept allows for a flexiblephase switching, each reaction step can be carried out insolution or attached to the hydrophobic carrier phase. Theconcept has been employed for repetitive glycosylationsyielding oligosaccharides attached to a hydrophobicanchor. In the next step, a library of immuno-stimulatingrhamnolipids has been prepared. These molecules areinteresting in that they are low molecular weight com-pounds which stimulate the innate immune system in asimilar way to bacterial lipopolysaccharides (LPS). UnlikeLPS, these rhamnolipids do not activate the cytokine

excretion via the Toll-like receptors (Tlr-II and -IV) by a hitherto unknown mechanism.

The main focus of the synthetic efforts was in the area ofprotease inhibitors. Stabilized phosphoranes were foundto be an efficient tool for polymer-supported C-acylationreactions. With this concept, it was possible to constructthe central element of protease inhibitors, the isostericcore, directly on the solid support. As a consequence, allsubstituents of peptide isoster inhibitors now can be intro-duced and varied flexibly. This variability is a new oppor-tunity especially in respect to the central side chain, theP1-site, and thus could be employed to investigate theeffect of P1-site variations on specificity and selectivity ofprotease inhibitors. In a model study, conducted with theplasmepsin II, the malaria-linked aspartyl protease of plas-modium falciparum, we discovered that P1-site mutationsenhanced the affinity of norstatine inhibitors by the factorof 60 compared to the natural, i.e. substrate-derived sidechain. Further protease projects yielded reversible inhibi-tors of the SARS-main protease (in cooperation with R. Hilgenfeld, Lübeck) and of industrial targets (patent filedwith Boehringer Ingelheim Pharma in May 2004).

A future keystone of our research will be the developmentof novel screening concepts. In this area we have initia-ted a project on primary screen techniques together withthe Screening Unit. Several collaborative projects withinthe FMP and on-Campus have been started as well.

Group members

Dr. Samer Al-Gharabli Dr. Ludmila Perepellichenko*Dr. Michael BarthDr. Syed Tasadaque Ali Shah*Dr. Steffen Weik Jörg Bauer (Doctoral student)Adeeb El-Dashan (Doctoral student)Viviane Uryga-Polowy (Doctoral student)*Franziska Meier (Student)*

FIGURE 1

Synthetic methods& analysis Modelliung/

Structural biology

(Focussed)libraries

Screeningconcepts


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External funding

Deutsche Forschungsgemeinschaft„Reaktive Intermediate in polymeren Gelen, ihre Anwen-dung in parallelen Synthesen von Proteaseinhibitorensowie deren biochemische Evaluierung“ (Ra895-2)Jörg Rademann

Deutsche Forschungsgemeinschaft„Diversitätsorientierte Synthese von Rhamnolipiden“(Ra895-3)Jörg Rademann

Deutsche Forschungsgemeinschaft Teilprojekt im Graduate College „Chemistry in Interpha-ses“Jörg Rademann

Land Baden-Württemberg „Reaktionskaskaden“ (Landesforschungsschwerpunkt)Jörg Rademann

Boehringer Ingelheim PharmaJörg Rademann

Merck BiosciencesJörg Rademann


Rademann J (2003) Advanced polymer reagents based onactivated reactants and reactive intermediates: Powerfulnovel tools in diversity-oriented synthesis. In: Methods inEnzymology 369, Bunin BA, Morales G (eds), AcademicPress San Diego, 366-390

Bauer J, Rademann J (2003) Trimellitic anhydride linker(TAL) – highly orthogonal conversions of primary aminesemployed in the parallel synthesis of labeled carbohydra-te derivatives (including 5 pages supplemental material).Tetrahedron Lett 44, 5019 - 5023

Weik S, Rademann J (2003) A Phosphorane as SupportedAcylanion Equivalent. Linker Reagents for Smooth andVersatile C-C-Coupling Reactions. Angew Chem Int Ed 42,2491-2494

Barth M, Rademann J (2004) Tailoring Ultraresins based onthe cross-linking of polyethylene imines. Comparativeinvestigation of the chemical composition, the swelling,the mobility, the chemical accessibility, and the perfor-mance in solid phase synthesis of very high-loaded resins.J Comb Chem 6, 340-349

Barth M, Ali Shah T, Rademann J (2004) High LoadingPolymer Reagents based on Polycationic Ultragels. Poly-mer-Supported Reductions and Oxidations with IncreasedEfficiency. Tetrahedron – Combinatorial Chemistry Sympo-sium in Print 60, 8703-8709

Rademann J (2004) Organic protein chemistry: drug disco-very through the chemical modification of proteins. AngewChem Int Ed 43, 4554-4556

Barth M, Fischer R, Brock R, Rademann J (2005) Reversiblecross-linking of hyperbranched polymers: A strategy forthe combinatorial decoration of multivalent scaffolds.Angew Chem Int Ed 44, 1560-1563

Bauer J, Rademann J (2005) Hydrophobically assistedphase synthesis: the flexible combination of solid-phaseand solution-phase reactions employed for oligosaccha-ride Preparation. J Am Chem Soc 127, 7296-7297

Collaborations

G. Klebe, University of MarburgR. Brock, University of TübingenR. Hilgenfeld, University of LübeckU. Zähringer, K. Brandenburg, H. Heine, FZ BorstelT. Mayer, MPI Martinsried

SCREENING UNIT Group Leader: Dr. Jens Peter von Kries

SCREENING FOR BIOACTIVE SMALLMOLECULES IN AN ACADEMIC SET UP -CHEMICAL BIOLOGY INITIATIVE

Beside their medical potential as drugs which cure dis-eases, bioactive small molecules may also serve as power-ful tools for analysis of complex biological systems. Syste-matic screening of large compound libraries for bioactivesmall molecules which serve as molecular „switches“ inbiological networks may become a key technology inChemical Biology. Therefore, the “Forschungsinstitut fürMolekulare Pharmakologie“ provides a Screening Unit foracademic research groups which has been functionalsince autumn 2004. Furthermore, the institute supplies thecentral compound collection of the German Initiative forChemical Biology: ChemBioNet.

Biological networksAbout 30,000 genes determine the development and stabi-lity of a human organism. They code for proteins of theprotein synthesis machinery, ion channels, receptors,kinases, adaptor- or scaffolding proteins of cellular com-munication pathways, cell adhesion molecules or proteinsof the immune system. Their correct function is maintai-ned by complex interactions including enzymatic modifi-cations, like phosphorylation or proteolytic processing. Forinstance, adaptor proteins may serve as scaffolds aftermodification by phosphorylation which then in turn recruitenzymes and their corresponding substrates for furthersignal transduction.

Comparative genomics has shown that evolution does notsolely correspond to a growing number of genes, but cor-responds to an increase in the complex combination ofregulatory modules in multifunctional proteins or genes.Perturbation of these complex interaction networks, i.e. bymutation, viral infection or environmental influences, mayresult in diseases like cancer, cardiac disease or diabetes.

Classical methods for analysisMany proteins have been positioned into pathways usingclassical genetic methods, which are based on loss offunction by mutation and on complementation studies todefine for example the position of a given protein in a path-way. Biochemical in vitro assays measure enzyme activ-ities or characterize binding domains of proteins. Methodsin modern molecular biology comprise overexpressionexperiments of wild-type or mutant proteins in cell cultu-re. Many of those strategies result in deletion of all func-tions of a given protein (antisense) or result in perturba-

tion of many functions, if the overexpressed domain con-tains multiple binding sites for other factors. Contributionof individual interactions cannot be determined from theseexperiments. Although some protein functions have beenidentified in the past, only a small part of the complex inter-actions and functions of the proteins encoded in thehuman genome is known as of yet.

Small molecules: new tools for analysisThe functional characterization of the proteins encoded inthe humane genome and those of a growing number ofmodel organisms present a big challenge for the biosci-ences. Chemistry may become a driving force for solution,as has been shown by recent publications presenting inhi-bitors of protein interactions and of enzymatic activities ofproteins. Molecular “switches“ may be generated by che-mical synthesis which modulate the activity of proteins orgenes. The combination of biological and chemicalmethods may allow the investigation of the function of pro-teins which have, until now, defied functional analysis.

Small bioactive molecules often bind in hydrophobic pock-ets of the surface of proteins. This hydrophobic characteralso favors crossing of cellular membranes. As smallmolecules extend on only small regions of a protein sur-face, they potentially inhibit only individual and not allfunctions of a protein. Therefore small molecules offer anideal tool for identification of individual contribution ofinteractions towards function of multiprotein complexes,i.e. in biological signaling networks. Furthermore, selec-tive interference with protein binding or enzyme functionallows the comparison with the consequence of loss ofspecific functions in human diseases. Moreover, the datacollection and analyses of interactions of small moleculeswith proteins provides insights into substrate recognitionmechanisms and will allow more effective de novo designof agonists or antagonists in the future.

Small molecules and structural biologySmall molecules, which resemble natural substrates ofenzymes (like ATP), but cannot be processed, fix dynamicstructures of enzymes for x-ray analysis of crystals. Theyallow the analysis of the active state of an enzyme. This isan important feature as many enzymes in cancer becomepermanent active after mutation. Therefore, this fixedstructure provides an ideal starting point for the design ofinhibitors. Alternatively, this information has been used todesign an enzyme-specific cosubstrate (ATP) which is onlyrecognized by a specifically mutated enzyme for labelingof its specific downstream target proteins. In general,small molecules may help to stabilize protein structures,which cannot be structurally analyzed in their absence.

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Small molecules and protein familiesPharmaceutical companies prefer to inhibit enzyme activ-ities for drug development.

Inhibition of protein interactions is not a favored project,because it has been shown to be extremely difficult. Onecause for problems targeting protein interactions are theextensive interaction surfaces, which cannot be comple-tely blocked by small molecules. But mutagenesis studiesof many interaction surfaces demonstrate that essentialcontact points cluster in a small region of the interactionsurface. This region is defined as a hot spot of interaction.As substitutions of single amino acid residues in theseregions (point mutants) abrogate the complex formation ofthe proteins, small molecules, which bind there, potential-ly interfere in the same manner. In protein families thesehot spots show significant variation in amino acid sequen-ce. Therefore, after modification small molecules mayeven discriminate between family members and allow thetargeting of individual interactions. In summary smallmolecules present an ideal tool for analysis of the complexfunction of the human genome.

Concept of the Screening UnitThree main strategies of screening are supported: (1) Screening with moderate “HTS” using standard ELISAor fluorescence techniques. The screening lab of theScreening Unit is well equipped with a liquid handlingrobot and automatic dispensers, washers and differentreaders to support most standard screening techniques.

(2) Parallel to this, a spot synthesis of peptide librariesderived from the minimal binding domains, containing per-mutated peptides is offered. The screening of this peptidelibrary characterizes the hot spots for interactions

(3). Use of virtual drug screening for the selection of com-pounds to be ordered for each project. The protein sur-faces will be analyzed and used for computer-aideddocking of virtual libraries into pockets of hot spots orcatalytic sites.

ChemBioNet : chemistry and screening faci l i -t ies for Chemical BiologyThe German Initiative for Chemical Biology, named Chem-BioNet, is currently organizing an academic network com-bining the individual screening initiatives of many researchinstitutions in close contact with industrial partners (for

FIGURE 1Screening robots support to screen the 20.000 compounds of the FMP library in less than one reach

detailed information see: www.chembionet.de) for supportof academic screening projects. This screening networkwill also build up a central shared compound library at theFMP and a database of combined bioactivity profiles andchemical data of compounds (see www.chembionet.depage “Datenbank”). The network will be supported by che-mistry for the modification of molecules and by the exper-tise of the screening centers for the setup of robustscreening assays. The research projects will identify anduse bioactive compounds for functional analysis of bio-logical networks in development or in disease situationslike cancer, cardiac disease, autoimmune reactions orothers. Beside the established role of small molecules foridentification and characterization of protein functions,these compounds may serve for the development of newdrugs for interference with human diseases.

Competi tor or partner of pharmaceutical com-panies?Screening in an academic setup has the advantage thatno obvious disease relevance is a prerequisite for usage ofsmall molecules for interference and characterization ofbiological functions. A specific inhibitor of an individualprotein function is a valuable tool, even when it “only” pro-vides a molecular switch for characterization of a functi-on, without any perspective for serving as a new drug. Thisindependence from commercial aspects allows free col-lection of chemical and biological data of bioactive mole-cules. These data are not limited to a few types of enzy-mes as they include a much broader panel of targets.Therefore this database may provide important cluestowards the design of new classes of bioactive molecules.

Another advantage of the independence from commerci-al aspects is the chance to identify and characterize themode of action of potential drugs years before the disea-se relevance becomes established. At this special pointthe interests of pharmaceutical companies and academicScreening Units could partially overlap, since academicinstitutions do not have the resources for successful drugdevelopment, and pharmaceutical companies do not wantto focus on protein interactions as targets. Pharmaceuti-cal companies could get rights of privileged usage ofpatents and sponsor the academic units with know how ormoney for library enlargement. In summary, this partner-ship would benefit people suffering from a disease forwhich no drug is available yet.

Current academic projects : Mycobacteriumtuberculosis enzyme inhibitor screensInfection with Mycobacterium tuberculosis, the causativebacterium of human tuberculosis, results predominantly inan asymptomatic persistent infection, often referred to aslatency. Infected individuals are at risk over their lifetimeto convert their asymptomatic infection into what is calledreactivation tuberculosis, a disease state both potentiallyfatal and highly contagious. The long persistence of thebacteria in the host during asymptomatic infection in theface of a robust host immune response poses fundamen-tal biological questions. The bacteria could be in a nonre-plicative, metabolically and transcriptionally inactive anddormant state. Recent molecular genetic approaches haveyielded Mycobacterium proteins and lipids important forvirulence and persistence. Hence, it can be argued thatthe bacteria manipulate the host immune response toensure their persistence.

It is estimated that nearly 2 billion people currently sufferfrom latent Mycobacterium tuberculosis infection. Al-though the key front-line antituberculosis drugs are effec-tive in treating individuals with acute tuberculosis, thesedrugs are ineffective in eliminating M. tuberculosis duringthe persistent stages of latent infection. Consequently,therapeutics that directly target persistent bacilli areurgently needed.

The “Structural Proteomics Consortium” selected proteinsfrom Mycobacterium tuberculosis on the basis of geneablation experiments (MPI für Infektionsbiologie, Berlin)resulting in reduced infectiveness. The structures ofselected proteins are solved at DESY in Hamburg (EMBLand MPG research groups) and screened for small mole-cule inhibitors at the Screening Unit in Berlin and by Com-binature (NMR-screening, Berlin).

Two primary screens using the 3-isopropylmalate dehydro-genase (IPMDH, enzyme for biosynthesis of branchedchain amino acids) and sterol 14?-demethylase (CYP51)resulted in identification of inhibitors with IC50-values inthe micromolar range (Rajesh et al. 2005). Two structuralclasses of inhibitors for CYP51 resemble already publishedcompounds as estriol (IC50: 100 µM) or 4-phenylimidazole(IC50: 1 mM) demonstrating the reliability of this screen be.One of the CYP51 antagonists inhibits growth of tuberculo-sis bacteria in human macrophages (Stefan HE Kaufmann,MPI-IB, Berlin). Therefore this compound may be used fordevelopment of new drugs against tuberculosis infections.The novel compound classes identified will be cocrystal-lized to analyze substrate recognition mechanisms in thecatalytic site of the enzyme. The IPMDH inhibitors havebeen identified in a dual approach. First, a compound libra-

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ry of about 37,000 compounds was docked into the cataly-tic site of the enzyme using the computer program GOLDand default library screening settings. From 30 virtual high-score compounds, 10 were selected due to the proposednumber of hydrogen bonds in the site and tested in theenzyme assay. One compound demonstrated the highestaffinity of all inhibitors identified (IC50: 35 µM). Second, theFMP small molecule library of 20,000 compounds wasscreened and inhibitors of IPMDH identified (IC50: 75-250µM). Cocrystallization experiments of inhibitors withrespective enzymes have been already started. Theirstructures serve as an input for ordering related com-pound structures to identify inhibitors with higher affinityfor analysis in model systems which measure infective-ness of treated bacteria. These projects may result in theidentification of small molecules which enable to identifythe biological mechanisms of Mycobacterium tuberculo-sis to escape from immune defence. Beside this result, thecompounds could potentially be of use in the developmentof drugs against tuberculosis infections in partnership withpharmaceutical companies.

The Screening Unit is supported by a grant of the BMBF(0312992J, “Mycobacterium tuberculosis Strukturproteo-mik Konsortium”).

Group members

DC Angelika Ehrlich**Christoph Erdmann (Technical assistance)*

Collaborations

Matthias Willmanns, EMBL, HamburgManfred S. Weiss, EMBL, HamburgHans Bartunik, MPG, HamburgW. Birchmeier, MDC, Berlin


SCIENTIFIC AND TECHNICAL SERVICES

MICRODIALYSISGroup Leader: Dr. Regina M. Richter

DIRECT MEASUREMENT OF IN VIVOPROCESSING OF BRAIN NEUROPEPTIDESUSING MICRODIALYSIS

Elevated cerebral levels of a variety of neuropeptides arebelieved to be risk factors for the development of diseasesthat are related to the stress syndrome, cardiovasculardysfunction and, ultimately to neurodegenerative proces-ses such as Alzheimer’s disease (AD). Specifically, exces-sive accumulation and deposition of β-amyloid peptides(Aβ) form senile plaques in AD brains which are conside-red to be one hallmark of the disease. Emerging evidencesuggests that Aβ accumulation is not in all AD cases asso-ciated with increased Aβ production. Impaired clearanceof Aβ is, therefore, considered to be a critical factor in thedevelopment of AD pathology. However, the mechanismsof the in vivo clearance of these critical neuropeptides arestill not understood, and elimination of Aβ from tissues andplasma by activation of degrading proteases appears tohave considerable therapeutic potential. Thus, the majorfocus of research in our laboratory is to understand theproteolytic mechanism of in vivo Aβ degradation.

For all investigated peptides we identified primary cleava-ge sites and involved proteases by using specific protea-se inhibitors. The impact of structural parameters on Aβ

clearance was explored in greater detail by using doubleD-amino acid replacement sets and N-C inverted peptides.In addition, other factors such as astrocytes which mayhave a direct role in Aβ degradation have been studied. Toaddress specific questions of peptide processing, weinclude transgenic and gene-targeted mouse lines that ei-ther overexpress or carry deletions of genes of interest.Our experimental tools comprise the use of reverse micro-dialysis both to introduce neuropeptides or inhibitors intothe brain of awake rodents, as well as to collect metabo-lic fragments. In collaborative work, advanced mass spec-trometric techniques are applied to monitor the clearanceof these neuropeptides close to real-time in dialysates ofa few microliters volume. Further efforts are directedtowards quantitative analysis of the peptide fragments.

Work report : Major objectives of the laboratoryNormally soluble β-amyloid peptides (Aβ), generated byproteolytic cleavage of the β-amyloid precursor protein(APP), is a mainly 40-42 amino acid species which mayextend to Aβ residue 43/46 in neuritic plaques (Zhao et al.,JBC 49, 50647, 2004). Although multiple proteases such asneutral endopeptidase (NEP; neprilysin), insulin-degrading

enzyme (IDE; insulysin) and angiotensin-converting enzy-me (ACE) have recently been identified to degrade extra-cellular Aβ, the proteolytic pathways are less well under-stood. In particular, in vivo studies are rare and differevidently from in vitro reports (for review Hersh, CurrPharm Des 9, 449, 2003). Reverse microdialysis in combina-tion with advanced mass spectrometric techniques is usedto study the clearance of these neuropetides in the brainof rodents (Figure 1).

Using these methods, we have recently demonstrated thepathways of in vivo processing of several Aβ species byNEP using specific protease inhibitors. Current studiesfocus on the interplay of structural parameters and protea-se activity as well as on the contribution of other candida-te proteases to the regulation of brain Aβ levels using rele-vant protease knockout and overexpressing mouse lines.Another project explores whether astrocytes could have adirect role in Aβ degradation either by Aβ-induced activa-tion or chemotactic migration to Aβ deposits (Wyss-Corayet al., Nature Med 9: 453-456, 2003). Astrocyte motility wasdemonstrated to be controlled by the ryanodine type 3receptor (RyR3) (Matyash et al., FASEB J 16: 84-86, 2002).These findings encouraged us to test the hypothesis thatextracelluar Aβ degradation is reduced in RyR3 knockoutmice associated with impaired astrocyte motility (in collabo-ration with H. Kettenmann, MDC, Berlin). Finally, studies areunderway directed at understanding the principles of exten-sive amyloid deposition in cerebral vessels leading to cere-bral amyloid angiopathy. While chronically reduced micro-circulation is believed to impair the clearance of circulatingAβ, the reported vasoconstrictor action of circulating Aβ

(Niwa et al., Am. J. Physiol 281, H2417, 2001) is still contro-versial. In order to address these questions, we have initia-ted collaborative work with M.J. Mulvany, University ofAarhus, Denmark, to explore the direct contractile effectsof Aβ in microvessels, as well as the possibility that theresulting hypoperfusion results in accumulation of Aβ.

A second area of research concerns the generation of bio-active angiotensin peptides from angiotensin I. In particu-lar, angiotensin (1-7) [Ang-(1-7)] is thought to mimic andoppose the multiple actions of angiotensin II. Both the roleof substrate availability and the metabolic pathways yield-ing to the formation of Ang-(1-7) remain unclear. Usingprotease knockout mouse models and protease inhibitors,we have elucidated the major pathways of Ang-(1-7) for-mation including the involved proteases in vivo.

Main objective 1. Impact of structural parameters on Aβ

processing Little is known about the impact of structural parameterson the catabolic pathways of Aβ peptides. Therefore, we

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FIGURE 2Comparison of MALDI mass spectra of Aβ(1-40) peptide fragments obtained fromwildtype (upper) and NEP knockout mice(bottom). The peptide was infused intothe hippocampus of the animals at a verylow flow rate. Relative signal intensity isgiven in arbitary units; m/z = mass-to-charge ratio.

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tested the hypothesis that both the sequence of aminoacids and the secondary structure may play a substantialrole in the processing of Aβ. Thus, two N-C inverted Aβ

chains, (42-1) and (40-1), and a double D-amino acid repla-cement set of Aβ were involved in the paradigm to studytheir proteolytic cleavage pattern. The two inverted Aβ

species serve as inactive controls because of lacking neu-rotoxic properties in cell cultures (Simmons et al., MolPharmacol 45, 373, 1994). In contrast to regular Aβ, theinverted Aβ species were cleaved to only a few short C-terminal fragments at the peptide bond His27-His28, Leu24-His25 and Phe22-Val23 in the (40-1) chain (Richter & Kraus,Soc Neurosci Abstr # 409.3, 2003). The cleavage site at His-His is considered to be a preferred target of IDE, whereascleavage sites at Phe-Phe and in the N-terminal region ofthe N-C inverted molecule are thought to be caused byNEP. The lack of cleavage sites caused by NEP suggeststhat only IDE but not NEP was involved in the degradationof Aβ under these experimental conditions.

Systematic double D-amino acid replacement of adjacentamino acids within the Aβ(1-42) molecule was used to studythe impact of the secondary structure on Aβ clearance. Incontrast to former reports, more recent studies revealedthat double D-amino acid substitution efficiently inhibitedthe peptide degradation primarily around the replacedamino acids. In aCSF, a complete protection of the molecu-le against enzymatic degradation indicating a folding of themolecule was not found. These results are strongly suppor-ted by findings obtained with Aβ dissolved in buffer andsubsequently analyzed with CD and ITC methods (S. Keller, unpublished data). These results suggest that theproteolytic clearance of Aβ peptides in vivo depends highlyon both the primary and secondary structure of the mole-cule and differs significantly from that ex vivo.

Main objective 2. Proteases involved in Aβ clearanceFirst, we explored the two principal regulators of Aβ clea-rance: NEP and IDE. The postulated primary cleavage siteat positions 33/44 (Gly-Leu) and the N-terminal directedladder-like degradation in rat hippocampus were success-fully blocked by the NEP inhibitors phosphoramidon andthiorphan, suggesting a major role of this particular pro-tease in Aβ clearance. However, compared to the litera-ture our in vivo findings indicate additional metabolic path-ways of Aβ. For example, the intermediate fragment(10-37), identified as the major product of Aβ(1-42) clea-vage by NEP (Iwata et al., Nature Med 6, 143, 2000), wasnot detected in our close to real-time experiments. More-over, separate metabolic studies revealed a very rapiddegradation of the fragment which does not correspond tothe Aβ(10-37) accumulation in catabolic studies on radio-labeled Aβ reported by Iwata and collegues. Also, nume-

rous mass spectra displayed an additional cleavage site atposition 34/35 (Leu-Met) attributed to a matrix metallopro-teinase-9 (MMP-9) which is believed to prevent plaqueformation (Backstrom et al., J Neurosci 16:7910, 1996).

To study the particular role of IDE in Aβ degradation, weextended our studies to awake NEP knockout versus wild-type mice. The in vivo profile of hippocampal Aβ proces-sing revealed cleavage patterns which match well to invitro findings from Mukherjee and colleagues (J Neurosci20:8745-8749, 2000) in so far as short N-terminal fragments(1-11 to 1-16) dominated in mass spectra while the C-ter-minal region remained intact (Figure 2) (Richter et al., SocNeurosci Abstr # 220.6, 2004). These results provide strongevidence that the major proteases NEP and IDE contribu-te to the clearance of Aβ by cleavage of differing peptidebonds in distinct regions within the peptide chain.

Group members

Antje Muschter (Technical assistance)* **Christian Wolff (Technical assistance)**

External funding

Schering AG-Ri1; Regina Richter


Richter RM, Kraus M (2003). Profile and Inhibition of the invivo Degradation of Alzheimer’s β-Amyloid Peptides in RatBrain. Soc Neurosci Abstr # 409.3

Richter RM, Heyne A, Schuchardt S (2004). Degradation ofAmyloid β-Peptide in the Hippocampus of a NEP knockoutMouse Model. Soc Neurosci Abstr # 220.6

Collaborations

Hersh, L.B. (Dept. Molec. and Cellular Biochemistry, Uni-versity of Kentucky, Lexington, KY, USA)Degradation of β-amyloid peptides in transgenic andknockout mouse models

Kettenmann, H. (Dept. Cellular Neuroscience, MDC forMolecular Medicine, Berlin, Germany)Clearance of β-amyloid peptides in the brain of RyR3-knockout mice

Mulvany, M.J. (Dept. Pharmacology, Aarhus University,Aarhus, Denmark)Cerebral hypoperfusion and beta-amyloid plaque formation

Schuchardt, S. (PLANTON GmbH), KielMALDI-TOF and MALDI-MS/MS analysis of the in vivometabolism of neuropeptides


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DNA SEQUENCINGGroup Leader: Dr. Erhard Klauschenz

The DNA Sequencing Service Group conducts DNAsequencing for the molecular research groups of the FMP.It comprises a scientist and a technician (part-time); twoautomated sequencers are available (ABI 377 and ABI310).

At present about 100 samples are processed weekly. TheDepartment of Signal Transduction/Molecular Medicinesubmits about 60% of the samples, the Junior ResearchGroup Cellular Signal Processing (Vinkemeier) 30%, withoccasional samples from other groups.

In addition to the service function, the group continues towork on the molecular analysis of the vasopressin V2receptor genes of patients (and their families) sufferingfrom congenital nephrogenic diabetes insipidus (NDI).

Group members

Barbara Mohs (Technical assistance)**

ADDITIONAL SERVICES

Scientif ic workshop

Jürgen MevertMichael UschnerHolger PanzerHelmut BlickStefanie Wendt

Safety Officer

Dr. Hans-Ulrich Heyne

Central glassware washing facili ty

Uwe Hackel †

Animal housing

Dr. Regina M. RichterEva LojekPetra Göritz

Academic l ibrary

Renate Peters

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PUBLIC RELATIONS AND THE MEDIADr. Björn Maul

The Forschungsinstitut für Molekulare Pharmakologie(FMP) uses public funds to conduct the major part of itsresearch into the basis of the effects of substances on theorganism. In order to give the taxpayer, who provides thefunds, some insight into how these resources are utilized,the FMP presents its work to the general public in an easi-ly understood form.

In 2003 and 2004 the FMP successfully participated invarious public exhibitions and presentations. Scientistsfrom the Institute used exhibits of interest to the laypersonto introduce their fields of research at the Science Fair ofthe Free University Berlin, at the “Müncher Wissen-schaftstage”, and at the presentation "Window on Sci-ence" held in the arcades at Potsdamer Platz.

The FMP also participated in the exhibition "Swimming lab(MS Chemie)" held on a ship in summer of the Year of Che-mistry, 2003. The ship, which was initiated by the science-to-public platform “Wissenschaft im Dialog”, moored in 25cities along the Rhine River and attracted more than 40,000visitors.

In April 2004 the FMP, together with ten other scientificinstitutions from Berlin and the German Human GenomeProject (DHGP), celebrated the fiftieth anniversary of thediscovery of the DNA helix structure with an exhibition inthe Berlin Museum of Natural History (Naturkundemu-seum) that received striking attention. About 40,000 peoplepaid a visit to the exhibition, which was the only one of itskind in all of Germany.

The FMP constantly strives to inspire enthusiasm forscientific research in young people of school age. Scien-tists regularly visit schools, presenting easily understoodlectures to promote discussion with pupils and teachingstaff. In addition, the FMP once a year awards a practicalcourse in the Life Science Learning Lab of the Berlin-BuchCampus to a winner of one of the country-wide competiti-ons in "Youth in Research.“ In 2004, the college studentSylke Höhne from Chemnitz won the prize and was hostedby the FMP in Berlin.

In each of the two reporting years, the FMP took theopportunity to present itself to the general public duringthe Berlin Long Night of the Sciences. At this event, morethan 1000 visitors came to isolate their own DNA from their

Hans-Olaf Henkel, President of the Leibniz Association, during theinauguration of the 900 Mhz spectrometerHans-Olaf Henkel, Präsident der Leibniz-Gemeinschaft, zur Einweihungdes 900 MHz-Spectrometers

Visit of the Federal President on Berlin-Buch Campus 2004Besuch des Bundespräsidenten auf dem Campus Berlin-Buch 2004

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PRESSE- UND ÖFFENTLICHKEITSARBEITDr. Björn Maul

Das Forschungsinstitut für Molekulare Pharmakologie(FMP) betreibt den überwiegenden Teil seiner Forschungüber die Grundlagen der Wirkung von Stoffen auf denOrganismus mit Geldern der öffentlichen Hand. Um demSteuerzahler, der die Gelder aufbringt, Einblick zu geben,wofür diese Mittel verwendet werden, stellt das FMPseine Arbeit in leicht verständlicher Form der breitenÖffentlichkeit vor.

In den Jahren 2003 und 2004 beteiligte sich das FMPerfolgreich an verschiedenen öffentlichen Ausstellungenund Präsentationen. Wissenschaftler des Instituts stelltenihre Forschungsgebiete anhand von für Laien eingängigenExponaten auf der Science Fair der Freien Universität Ber-lin, auf der Präsentation „Schaufenster der Wissenschaft“in den Potsdamer-Platz-Arkaden und auf den MünchnerWissenschaftstagen vor.

Das FMP beteiligte sich am Chemieschiff (MS Chemie) imSommer des Jahres der Chemie 2003. Diese von der „Wis-senschaft im Dialog” initiierte, schwimmende Ausstellunglief 25 Städte entlang des Rheins an und wurde von mehrals 40,000 Menschen besucht.

Im April 2004 beging das FMP zusammen mit zehn wei-teren wissenschaftlichen Einrichtungen aus Berlin undBrandenburg und dem Deutschen Humangenom-Projekt(DHGP) den 50. Jahrestag der Entdeckung der DNA-Helixmit einer Sonderausstellung im Berliner Naturkunde-museum. Etwa 50,000 Menschen besichtigten die Austel-lung, die die einzige ihrer Art in Deutschland war.

Das FMP versucht konsequent, junge Menschen bereitsim Schulalter für die naturwissenschaftliche Forschung zubegeistern. Regelmäßig besuchen Wissenschaftler Schu-len und stellen sich dort mit leicht verständlichen Vorträ-gen der Diskussion mit Schülern und Lehrern. Das Institutunterstützt den Wettbewerb „Jugend forscht“. Es vergibteinmal im Jahr einen Praktikumskurs im Gläsernen Labordes Campus als Preis für einen ausgewählten Landeswett-bewerb der Aktion. In 2004 erhielt die Abiturientin SylkeHöhne aus Chemnitz den Preis und war eine Woche langGast des FMP.

In jedem der beiden Berichtsjahre nahm das FMP dieGelegenheit wahr, sich in der Berliner Langen Nacht derWissenschaften einer breiten Öffentlichkeit zu präsentie-ren. Insgesamt kamen mehr als 1000 Menschen zu dieserVeranstaltung an das FMP, insbesondere um den Mit-machkurs „Meine DNA“ zu absolvieren und die eigene

Window on science, Potsdamer Platz in Berlin: Opening lectureSchaufenster der Wissenschaft am Potsdamer Platz 2003: Eröffnungs-vortrag

Exhibition “50th anniversary of the DNA helix structure” in the museumof Natural HistoryAusstellung „50 Jahre Doppelhelix“ im Naturkundemuseum

saliva. This special offer was first made in 2002 and beca-me a real attraction over the years.

The FMP greeted delegations from Germany and abroadand introduced the participants – government delegations,journalists, students and school pupils – to its work andthat of the entire Campus. In collaboration with BBB Cam-pus Management GmbH, the directorate consultants brief-ed the numerous visitors on content and organization.With the cooperation of Institute scientists, short lectures,tours of the laboratories and discussions were available.

To give the media the opportunity to report significantevents at the FMP, press releases were researched, writ-ten and issued. The public relations consultant as well asscientists gave interviews, e.g. for Deutschlandfunk, theHessian and Berlin broadcasters HR and RBB, as well asRadio Eins, Radio Multi-Kulti, Korea TV, and the audio:linkInternet radio station. The FMP has been reported in themajor Berlin daily newspapers. The national as well as theregional press have published reports on FMP scientistsand their research. FMP consultants and scientists havemade several contributions to brochures, various periodi-cals, e.g. for the Leibniz Association, the Berlin ResearchAssociation, the German Human Genome Project and for

the Berlin Buch Campus. The public relations consultantsupported telecasts like “nano” (3sat), “Quarks & Co.”(West German Broadcast WDR), and “ZDF-Expeditionen”(ZDF) as an adviser and by providing broadcastingmaterial.

The FMP gave independent presentations at the Parlia-mentary Evenings of the Leibniz Association in Brussels in2003 and the Forschungsverbund Berlin e.V. (BerlinResearch Association) in Berlin in 2004. The Institutebrought the research of its scientists to the attention of theinternational biotech community with a presentation at theinternational convention BIO 2004.

Personnel

Ulrike Lauterjung (Assistant)

“My DNA“ – The Long Night of the Sciences 2003 at the FMP„Meine DNA“ – Lange Nacht der Wissenschaften 2003 am FMP

Exhibition “Swimming lab“ 2003Ausstellung auf der MS Chemie 2003

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DNA aus einer Speichelprobe zu isolieren. Dieses beson-dere Angebot wurde im Jahr 2002 vom FMP initiiert undhat sich zu einer echten Attraktion entwickelt.

Das FMP empfing Delegationen aus dem In- und Auslandund machte die Teilnehmer – Regierungsdelegationen,Journalisten, Studenten und Schüler – mit seiner und derArbeit des gesamten Campus bekannt. Das Direktoratbereitete, oft in Zusammenarbeit mit der BBB Campusma-nagement GmbH, die zahlreichen Besuche inhaltlich undorganisatorisch vor. Zusammen mit den Wissenschaftlerndes Hauses wurden kurze Vorträge, Führungen durch dieLabore und Gespräche angeboten.

Um den Medien Anlass zur Berichterstattung über wichti-ge Ereignisse am FMP zu geben, wurden Pressemitteilun-gen recherchiert, verfasst und veröffentlicht. Der Referentfür Öffentlichkeitsarbeit vermittelte und gab Interviews,z. B. für den Deutschlandfunk, den Hessischen Rundfunk(HR), den Rundfunk in Berlin-Brandenburg (RBB), RadioEins, Radio Multi-Kulti, Korea TV und die audio:link Inter-net-Radiostation. Über das FMP und die FMP-Wissen-schaftler und ihre Forschung wurde in der überregionalenund regionalen Presse berichtet, so auch in Artikeln wich-

tiger Tageszeitungen der Berliner Presselandschaft. Refe-renten und Wissenschaftler des FMP verfassten Beiträgefür Broschüren und verschiedene Periodika, z. B. der Leib-niz-Gemeinschaft, des Forschungsverbunds Berlin e. V.,des Deutschen Humangenomprojekts und des CampusBerlin-Buch.

Der Referent für Öffentlichkeitsarbeit unterstützte ver-schiedene Fernsehpoduktionen, wie „nano” (3sat),„Quarks & Co.” (Westdeutscher Rundfunk) und „ZDF-Expeditionen” (ZDF) als Berater und mit audiovisuellemMaterial.

Das FMP beteiligte sich mit eigenständigen Präsentatio-nen an den Parlamentarischen Abenden der Leibniz-Gemeinschaft in Brüssel 2003 und des Forschungsver-bunds Berlin e. V. in Berlin 2004. Das Institut machte dieinternationale Biotech-Community auf die Forschung sei-ner Wissenschaftler mit einer Präsentation auf der BIO2004 aufmerksam.

Parliamentary Evening of the Forschungsverbund Berlin 2004Parlamentarischer Abend des Forschungsverbunds Berlin 2004

Science Fair 2004Science Fair 2004

VERWALTUNGLeiter: Thomas Ellermann

In der Verwaltung des FMP sind neben dem Verwaltungs-leiter sechs Mitarbeiterinnen beschäftigt (drei Vollzeit unddrei Teilzeit) beschäftigt. Nach dem positiven Abschlussdes letzten Ausbildungszyklus hat sich das FMP außerdementschlossen, erneut eine Bürokommunikations-Kauffrauauszubilden.

Ein Tätigkeitsschwerpunkt der Verwaltung im Berichtszeit-raum war die Vorbereitung zur Umstellung von der Fehlbe-darfsfinanzierung zu einer leistungs- und ergebnisorien-tierten Finanzierung des Instituts. Die leistungs- undergebnisorientierte Finanzierung ist erstmalig auf dasHaushaltsjahr 2006 anzuwenden.

Die Bund-Länder-Kommission für Bildungsplanung undForschungsförderung (BLK) fordert für die Einrichtungender Leibniz Gemeinschaft die Einführung eines Programm-budgets spätestens zum Haushaltsjahr 2006. Grundlage istder Beschluss der Regierungschefs des Bundes und derLänder vom Herbst 1997 zur „Sicherung und Qualität derForschung“.

Die im FMP 2002 implementierte und seit 2003 voll produk-tive Kosten- und Leistungsrechnung (KLR) mit ihren zweiHierarchieebenen war die Voraussetzung für die Einfüh-rung des Programmbudgets. In der vom Forschungsver-bund Berlin e. V. eingerichteten Projektgruppe hatte dasFMP 2004 die Rolle des Musterinstituts für alle Einrichtun-gen des FVB übernommen. Orientiert an den Vorgaben derBLK zu den „Mindestanforderungen an Programmbud-gets“ konnte ein Standardverfahren für den finanzwirt-schaftlichen Teil des Programmbudgets für die Institutedes FVB erarbeitet werden. Zunächst wurde mit Daten derKLR vom Jahr 2004 ein Programmbudget für das Haus-haltsjahr 2006 aufgestellt. Parallel wurde in Abstimmungmit dem Zuwendungsgeber Land Berlin ein Wirtschafts-plan erstellt.

Auf Wunsch der Wissenschaftler des FMP wurde die elek-tronische Bestellübermittlung eingeführt. Voraussetzungdafür war die erfolgreiche Implementierung der SAP-Ver-sion 4.6. Wissenschaftlich Beschäftigte haben jetzt dieMöglichkeit, ihre Beschaffungsanträge in digitaler Formim Intranet des Instituts zu hinterlegen. Softwareseitigkönnen buchungsrelevante Daten nach Prüfung über eineSchnittstelle in eine „Bestellanforderung“ (BANF) desSAP-Systems übernommen werden. Der Buchungsvor-

ADMINISTRATIONHead: Thomas Ellermann

In addition to the Administrative Director, the FMP admini-strative staff comprises six employees (three full-time andthree part-time). After the positive conclusion of the lasttraining cycle, the FMP has decided to take on a newoffice communication trainee.

A main focus of activity of the administration during thereporting period was the preparation for the conversionfrom the cameralistic system of accounting to a cost andperformance accounting system (CPA) for the institute.This CPA system will first be applied to the 2006 budgetyear.

The Joint Federal-State Commission for Education Plan-ning and Research Promotion demands that the instituti-ons of the Leibniz Association introduce program budgetsat the latest for the budget year 2006. The basis for this isthe decision of the heads of the federal and state govern-ments from autumn 1997 on “Securing and Quality ofResearch”.

The cost and performance accounting system (CPA) withits two hierarchical levels, which was implemented in 2002and has been operative since 2003, was the prerequisitefor the introduction of the program budget. In the projectgroup established by the Berlin Research Association(Forschungsverbund Berlin e.V., FVB) the FMP assumed in2004 the role of pilot institute for all institutes of the FVB.Following the directives of the Joint Federal-State Com-mission on “minimum requirements on program budgets”,a standard procedure for the fiscal part of the programbudget could be developed for the institutes of the FVB.Initially, with the data from the CPA from the year 2004, aprogram budget for the budget year 2006 was prepared.Parallel to that, a financial plan was created in coordina-tion with the State of Berlin as funding allocator.

At the request of FMP scientists, an electronic orderingsystem was introduced. A prerequisite for that was thesuccessful implementation of SAP version 4.6. Researchstaff members can now submit their purchase requests indigital form via the intranet of the institute. After verifica-tion of the entered data, data relevant to the transaction is

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transferred into SAP via an interface, and an SAP purcha-se request is created. With this procedure, the administra-tion’s transaction workflow is separated from the rest ofthe organization and cannot be accessed by third parties.The aim is to avoid error-prone and time-consuming dou-ble entries, e.g. in connection with chemical designations.Orders usually leave the institute on the same day theywere entered using the intranet. Expendable material isoften delivered within 24 hours of the order by the scien-tist, depending on the supplier. Therefore, there is no needfor costly warehousing of expendable material at the FMP.

Since 2002, together with the Max Delbrück Center forMolecular Medicine, the FMP has begun three buildingprojects and has in part completed them in the periodcovered by the report. In 2003 the new lab building NMR IIopened with 200 m2 floor space used by the FMP, and in2004 the 900 MHz NMR spectrometer started operation.After completion, together with the MDC, of the animallaboratory building Erwin Negelein House at the end of2004, the FMP has new animal laboratories with a total of385 m2 at its disposal. Organizing an economically sound

usage of these new resources in the coming years pre-sents great challenges for the FMP administration. Costsfor laboratory animal care will greatly increase.

The new Medicinal Genomics Research Building is due toopen in 2006. Five work groups with a total of 40 employeesfrom the FMP will use the 1.094 m2 of floor space allottedto the institute. In the new laboratory buildings labs arebeing fitted out with extensive technical equipment.

Personnel

Silvia Mauks (Personnel Manager) Birgit Sperling (General Administration)* **Christel Otto (General Administration) Claudia Messing (General Administration)**Kerstin Brauße (General Administration)**Gabriele Schumacher (Secretary)Dana Hausbeck (Trainee)*Josephine Passow (Trainee)*

gang der Verwaltung ist bei diesem Verfahren immer vomZugriff Dritter abgekoppelt. Ziel ist es fehlerträchtige undzeitaufwendige Doppelerfassungen, beispielsweise derChemikalienbezeichnung, zu vermeiden. Bestellungen ver-lassen das Institut in der Regel tagesgenau. Verbrauchs-mittel werden, abhängig vom Lieferanten, nicht selteninnerhalb von 24 Stunden nach der Bestellung durch denWissenschaftler ausgeliefert. Eine aufwendige Lagerhal-tung von Verbrauchsmitteln ist so am FMP nicht nötig.

Das FMP hat gemeinsam mit dem Max-Delbrück-Centerfür Molekulare Medizin seit 2002 drei Baumaßnahmenbegonnen und zum Teil im Berichtszeitraum abgeschlos-sen. In dem 2003 neu errichtete Labor-Gebäude „NMR II“mit 200 m2 vom FMP genutzter Fläche wurde 2004 das 900MHz-NMR-Spektrometer in Betrieb genommen. NachAbschluss der Baumaßnahme des gemeinsam mit demMDC erstellten Tierlaborgebäudes „Erwin-Negelein-Haus“ Ende 2004 kann das FMP neue Tierlabore mit insge-samt 385 m2 nutzen. Die Organisation eines wirtschaft-lichen Umgangs mit diesen neuen Ressourcen in dennächsten Jahren stellt die Verwaltung des FMP vor großeAnforderungen. Der Mittelaufwand für Tierhaltung wirdsich stark erhöhen.

Der Neubau „Medizinische Genomforschung“ wird vor-aussichtlich in 2006 bezogen werden. Fünf Arbeitsgruppenmit insgesamt 40 Mitarbeitern aus dem FMP werden dendem Institut zuzurechnenden Flächenanteil von 1.094 m2

nutzen. In den neuen Laborgebäuden entstehen Laborflä-chen mit aufwendigen technischen Ausstattungen.


COMPUTER-SERVICELeiter: Thomas Jahn

Zur Unterstützung der wissenschaftlichen Arbeit der For-schungsgruppen und der administrativen Infrastruktur(Verwaltung, Bibliothek u. a.) ist eine Arbeitsgruppe EDVzentral angesiedelt, die für folgende Bereiche verantwort-lich ist:• Bereitstellung von Kommunikationswegen und -res-

sourcen• Betrieb und Ausbau aller Netzwekkomponenten• zentrale Datenspeicherung, -sicherung und -austausch,

Printservices• Grundausstattung PC’s (Hard- und Softwarekoordinie-

rung)• technische Unterstützung bei Publikationserstellung

und -präsentationen

Das Rückgrat für alle Kommunikationswege bildet eineleistungsfähige Netzinfrastuktur, bestehend aus einerflexiblen strukturierten Netzwerkverkabelung und einemaktiven Netzwerk (modulare Layer 2/3-Switches).

Basis für alle EDV-Dienstleistungen ist ein heterogenerServerpool (NetWare 6 Cluster, LINUX und Windows 2kServer).

Durch die Arbeitsgruppe EDV werden die üblichen Inter-netdienste angeboten (eMail, www (Intranet), ftp-Server).Durch Kooperation mit der BBB werden die Services www(für FMP extern) und eMail (MX-Service) bereitgestellt.Der Internetprovider des FMP ist der DFN Verein. Der FVBerlin betreibt eine SAP-R/3-Serverarchitektur für Verwal-tungsaufgaben die clientseitig im FMP genutzt wird.

In der Bibliothek und in einem Grafikraum stehen PC-Arbeitsplätze mit entsprechender Peripherie für die Gra-fik- und Bildverarbeitung zur Verfügung.

Eine EDV-Kommission berät über Investitionen und per-spektivische Entwicklungen im IT-Bereich.

Für die nächste Zukunft bereitet die Gruppe Maßnahmenzur Erhöhung der Security vor, plant Schritte zur Server-konsilidierung und evaluiert künftige Thin-Client-Applika-tionen zur Senkung des administrativen Aufwandes.


COMPUTER SERVICESHead: Thomas Jahn

The EDP Service Group is centrally located to support thescientific work of the Research Groups and the admini-strative infrastructure (Administration, Library, etc.) and isresponsible for the following areas:• Provision of communication channels and resources • Operation and extension of network components • Central data storage, backup and interchange, print ser-

vices• Standard equipment for PCs (hardware and software

co-ordination)• Technical support in preparing publications and presen-

tations

The backbone of all communication channels forms anefficient network infrastructure consisting of flexible,structured network connections and an active network(modular layer 2/3 switches).

All EDP services are based on a heterogenous server (Net-Ware 6 Cluster, LINUX and Windows 2k Server). The usualInternet services are made available by the EDP Group

(e-mail, www (Intranet), ftp-Server). The services www (forFMP externally) and e-mail (MX-Service) are providedthrough cooperation with the BBB. The DFN Association isthe Internet provider for the FMP. The Berlin Research Asso-ciation (FV Berlin) operates SAP-R/3 server architecture foradministrative tasks, used on the client side in the FMP. PCwork stations with corresponding peripherals for graphicsand image processing are available in the library and in agraphics room.

An EDP Commission advises on investments and develop-mental perspectives in the IT field.

The next tasks of the Group will be implementing measuresto increase security, server consolidation and the evalua-tion of future Thin Client Applications to reduce admini-strative overheads.

Personnel

Ingrid Hermann (Software Administration) Hans-Werner Pisarz (Service Engineer)Alexander Heyne (Student)**Björn Schümann (Student)* **

APPENDIX

PEER REVIEWED ARTICLES 2003ORIGINALARBEITEN 2003

SECTION STRUCTURAL BIOLOGY

Protein Structure

Castellani F, van Rossum BJ, Diehl A, Rehbein K, Oschkinat H (2003) Determination of solid-state NMRstructures of proteins by means of three-dimensional 15N-13C-13C dipolar correlation spectroscopy and chemicalshifts analysis. Biochemistry 42, 11476-11483

Chevelkov V, van Rossum BJ, Castellani F, Rehbein K,Diehl A, Hoh M, Steuernagel S, Engelke F, Oschkinat H,Reif B (2003) 1H detection in MAS solid-state NMR spec-troscopy of biomacromolecules employing pulsed fieldgradients for residue solvent suppression. J Am Chem Soc125, 7788-7789

Labudde D, Leitner D, Krüger M, Oschkinat H (2003) Pre-diction algorithm for amino acid type with its secondarystructure in proteins (PLATONS) using chemical shifts. JBiomol NMR 25, 41-53

Ladizhansky V, Jaroniec CP, Diehl A, Oschkinat H, GriffinRG (2003) Measurement of multiple psi torsion angles inuniformly 13C, 15N-labeled alpha-spectrin SH3 domainusing 3D 15N-13C-13C-15N MAS dipolar-chemical shiftcorrelation spectroscopy. J Am Chem Soc 125, 6827-6833

Pires JR, Hong X, Brockmann C, Volkmer-Engert R,Schneider-Mergener J, Oschkinat H, Erdmann R (2003)The ScPex13p SH3 domain exposes two distinct bindingsites for Pex5p and Pex14p. J Mol Biol 326, 1427-1435

Reif B, van Rossum B, Castellani F, Rehbein K, Diehl A,Oschkinat H (2003) Characterization of 1H-1H distances ina uniformly 2H, 15N-labeled SH3 domain by MAS solid-state NMR spectroscopy.J Am Chem Soc 125, 1488-1489

Strauss H (2003) A device for facilitating the use of theFrench Press. Analytical Biochemistry 321, 276-277

Toepert F, Knaute T, Guffler S, Pires JR, Matzdorf T,Oschkinat H, Schneider-Mergener J (2003) CombiningSPOT Synthesis and Nature Peptide Ligation to CreateLarge Arrays of WW Domains. Angew Chem Int Ed 42,1136-1140

Van Rossum B, Castellani F, Pauli J, Rehbein K, HollanderJ, de Groot H, Oschkinat H (2003) Assignment of amideproton signals by combined evaluation of HN, NN andHNCA MAS-NMR correlation spectra. J Biomol NMR 25,217-223

Zimmermann J, Kühne R, Volkmer-Engert R, Jarchau T,Walter U, Oschkinat H, Ball LJ (2003) Design of N-substi-tuted Peptomer Ligands for EVH1 Domains. J Biol Chem278, 36810-36818

Solution NMR

Hupfer M, Rübe B, Schmieder P (2003) Origin and diage-nesis of polyphosphate in lake sediments: A 31P-NMRstudy. Limnol Oceanogr 49, 1-10

Otte L, Wiedemann U, Schlegel B, Pires JR, BeyermannM, Schmieder P, Krause G, Volkmer-Engert R, Schneider-Mergener J, Oschkinat H (2003) WW domain sequenceactivity relationships identified using ligand recognitionpropensities of 42 WW domains. Protein Sci 12, 491-500

Structural Bioinformatics


Molecular Modelling

Freund C, Kühne R, Park S, Thiemke K, Reinherz EL,Wagner G (2003) Structural investigations of a GYF domaincovalently linked to a proline-rich peptide. J Biomol NMR27, 143-149

Karges B, Karges W, Mine M, Ludwig L, Kühne R, MilgromE, de Roux N (2003) Mutation Ala171Thr stabilizes thegonadotropin releasing hormone receptor in its inactiveconformation, causing familial hypogonadotropic hypogo-nadism. J Clin Endocrinol Metab 88, 1873-1879

Zimmermann J, Kühne R, Volkmer-Engert R, Jarchau T,Walter U, Oschkinat H, Ball LJ (2003) Design of N-substi-tuted Peptomer Ligands for EVH1 Domains. J Biol Chem278, 36810-36818

Solid State NMR

Chevelkov V, van Rossum BJ, Castellani F, Rehbein K,Diehl A, Hoh M, Steuernagel S, Engelke F, Oschkinat H,Reif B (2003) 1H detection in MAS solid-state NMR spec-troscopy of biomacromolecules employing pulsed fieldgradients for residue solvent suppression. J Am Chem Soc125, 7788-7789

Narayanan S, Bösl B, Walter S, Reif B (2003) Importance oflow oligomeric weight species for prion propagation in theyeast prion system Sup35/Hsp104. Proc Natl Acad SciU.S.A. 100, 9286-9291

FMP-authors in bold.

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Reif B, Griffin RG (2003) 1H detected 1H, 15N correlationspectroscopy in rotating solids. J Magn Reson 160, 78-83

Reif B, van Rossum B, Castellani F, Rehbein K, Diehl A,Oschkinat H (2003) Characterization of 1H-1H distances ina uniformly 2H, 15N-labeled SH3 domain by MAS solid-state NMR spectroscopy. J Am Chem Soc 125, 1488-1489

Protein Engineering

Freund C, Kühne R, Park S, Thiemke K, Reinherz EL,Wagner G (2003) Structural investigations of a GYF domaincovalently linked to a proline-rich peptide. J Biomol NMR27, 143-149

SECTION CELLULAR SIGNALLING /MOLECULAR GENETICS

Cellular Signalling

Tamma G, Klussmann E, Procino G, Svelto M, RosenthalW, Valenti G (2003) cAMP induced AQP2 translocation isassociated with RhoA inhibition through RhoA phosphory-lation and interaction with RhoGDI. J Cell Sci 116, 1519-1525

Lorenz D, Krylov A, Hahm D, Hagen V, Rosenthal W, Pohl P, Maric K (2003) Cyclic AMP is sufficient for trigger-ing the exocytic recruitment of aquaporin-2 in renal ephi-thelial cells. EMBO Rep 4, 88-94

Protein Trafficking

Engelsberg A, Hermosilla R, Karsen U, Schülein R, DorkenB, Rehm A (2003) The Golgi protein RCAS1 controls cellsurface expression of tumor-associated O-linked glycanantigens. J Biol Chem 278, 22998-23007

Anchored Signalling

Tamma G, Klussmann E, Procino G, Svelto M, RosenthalW, Valenti G (2003) cAMP induced AQP2 translocation isassociated with RhoA inhibition through RhoA phosphory-lation and interaction with RhoGDI. J Cell Sci 116, 1519-1525

Tamma G, Wiesner B, Furkert J, Hahm D, Oksche A,Schaefer M, Valenti G, Rosenthal W, Klussmann E (2003)The prostagladin E2 analogue sulprotone antagonizesvasopressin-induced antidiuresis through activation ofRho. J Cell Sci 116, 3285-94

Schmitt R, Klussmann E, Kahl T, Ellison DH, Bachmann S(2003) Renal expression of sodium transporters and aqua-porin-2 inhypothyroid rats. Am J Physiol Renal Physiol 284,1097-104

Storm R, Klussmann E, Geelhaar A, Rosenthal W, Maric K(2003) Osmolality and solute composition are strong regu-lators of AQP2 expression in renal principal cells. Am JPhysiol Renal Physiol 284, 189-98

Cellular Imaging

Geissler D, Kresse W, Wiesner B, Bendig J, KettenmannH, Hagen V (2003) DMACM-Caged Adenosine Nucleotides:Ultrafast Phototriggers for ATP, ADP, and AMP Activatedby Long-Wavelength Irradiation. ChemBiochem 4, 162-170

Hagen V, Frings S, Bendig J, Lorenz D, Wiesner B, KauppUB (2003) Fluoreszenzspektroskopische Quantifizierungder Freisetzung von cyclischen Nukleotiden aus photo-aktivierbaren [Bis(carboxymethoxy)-cumarin-4-yl]methy-lestern in Zellen. Angew Chem 114, 3775-3777

Hagen V, Frings S, Wiesner B, Helm S, Kaupp UB, BendigJ (2003) [7-(Dialkylamino)coumarin-4-yl]methyl-cagedcompounds as ultrafast and effective long-wavelengthphototriggers of 8-Bromo-substituted cyclic nucleotides.ChemBiochem 4, 434-442

Kamp G, Büsselmann G, Jones N, Wiesner B, LauterweinJ (2003) Energy metabolism and intracellular pH in boar(Sus scrofa) spermatozoa. Reproduction 126, 517-525


Wiesner B, Roloff B, Fechner K, Slominski A (2003) Intra-cellular calcium measurements of single human skin cellsafter stimulation with corticotropin-releasing factor andurocortin using confocal laser scanning microscopy. J CellSci 116, 1261-1268

Meyer T, Marg A, Lemke P, Wiesner B, Vinkemeier U(2003) DNA binding controls inactivation and nuclearaccumulation of the transcription factor. Stat1 Genes Dev17, 1992-2005


Molecular Cell Physiology

Wallukat G, Podlovski S, Nissen ER, Morwinski R, CsonkaC, Tosaki A, Blasig IE (2003) Functional and structural cha-racterization of anti-beta1-adrenoceptor auto-antibodiesof spontaneously hypertensive rats. Mol Cell Biochem 251,67-75

Schroeter ML, Abdul-Khaliq H, Fruhauf S, Hohne R, SchickG, Diefenbacher A, Blasig IE (2003) Serum S100B is increa-sed during early treatment with antipsychotics and in defi-cit schizophrenia. Schizophrenia Res 62, 231-236

Biochemical Neurobiology


Biophysics

Urbánková E, Voltchenko A, Pohl P, Jezek P, Pohl EE (2003)Transport kinetics of uncoupling proteins, Analysis ofUCP1 reconstituted in planar lipid bilayers. J Biol Chem278, 32497-32500

Lorenz D, Krylov A, Hahm D, Hagen V, Rosenthal W, PohlP, Maric K (2003) Cyclic AMP is sufficient for triggering theexocytic recruitment of aquaporin-2 in renal ephithelialcells. EMBO Rep 4, 88-94

Molecular Genetics

Wieczorek G, Steinhoff C, Schulz R, Scheller M, VingronM, Ropers HH, Nuber UA (2003) Gene expression profile ofmouse bone marrow stromal cells determined by cDNAmicroarray analysis. Cell Tissue Res 311, 227-237

Barmeyer C, Horak I, Zeitz M, Fromm M, Schultzke JD(2003) The interleukin-2-deficient mouse model. Patho-biology 70, 139-142

Cellular Signal Processing

Chen X, Bhandari R, Vinkemeier U, Van Den Akker F,Darnell JE Jr, Kuriyan J (2003) A reinterpretation of thedimerization interface of the N-terminal domains of STATs.Protein Sci 142, 361-365

Meyer T, Vinkemeier U, Meyer U (2003) MedizinischeImplikationen pharmakogenomischer Behandlungsstrate-gien. Ethik in der Medizin 12, 207-209

Meyer T, Vinkemeier U, Meyer U (2003) Evidence-basedmedicine - Was geht verloren? Ethik in der Medizin 14, 3-10



Peptide Synthesis

Carpino LA, Ionescu D, El-Faham A, Beyermann M, Henk-lein P, Hanay C, Wenschuh H, Bienert M (2003) Complexpolyfluoride additives in Fmoc-amino acid fluoride coup-ling processes. Enhanced reactivity and avoidance ofstereomutation. Org Lett 5, 975-977

Carpino LA, Imazumi H, El Faham A, Fernando JF, Zhang C,Lee Y, Foxman BM, Henklein P, Hanay C, Mugge C,Wenschuh H, Klose J, Beyermann M, Bienert M (2003)The uronium/guanidinium peptide coupling reagents:Finally the true uronium salts. Biopolymers 71, 349


Von Eggelkraut-Gottanka R, Klose A, Beck-Sickinger AG,Beyermann M (2003) Peptide (alpha)thioester formationusing standard Fmoc-chemistry. Tetrahedron Lett 44, 3551-3554

Wissmann R, Bildl W, Oliver D, Beyermann M, Kalbitzer HR,Bentrop D, Fakler B (2003) Solution structure and functionof the “Tandem inactivation domain” of the neuronal A-type potassium channel Kv1.4. J Biol Chem 278, 16142-16150

Kaupp UB, Solzin J, Hildebrandt E, Brown JE, Helbig A,Hagen V, Beyermann M, Pampaloni F, Weyand I (2003) Thesignal flow and motor response controlling chemotaxis ofsea urchin sperm. Nat Cell Biol 5, 109-117


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Mass Spectrometry

Bente M, Harder S, Wiesgigl M, Heukeshoven J, GelhausC, Krause E, Clos J, Bruchhaus I (2003) Developmentallyinduced changes of the proteome in the protozoan parasi-te Leishmania donovani. Proteomics 3, 1811-1829

Czupalla C, Nürnberg B, Krause E (2003) Analysis of classI phosphoinositide 3-kinase autophosphorylation sites bymass spectrometry. Rapid Commun Mass Spec 17, 690-696

Czupalla C, Culo M, Müller EC, Brock C, Reusch HP,Spicher K, Krause E, Nürnberg B (2003) Identification andCharacterization of the Autophosphorylation Sites of Phos-phoinositide 3-Kinase Isoforms Beta and Gamma. J BiolChem 278, 11536-11545

Beattie KA, Ressler J, Wiegand C, Krause E, Codd GA,Steinberg CEW, Pflugmacher S (2003) Comparative effectsand metabolism of two microcystins and nodularin in thebrine shrimp Artemia salina. Aquat Toxicol 62, 219-226

Karlsson K, Sipia V, Krause E, Meriluoto J, Pflugmacher S(2003) Mass Spectrometric Detection and Quantificationof Nodularin-R in Flounder Livers. Environ Toxicol Chem 18,284-288

Kraus M, Bienert M, Krause E (2003) Hydrogen exchangestudies on Alzheimer's amyloid-beta peptides by massspectrometry using matrix-assisted laser desorption/ioni-zation and electrospray ionization. Rapid Commun MassSpec 17, 222-228

Kleuss C, Krause E (2003) G-alpha-s is palmitoylated at theN-terminal glycine. EMBO J 22, 826-832

Synthetic Organic Biochemistry

Geissler D, Kresse W, Wiesner B, Bendig J, KettenmannH, Hagen V (2003) DMACM-Caged Adenosine Nucleotides:Ultrafast Phototriggers for ATP, ADP, and AMP Activatedby Long-Wavelength Irradiation. ChemBiochem 4, 162-170

Hagen V, Frings S, Bendig J, Lorenz D, Wiesner B, KauppUB (2003) Fluoreszenzspektroskopische Quantifizierungder Freisetzung von cyclischen Nukleotiden aus photo-aktivierbaren [Bis(carboxymethoxy)-cumarin-4-yl]methyl-estern in Zellen. Angew Chem 114, 3775-3777

Hagen V, Frings S, Wiesner B, Helm S, Kaupp UB, BendigJ (2003) [7-(Dialkylamino)coumarin-4-yl]methyl-cagedcompounds as ultrafast and effective long-wavelengthphototriggers of 8-Bromo-substituted cyclic nucleotides.ChemBiochem 4, 434-442

Kaupp UB, Solzin J, Hildebrandt E, Brown JE, Helbig A,Hagen V, Beyermann M, Pampaloni F, Weyand I (2003) Thesignal flow and motor response controlling chemotaxis ofsea urchin sperm. Nat Cell Biol 5, 109-117

Matsumoto M, Solzin J, Helbig A, Hagen V, Ueno S,Kawase O, Maruyama Y, Ogiso M, Godde M, Minakata H,Kaupp UB, Hoshi M, Weyand I (2003) A sperm-activatingpeptide controls a cGMP-signaling pathway in starfishsperm. Dev Biol 260, 314-324

Lorenz D, Krylov A, Hahm D, Hagen V, Rosenthal W, PohlP, Maric K (2003) Cyclic AMP is sufficient for triggering theexocytic recruitment of aquaporin-2 in renal ephithelialcells. EMBO Rep 4, 88-94

Medicinal Chemistry

Rademann J (2003) Advanced polymer reagents based onactivated reactants and reactive intermediates: powerfulnovel tools in diversity-oriented synthesis. Method Enzy-mol 369, 366-390

Weik S, Rademann J (2003) A phosphorane as supportedacylanion equivalent: linker reagents for smooth and ver-satile C-C coupling reactions. Angew Chem Int Ed 42, 2491-2494

Rademann J (2003) Trimellitic anhydride linker (TAL) –highly orthogonal conversions of primary amines employ-ed in the parallel synthesis of labeled carbohydrate deriva-tives. Tetrahedon Lett 44, 5019-5023


PEER REVIEWED ARTICLES 2004ORIGINALARBEITEN 2004

SECTION STRUCTURAL BIOLOGY

Protein Structure

Brockmann C, Leitner D, Labudde D, Diehl A, Sievert V,Büssow K, Kühne R, Oschkinat H (2004) The solution struc-ture of the SODD BAG-domain and a model of the SODD-BAG/HAP70 complex reveal additional SODD subfamily-specific electrostatic interactions. FEBS Lett 558, 101-106

Brockmann C, Diehl A, Rehbein K, Strauss H, SchmiederP, Korn B, Kuhne R, Oschkinat H (2004) The oxozidid sub-unit b8 from human complex I adopts a thioredoxin fold.Structure 12, 1645-1654

Gaiser OJ, Oschkinat H, Heinemann U, Ball LJ (2004) (1),(13) C and resonance assignments of C-terminal BRCTdomain from human BRCA1. J Biol NMR 30, 221-222

Gaiser OJ, Ball LJ, Schmieder P, Leitner D, Strauss H,Wahl M, Kühne R, Oschkinat H, Heinemann U (2004) Solu-tion structure, backbone dynamics, and association beha-vior of the C-terminal BRCT domain from the breast can-cer-associated BRCA1. Biochemistry 43, 15983-15995

Kahmann JD, Wecking DA, Putter V, Lowenhaupt K, KimYG, Schmieder P, Oschkinat H, Rich A, Schade M (2004)The solution structure of the N-terminal domain of E3Lshows a tyrosine conformation that may explain its redu-ced affinity to Z-DNA in vitro. Proc Natl Acad Sci U.S.A.101, 2712-2717

Krabben L, van Rossum BJ, Castellani F, Bocharov E,Schulga AA, Arseniev AS, Weise C, Hucho F, Oschkinat H(2004) Towards structure determination of neurotoxin IIbound to nicotinic acetylcholine receptor: a solid-stateNMR approach. FEBS Lett 564, 319-324

Mueller U, Bussow K, Diehl A, Bartl FJ, Niesen FH, Nyarsik L, Heinemann U (2004) Rapid purification and cry-stal structure analysis of a small protein carrying two ter-minal affinity tags. J Struct Funct Genomics 4, 217-225

Pahlke D, Leitner D, Wiedemann U, Labudde D (2004)COPS - Cis/trans peptide bond conformation prediction ofamino acids on the basis of secondary structure informa-tions. Bioinformatics 10, 27-28

Pope BJ, Zierler-Gould KM, Kuhne R, Weeds AG, Ball LJ(2004) Solution structure of human cofilin: rationalising thepH sensitivity of actin binding. J Biol Chem 279, 4840-4848

Soukenik M, Diehl A, Leidert M, Sievert V, Büssow K,Leitner D, Labudde D, Ball LJ, Lechner A, Nägler DK,Oschkinat H (2004) The SEP domain of p47 acts as a rever-sible competitive inhibitor of cathepsin L. FEBS Lett 576,358-362

Schleinkofer K, Wiedemann U, Otte L, Wang T, Krause G,Oschkinat H (2004) Comparative Structural and EnergeticAnalysis of WW Domain-peptide. J Mol Biol 344, 865-881

Waldmann H, Karaguni IM, Carpintero M, Gourzoulidou E,Herrm C, Brockmann C, Oschkinat H, Muller O (2004) Sulin-dac-Derived Ras Pathway Inhibitors Target the Ras-RaInteraction and Downstream Effectors in the Ras Pathway.Angew Chem Int Ed 43, 454-458

Wiedemann U, Boisguerin P, Leben R, Leitner D, KrauseG, Mölling K, Volkmer-Engert R, Oschkinat H (2004) Quanti-fication of PDZ Domain Specificity, Prediction of LigandAffinity and Rational Design of Super-Binding Peptides. JMol Biol 343, 703-718

Zierler-Gould KM, Pope B, Weeds AG, Ball LJ (2004) Letterto the Editor: backbone and sidechain 1H, 13 C and 15Nresonance assignments of human cofilin. J Biol NMR 29,429-430

Zimmermann J, Jarchau T, Walter U, Oschkinat H, Ball LJ(2004) 1H, 13C and 15N resonance assignment of thehuman Spred2 EVH1 domain. J Biol NMR 29, 435-436

Wüller S, Wiesner B, Löffler A, Furkert J, Krause G,Hermosilla R, Schaefer M, Schülein R, Rosenthal W,Oksche A (2004) Pharmacochaperones post-translational-ly enhance cell surface expression by increasing confor-mational stability of wild-type and mutant vasopressin V2receptor. J Biol Chem 279, 47254-47263

Solution NMR

Brockmann C, Diehl A, Rehbein K, Strauss H, SchmiederP, Korn B, Kuhne R, Oschkinat H (2004) The oxozidid sub-unit B8 from human complex I adopts a thioredoxin fold.Structure 12, 1645-1654


Kahmann JD, Wecking DA, Putter V, Lowenhaupt K, KimYG, Schmieder P, Oschkinat H, Rich A, Schade M (2004)The solution structure of the N-terminal domain of E3Lshows a tyrosine conformation that may explain its redu-ced affinity to Z-DNA in vitro. Proc Natl Acad Sci U.S.A.101, 2712-2717


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Scheich C, Leitner D, Sievert V, Leidert M, Schlegel B,Simon B, Letunic K, Bussow K, Diehl A (2004) Fast identi-fication on folded human protein domains expressed in E. coli suitable for structural analysis. BMC Structural Bio-logy 4, 4


Neumann S, Krause G, Claus M, Paschke R (2004) Structu-ral Determinants for G-Protein Activation and Selectivityin the Second Intracellular Loop of the Thyrotropin Recep-tor. Endocrinol 46, 477-485

Kaufmann R, Schulze B, Krause G, Mayr LM, Setmacher U,Henklein P (2004) Proteinase-activated receptors (PARs)– The PAR3 Neo-N-terminal peptide TFRGAP interactswith PAR1. Regulatory Peptides, in press

Kleinau G, Jaeschke H, Neumann S, Laettig J, Paschke R,Krause G (2004) Identification of a novel epitope in the TSHreceptor ectodomain acting as intramolecular signallinginterface. J Biol Chem 279, 51590-51600

Müller SL, Portwich M, Schmidt A, Utepbergenov DI,Huber O, Blasig IE, Krause G (2004) The tight junction pro-tein occludin and the adherens junction protein alpha-catenin share a common interaction mechanism. J BiolChem 280, 3747-3756


Schleinkofer K, Wiedemann U, Otte L, Wang T, Krause G,Oschkinat H (2004) Comparative Structural and EnergeticAnalysis of WW Domain-peptide. J Mol Biol 344, 865-881

Wiedemann U, Boisguerin P, Leben R, Leitner D, KrauseG, Mölling K, Volkmer-Engert R, Oschkinat H (2004) Quan-tification of PDZ Domain Specificity, Prediction of LigandAffinity and Rational Design of Super-Binding Peptides. JMol Biol 343, 703-718


Molecular Modelling

Baumgart S, Lindner Y, Kuhne R, Oberemm A, WenschuhH, Krause E (2004) The contributions of specific amino acidside chains to signal intensities of peptides in matrix-assi-sted laser desorption/ionization mass spectrometry. RapidCommun Mass Spec 18, 863-868

Brockmann C, Leitner D, Labudde D, Diehl A, Sievert V,Büssow K, Kühne R, Oschkinat H (2004) The solution struc-ture of the SODD BAG-domain and a model of the SODD-BAG/HAP70 complex reveal additional SODD subfamily-specific electrostatic interactions. FEBS Lett 558, 101-106

Brockmann C, Diehl A, Rehbein K, Strauss H, SchmiederP, Korn B, Kuhne R, Oschkinat H (2004) The oxozidid sub-unit B8 from human complex I adopts a thioredoxin fold.Structure 12, 1645-1654


Pope BJ, Zierler-Gould KM, Kuhne R, Weeds AG, Ball LJ(2004) Solution structure of human cofilin: rationalising thepH sensitivity of actin binding. J Biol Chem 279, 4840-4848

Solid State NMR

Chen Z, Reif B (2004) Measurements of residual dipolarcouplings in peptide inhibitors weakly aligned by transientbinding to peptide amyloid fibrils. J Biol NMR 29, 525-530

Chevelkov V, Chen Z, Bermel W, Reif B (2004) Resolutionenhancement in MAS solid-state NMR by application of13C homonuclear scalar decoupling during acquisition. JMagn Reson 172, 56-62

Ventura S, Zurdo J, Narayanan S, Parreno M, Mangues R,Reif B, Chiti F, Giannoni E, Dobson CM, Aviles FX, SerranoL (2004) Short amino acid stretches can mediate amyloidformation in globular proteins: the Src homology 3 (SH3)case. Proc Natl Acad Sci U.S.A. 101, 7258-7263

Protein Engineering

Heuer K, Kofler M, Langdon G, Thiemke K, Freund C (2004)Structure of a helically extended SH3 domain of the T celladapter protein ADAP. Structure 12, 603-610

Kofler M, Heuer K, Zech T, Freund C (2004) Recognitionsequences for the GYF domain reveal a possible splicoso-mal function of CD2BP2. J Biol Chem 279, 28292-28297


SECTION CELLULAR SIGNALLING / MOLE-CULAR GENETICS

Cellular Signalling

Gregan B, Schäfer M, Rosenthal W, Oksche A (2004) Fluo-rescence resonance energy transfer analysis reveals theexistence of endothelin A and endothelin B receptorhomodimers. J Cardiovasc Pharmacol 44, S30-33

Gregan B, Jürgensen J, Papsdorf G, Furkert J, SchaeferM, Beyermann M, Rosenthal W, Oksche A (2004) Ligand-dependent differences in the internalization and intracel-lular trafficking of endothelin A and endothelin B receptorheterodimers. J Biol Chem 279, 27679-27687

Hällbrink M, Oehlke J, Papsdorf G, Bienert M (2004) Up-take of cell-penetrating peptides is dependent on the pep-tide-to-cell-ratio rather than on peptide concentration.Biochim Biophys Acta 1667, 222-228


Hermosilla R, Oueslati M, Donalies U, Schönenberger E,Krause E, Oksche A, Rosenthal W, Schuelein R (2004)Disease-causing V2 Vasopressin Receptors are Retainedin Different Compartments of the Early Secretory Pathway.Traffic 12, 993-1005

Tsunoda AP, Wiesner B, Lorenz D, Rosenthal W, Pohl P(2004) Aquaporin-1, Nothing but a water channel. J BiolChem 279, 11364-11367

Wietfeld D, Heinrich N, Furkert J, Fechner K, BeyermannM, Bienert M, Berger H (2004) Regulation of the Couplingto Different G-Proteins of Rat Corticotropin-Releasing Fac-tor Receptor Type 1 (rCRFR1) in HEK293 Cells. J Biol Chem279, 38386-38394


Protein Trafficking

Droese J, Mokros T, Hermosilla R, Schuelein R, Lipp M,Hopken UE, Rehm A (2004) HCMV-encoded chemokinereceptor US28 employs multiple routes for internalization.Biochem Biophys Res Comm 322, 42-49


Neuschäfer-Rube F, Rehwald M, Hermosilla R, Schülein R,Rönnstrand L, Püschel G (2004) Identification of differentSer/Thr residues in the C-terminal domain of the humanEP4 reseptor for agonist-induced phosphorylation, beta-arrestin interaction and sequestration Biochemistry 379,573-585


Anchored Signalling


Cellular Imaging

Geissler D, Antonenko YN, Schmidt R, Keller S, KrylovaOO, Wiesner B, Bendig J, Pohl P, Hagen V (2004) (Couma-rin-4-yl)methyl Esters as Highly Efficient, Ultrafast Photot-riggers for Protons and Their Application to AcidifyingMembrane Surfaces. Angew Chem Int Ed 44, 1195-1198



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Mathas S, Lietz A, Anagnostopoulos I, Hummel F, WiesnerB, Janz M, Jundt F, Hirsch B, Johrens-Leder K, VornlocherHP, Bommert K, Stein H, Dorken B (2004) C-FLIP mediatesresistance of Hodgkin/Reed-Sternberg cells to deathreceptor-induced apoptosis. J Exp Med 199, 1041-1052





Haseloff RF, Blasig IE, Bauer HC, Bauer H (2004) In searchof the astrocytic factor(s) modulating blood-brain barrierfunction in brain capillary endothelial cells in vitro. Mol CellNeurosi, in press

Moser KV, Reindl M, Blasig IE, Humpel C (2004) Braincapillary endothelial cells proliferate in response to NGF,express NGF receptors and secrete NGF after inflammati-on. Brain Res 1017, 53-60

Müller SL, Portwich M, Schmidt A, Utepbergenov DI,Huber O, Blasig IE, Krause G (2004) The tight junction pro-tein occludin and the adherens junction protein alpha-catenin share a common interaction mechanism. J BiolChem 280, 3747-3756


Zassler B, Blasig IE, Humpel C (2004) Protein delivery ofcaspase-3 induces cell death in malignant C6 glioma andbrain capillary endothelial cells. J Neuro-Oncol 71, 127-134


Walter T, Stepan H, Pankow K, Gembard F, Faber R,Schultheiss HP, Siems WE (2004) Relation of ANP and BNPto their N-terminal fragments in fetal circulation: evidencefor enhanced neutral endopeotidase activity and resi-stance of BNP to neutral endopeptidase in the fetus. BJOG111, 452-455

Walter T, Stepan H, Pankow K, Becker M, Schultheiss HP,Siems WE (2004) Biochemical analysis of neutral endopep-tidase activity reveals independent catabolism of atrialand brain natriuretic peptide. Biol Chem 385, 179-184

Biophysics

Geissler D, Antonenko YN, Schmidt R, Keller S, KrylovaOO, Wiesner B, Bendig J, Pohl P, Hagen V (2004) (Couma-rin-4-yl)methyl Esters as Highly Efficient, Ultrafast Photo-triggers for Protons and Their Application to AcidifyingMembrane Surfaces. Angew Chem Int Ed 44, 1195-1198

Krylova O, Pohl P (2004) Ionophoric Activity of PluronicBlock Copolymers. Biochemistry 43, 3696-3703

Saparov SM, Pohl P (2004) Beyond the diffusion limit:Water flow throught the empty bacterial potassium chan-nel. Proc Natl Acad Sci U.S.A. 101, 4805-4809

Sun J, Pohl EE, Krylova OO, Krause E, Agapov II, Tonevitzky AG, Pohl P (2004) Membrane destabilization byricin. Eur Biophys J 33, 572-579


Molecular Genetics

Barmeyer C, Harren M, Schmitz H, Heinzel-Pleines U,Mankertz J, Seidler U, Horak I, Wiedenmann B, Fromm M,Schulzke JD (2004) Mechanisms of diarrhea in the inter-leukin-2-deficient mouse model of colonic inflammation.Am J Physiol 286, 244-252

Terszowski G, Waskow C, Conradt P, Lenze D, Koenigs-mann J, Carstanjen D, Horak I, Rodewald HR (2004) Pro-spective Isolation and global gene expression analysis ofthe colony-forming urit-erythrocyte (CFU-E). Blood 105,1937-1945

Zatovicova M, Tarabkova K, Svastova E, Gibadulinova A,Mucha V, Jakubickova L, Biesova Z, Ortova-Gut M,Parkkila S, Parkkila AK, Waheed A, Horak I, Pastorek J,Pastorekova S (2004) Monoclonal antibodies generated inCA IX-deficient mice recognize different domains of tumor-associated hypoxia-induced carbonic anhydrase IX. JImmunol Methods 282, 117-134FMP-authors in bold.

Cytokine Signalling

Rosenbauer F, Wagner K, Zhang P, Knobeloch KP, IwamaA, Tenen DG (2004) pDP4, a novel glycoprotein secreted bymature granulocytes, is regulated by transcription factorPU.1. Blood 103, 4294-4301

Schuh K, Cartwright EJ, Jankevics E, Bundschu K, Lieber-mann J, Williams JC, Armesilla AL, Emerson M, OceandyD, Knobeloch KP, Neyses L (2004) Plasma membrane Ca2+ATPase 4 is required for sperm motility and male fertility JBiol Chem 279, 28220-28226

Van Spriel AB, Puls KL, Sofi M, Pouniotis D, Hochrein H,Orinska Z, Knobeloch KP, Plebanski M, Wright MD (2004)A regulatory role for CD37 in T cell proliferation. J Immunol172, 2953-2961

Molecular Myelopoiesis

Heymann GA, Carstanjen D, Kiesewetter H, Salama A(2004) Polymorphism of the a4-subunit of VLA-4 integrinand bone marrow transplantation. Haematologica 89, 882-884

Terszowski G, Waskow C, Conradt P, Lenze D, Koenigs-mann J, Carstanjen D, Horak I, Rodewald HR (2004) Pro-spective Isolation and global gene expression analysis ofthe colony-forming urit-erythrocyte (CFU-E). Blood 105,1937-1945


Marg A, Shan Y, Meyer T, Meissner T, Brandenburg M,Vinkemeier U (2004) Nucleocytoplasmic shuttling bynucleoporins Nup153 and Nup214 and CRM1-dependentnuclear export control the subcellular distribution of latentStat1. J Cell Biol 165, 823-833


Meissner T, Krause E, Lödige I, Vinkemeier U (2004)Arginine Methylation of STAT1: A Reassessment. Cell 119,587-589

Meyer T, Hendry L, Begitt A, John S, Vinkemeier U (2004)A single residue modulates tyrosine dephosphorylation,oligomerization, and nuclear accumulation of stat tran-scription factors. J Biol Chem 279, 18998-19007


Peptide Synthesis

Cansarek P, Beyermann M, Koch KW (2004) Thermodyna-mics of apocalmodulin and nitric oxide synthase II peptideinteraction. FEBS Lett 577, 465-468

Carpino LA, Krause E, Sferdean CD, Bienert M, BeyermannM (2004) Dramatically enhanced N to O acyl migrationduring the trifluoracetic acid-based deprotection step insolid phase peptide synthesis. Tetrahedon Lett 46, 1361-1364

Carpino LA, Krause E, Sferdean CD, Schümann M, FabianH, Bienert M, Beyermann M (2004) Synthesis of “difficult“peptide sequences:application of a depsipeptide techni-que to the Jung Redemann 10- and 26-mers and the amy-loid peptide Abeta (1-42). Tetrahedron Lett 45, 7519-7523

Gregan B, Jürgensen J, Papsdorf G, Furkert J, SchaeferM, Beyermann M, Rosenthal W, Oksche A (2004) Ligand-dependent differences in the internalization and intracel-lular trafficking of endothelin A and endothelin B receptorheterodimers. J Biol Chem 279, 27679-27687


Klemm C, Schöder S, Glückmann M, Beyermann M, Krau-se E (2004) Derivatization of phorphorylated peptides withS- and N-nucleophiles for enhanced ionization efficiencyin matrix-assisted laser desorption/ionization mass spec-trometry. Rapid Commun Mass Spectrom 18, 2697-2705

Klose J, Wendt N, Kubald S, Krause E, Fechner K,Beyermann M, Bienert M, Rudolph R, Rothemund S (2004)Hexa-histidin tag position influences disulfide structurebut not binding behaviour of in vitro folded N-terminaldomain of rat corticotropin-releasing factor receptor type2a. Protein Sci 13, 2470-2475



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Peptide Lipid Interaction / Peptide Transport

Dathe M, Nikolenko H, Klose J, Bienert M (2004) Cycli-zation increases the antimicrobial activity and selectivityof tryptophan and arginine containing hexapeptides. Bio-chemistry 43, 9140-9150

Hällbrink M, Oehlke J, Papsdorf G, Bienert M (2004)Uptake of cell-penetrating peptides is dependent on thepeptide-to-cell-ratio rather than on peptide concentration.Biochim Biophys Acta 1667, 222-228

Kerth A, Erbe A, Dathe M, Blume A (2004) Infrared reflec-tion absorption spectroscopy of amphipathic model pepti-des at the air/water interface. Biophys J 86, 3750-3758


Wessolowski A, Bienert M, Dathe D (2004) Antimicrobialactivity of Arginine -& Tryptophan-rich hexapeptides: theeffects of aromatic clusters, D-amino acid substitution andcyclization. J Pept Res 64, 159-69

Peptide Biochemistry

Oehlke J, Wallukat G, Wolf Y, Ehrlich A, Wiesner B, Berger H, Bienert M (2004) Enhancement of intracellularconcentration and biological activity of PNA after conju-gation with a cell-penetrating synthetic model peptide. EurJ Biochem 271, 3043-3049


Mass Spectrometry

Baumgart S, Lindner Y, Kuhne R, Oberemm A, WenschuhH, Krause E (2004) The contributions of specific amino acidside chains to signal intensities of peptides in matrix-assi-sted laser desorption/ionization mass spectrometry. RapidCommun Mass Spec 18, 863-868

Carpino LA, Krause E, Sferdean CD, Schümann M, FabianH, Bienert M, Beyermann M (2004) Synthesis of “difficult“

peptide sequences:application of a depsipeptide techni-que to the Jung Redemann 10- and 26-mers and the amy-loid peptide Abeta (1-42). Tetrahedron Lett 45, 7519-7523



Klemm C, Schöder S, Glückmann M, Beyermann M, Krau-se E (2004) Derivatization of phorphorylated peptides withS- and N-nucleophiles for enhanced ionization efficiencyin matrix-assisted laser desorption/ionization mass spec-trometry. Rapid Commun Mass Spectrom 18, 2697-2705

Klose J, Wendt N, Kubald S, Krause E, Fechner K, Beyer-mann M, Bienert M, Rudolph R, Rothemund S (2004) Hexa-histidin tag position influences disulfide structure but notbinding behaviour of in vitro folded N-terminal domain ofrat corticotropin-releasing factor receptor type 2a. ProteinSci 13, 2470-2475

Meissner T, Krause E, Lödige I, Vinkemeier U (2004)Arginine methylation of STAT1: a reassessment. Cell 119,587-589


Rettig H, Krause E, Börner HG (2004) Atom transfer radicalpolymerization with polypeptide-initiators: a generalapproach to block copolymers of sequence-defined poly-peptides and synthetic polymers. Macromol Rapid Com-mun 25, 1251-1256

Sidorova MV, Molokoedov AS, Az’muko AA, KydryavtsevaEV, Krause E, Ovchinnikov MV, Bespalova ZD (2004) TheUse of hydrogen Peroxide for Closing Disulfide Bridges inPeptides. Russ J Bio Chem 2, 101-110

Sun J, Pohl EE, Krylova OO, Krause E, Agapov II, Tonevitz-ky AG, Pohl P (2004) Membrane destabilization by ricin. EurBiophys J 33, 572-579


Synthetic Organic Biochemistry

Geissler D, Antonenko YN, Schmidt R, Keller S, KrylovaOO, Wiesner B, Bendig J, Pohl P, Hagen V (2004) (Couma-rin-4-yl)methyl Esters as Highly Efficient, Ultrafast Photo-triggers for Protons and Their Application to AcidifyingMembrane Surfaces. Angew Chem Int Ed 44, 1195-1198

Medicinal Chemistry

Barth M, Rademann J (2004) Tailoring Ultraresins based onthe cross-linking of polyethylene imines. Comperativeinvestigation of the chemical composition, the swelling,the mobility, the chemical accessibility, and the perfor-mance in solid phase synthesis of very high loaded resins.J Comb Chem 6, 340-349

Barth M, Fischer R, Brock R, Rademann J (2004) Reversi-ble cross-linking of hyperbranched polymers: A strategyfor the combinatorial decoration of multivalent scaffolds.Angew Chem Int Ed 44, 1560-1563

Rademann J (2004) High Loading Polymer Reagents basedon Polycationic Ultragels. Polymer-Supported Reductionsand Oxidations with Increased Efficiency. Tetrahedon Lett60, 8703-8709

Rademann J (2004) Organic Protein Chemistry: Drug Dis-covery through the Chemical Modification of Proteins.Angew Chem Int Ed 116, 4654-4656

Smerdka J, Rademann J, Jung G (2004) Polymer-boundalkyltriazenes for mild racemization-free esterification ofamino acid and peptide derivatives. J Pept Sci 10, 603-611

Sorg G, Thern B, Mader O, Rademann J, Jung G (2004) Pro-gress in the preparation of peptide aldehydes via polymersupported IBX oxidation and scavenging by threonyl resin.J Pept Sci 11, 142-152

Number of original articles published in peer reviewed journals ordered by impact factorAnzahl der Originalarbeiten geordnet nach Impact-Faktor

Impact factor 1999 2000 2001 2002 2003 2004< 3 14 17 24 16 23 283 - 4.5 13 8 7 11 6 164.5 - 7 9 8 10 12 12 22> 7 16 10 12 19 13 14total 52 43 53 58 54 80


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REVIEWS 2003 / 2004ÜBERSICHTSARBEITEN 2003 / 2004

Abdul-Kaliq H, Schubert S, Stoltenburg-Didinger G, Hueb-ler M, Troitsch D, Wehsack A, Boettcher W, Schwaller B,Crausaz M, Celio M, Schroeter ML, Blasig IE, Hetzer R,Lange PE (2003) Release patterns of astrocytic and neuro-nal biochemical markers in serum during and after expe-rimental settings of cardiac surgery. Neurol Neurosci /Molecular Markers of Brain Damage 21, 141-150

Ball LJ, Kühne R, Schneider-Mergener J, Oschkinat H(2003) Recognition of proline rich motifs (PRMs) by smalladaptor domains: An important class of protein-proteininteractions in signal transduction. Angew Chem Int Ed 44,2852-2869

Meyer T, Vinkemeier U (2004) Nucleocytoplasmic shuttlingof STAT transcription factors. Eur J Biochem 271, 4606-4612

Oehlke J, Lorenz D, Wiesner B, Bienert M (2004) Studieson the cellular uptake of substance P- and lysine-rich,KLA-derived model peptides. J Mol Recognit 17, 1-10

Oehme P, Schmidt J, Hinkel U (2004) Vitamin K und seineFunktionen im Körper. Pharmazie 29-31

Oehme, P (2004) Theoretische und klinische Aspekte derSucht. Meine Begegnungen mit der Suchtforschung.Medizin und Gesellschaft 51, 109-119

Pohl P (2004) Combined transport of water and ionsthrough membrane channels. Biol Chem 385, 921-926

Rademann, J (2004) Organic protein chemistry: drug dis-covery through the chemical modification of proteins.Angew Chem Int Ed 43, 4554-4556

Schülein R (2004) The early stages of the intracellulartransport of membrane proteins: clinical and pharmacolo-gical implications. Rev Physiol Biochem Pharmacol 151,45-91

Vinkemeier U (2004) Getting the message across, STAT.Design principles of a molecular signaling circuit. J CellBiol 167, 197-201

CONTRIBUTIONS IN MONOGRAPHS 2003 / 2004BEITRÄGE ZU SAMMELWERKEN 2003 / 2004

2003

Haseloff RF, Krause E, Blasig IE Proteomics of the brain endothelium: Separation of pro-teins by two-dimensional gel electrophoresis and identifi-cation by mass spectrometryIn: Methods of Molecular Medicine: The blood-brain bar-rier - Biology and research protocols (Ed.: Nag S) 89, 465-477, Humana Press 2003, Totowa, NJ, USA

Klussmann E Protein kinase AIn: Online pharmacology reference database, Elsevier Sci-ence Inc 2003, Amsterdam, The Netherlands

Krause E ProteomicsIn: Encyclopedic Reference of Molecular Pharmacology(Eds.: Rosenthal W, Offermanns S) 766-770, Springer Ver-lag 2003, Heidelberg, Germany

Krause G Molecular ModellingIn: Encyclopedic Reference of Molecular Pharmacology(Eds.: Rosenthal W, Offermann S) 603-609, Springer Verlag2003, Heidelberg, Germany

Meyer T, Vinkemeier U JAK/STAT PathwayIn: Encyclopedic Reference of Molecular Pharmacology,(Eds.: Rosenthal W, Offermanns S) 527-530, Springer Ver-lag 2003, Heidelberg, Germany

Oksche A EndothelinsIn: Encyclopedic Reference of Molecular Pharmacology(Eds.: Rosenthal W, Offermanns S) 339-345, Springer Ver-lag 2003, Heidelberg, Germany

Schmidt A, Utepbergenov DI, Krause G, Blasig IE (2003)Direct demonstration of the association of the blood-brainbarrier proteins ZO-1 and occludin using surface plasmonresonance spectroscopy-effect of SIN-1In: Blood-Spinal Cord and brain barriers in health anddisease (Eds.: Sharma HS, Westmann J), Elsevier Science/Academic Press 2003, San Diego, USA

Schülein R, Rosenthal W Protein Trafficking and Quality ControlIn: Encyclopedic Reference of Molecular Pharmacology(Eds: Rosenthal W, Offermanns S) 758-762, Springer Ver-lag 2003, Heidelberg, Germany


2004

Bauer HC, Bauer H, Haseloff RF, Blasig IE The role of glia in the formation and function of the blood-brain barrierIn: Neuroglia 2nd Edition (Eds.: Ramson B, Kettenmann H)325-333, Oxford University Press 2004, Oxford, UK

Freund C The Gyf DomainIn: Modular Protein Domains (Eds.: Cesareni G, Gimona M,Sudol M, Yaffe M) 107, Wiley-VHC 2004, Weinheim, Germa-ny

Hagen V Uncaging and photoconversion/activationIn: Encyclopedic Reference of Genomics and Proteomicsin Molecular Medicine (Eds.: Ganten D, Ruckpaul K) inpress, Springer Verlag 2004, Heidelberg, Germany

Kuehne R, Krause G, Rosenthal W Entdeckungsstrategien in der WirkstoffforschungIn: Handbuch der Psychopharmakologie (Eds: Holsboer F,Gruender G, Benkert O) in press, Springer Verlag 2004, Hei-delberg, Germany

Klussmann E Protein Kinase AIn: xPharm 1.0 (Eds.: Enna SJ, Bylund DB), Elsevier 2004,Amsterdam, The Netherlands

Krause EMass Spectrometry: MS/MSIn: Encyclopedic Reference of Genomics and Proteomicsin Molecular Medicine (Eds.: Ganten D, Ruckpaul K) inpress, Springer Verlag 2004, Heidelberg, Germany

Oehme, P Zwischen Wissenschaft und PolitikIn: Sitzungsbericht der Leibniz-Sozietät (Ed.: Steiger KP)67, Trafo-Verlag 2004, Berlin, Germany

Oehme P, Göres E, Rosenthal W, Ganten D Pharmakologische Institutionen Berlin-Buch und Berlin-FriedrichsfeldeIn: Geschichte und Wirken der pharmakologischen, kli-nisch-pharmakologischen und toxikologischen Institute(Ed.: Philippou A) 698-711, Berenkamp Verlag Innsbruck2004, Austria

Oksche A, Pohl P, Krause G, Rosenthal WMolecular biology of diabetes insipidusIn: Encyclopedia of Molecular Cell Biology and MolecularMedicine (Ed.: Meyers RA) 301-324, Wiley-VCH 2004,Weinheim, Germany

Rademann J Combinatorial Chemistry - Concepts and Methods forTasks of Molecular OptimizationIn: Encyclopedic Reference of Molecular Pharmacology(Eds.: Offermanns S, Rosenthal W) 257-261, Springer Ver-lag 2004, Heidelberg, Germany

Rademann JSolid phase synthesis (SPS) and polymer-assisted solutionphase (PASP) synthesisIn: Highlights in Bioorganic Chemistry (Eds.: WennemersH, Schmuck C) 290-293, Wiley-VCH 2004, Weinheim, Ger-many

Rademann J Novel polymer- and linker reagents employed for the pre-paration of protease inhibitor librariesIn: Highlights in Bioorganic Chemistry (Eds.: WennemersH, Schmuck C) 277-290, Wiley-VCH 2004, Weinheim, Ger-many

Rademann J Inhibition of ProteasesIn: Highlights in Bioorganic Chemistry (Eds.: WennemersH, Schmuck C) 293-295, Wiley-VCH 2004, Weinheim, Ger-many

Rosenthal W, Seyberth H Besonderheiten der Arzneimitteltherapie im Kindesalter In: Pharmakotherapie/Klinische Pharmakologie, 12thEdition (Eds.: Lemmer B, Brune K) 507-516, Springer Verlag2004, Heidelberg and Berlin, Germany

Schmidt A, Utepbergenov DI, Krause G, Blasig IE Direct demonstration of association between the blood-barrier proteins ZO-1 and occludin using surface plasmonresonance spectroscopy - Effect of SIN-1In: Blood-Spinal Cord Barriers in Health and Disease (Ed.:Sharma HS) 11-17, Elsevier Verlag/Academic Press 2004,Heidelberg, Germany

Zimmermann J EVH1/WH1 domainsIn: Modular Protein Domains (Eds.: Cesareni G, Gimona M,Sudol M, Yaffe M) 73-102. Wiley-VHC 2004, Weinheim,Germany

MONOGRAPHS 2003 / 2004MONOGRAPHIEN 2003 / 2004

Offermanns S (Eds.), Rosenthal WEncyclopedic Reference of Molecular PharmacologySpringer Verlag 2004, Heidelberg, Germany


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MEMBERSHIPS IN EDITORIAL BOARDS2003 / 2004MITGLIEDSCHAFTEN IN EDITORIALBOARDS 2003 / 2004

Bienert, MichaelJournal of Receptors and Signal TransductionTaylor and Francis, PhiladelphiaMember since 2003

Blasig, IngolfGliaWiley-Liss, New YorkMember since 2004

Oschkinat, HartmutJournal of Structural and Functional GenomicsKluwer Academic Publishers, Netherlands

Rademann, JörgJournal of Combinatorial ChemistryAmerican Chemical SocietyMember since 2002

Rosenthal, WalterJournal of Molecular MedicineSpringer Verlag, HeidelbergMember 2001-2004

Rosenthal, WalterHandbook of Experimental Pharmacology SeriesSpringer Verlag, Heidelberg, Germany

INVITED TALKS 2003 / 2004EINGELADENE VORTRÄGE 2003 / 2004

Becker M (2004)NEP-deficient mice - a new model for the late onset humanobesity?Peptidase-Workshop, EMC Rotterdam. Rotterdam, TheNetherlands

Beyermann M (2003) In vitro folding, disulfide pattern, and characterization ofthe first extracellular domain of rat corticotropin-releasingfactor receptor6th German Peptide Symposium. Berlin, Germany

Bienert M (2003)Cellular Uptake of Model Amphipathic Peptides, CellularTransport Strategies for Targeting of Epitopes, Drugs andReporter Molecules – CelltargetWorkshop. Budapest, Hungary

Bienert M (2003)Cellular Uptake of Peptides: Design and Synthesis of Pep-tide-Derived Carriers for the Delivery of Biologically Acti-ve Compounds into CellsOpening lecture - EU-Project QLK3-CT-2002-01989: Targetspecific delivery systems for gene therapy based on cell-penetrating peptides. Stockholm, Sweden

Bienert M (2004)Design, Synthesis and Characterization of Beta-Sheet-For-ming Peptides: The FBP 28 WW Domain10th Akabori Conference. Awaji, Japan

Blasig IE (2003)Phosphorylation and Protein-Protein InteractionsGordon Research Conference: Barriers of the Brain. Tilton,USA

Blasig IE (2003) Interaction of tight junction proteins - Oligomerization ofZO-1 and recruitment of occludinCVB 2003. Amarillo, USA

Blasig IE (2003)Hinge region of the SH3-GuK unit of ZO-1 is regulated byoccludin and oligomerization of ZO-16th Symposium Signal Transduction of the Blood-BrainBarriers. Szeged, Ungarn

Blasig IE (2004)Struktur, Funktion und Dichtheit der Blut-HirnschrankeHahn-Meitner-Institut. Berlin, Germany

Carstanjen D (2003)Die Rolle des Interferon induzierten Transkriptionsfaktors,ICSBP, in der Reifung und Funktion von Zellen des myelo-poetischen SystemsCharitè Universitätsmedizin, Campus Benjamin Franklin.Berlin, Germany

Dathe M (2004)Apolipoprotein E-derived peptides and drug delivery to thebrain7th Symposium Signal Transduction of the Blood BrainBarriers. Potsdam, Germany

Freund F (2003)Molecular recognition of proline-rich sequences by theGYF domainMax-Planck-Institut für Biophysikalische Chemie. Göttin-gen, Germany

Freund F (2004)Struktur-Funktionsbeziehungen wichtiger T-Cell-ProteineBMBF Bundesministerium für Bildung und Forschung.Berlin, Germany

Freund F (2004)Proline-rich sequence recognition by GYF domainsUniversität des Saarlandes. Saarbrücken, Germany

Freund F (2004)Molecular RecognitionMax-Planck-Institut für Biochemie. München, Germany

Freund F (2004)Proline-rich sequence recognition by GYF domainsJohann Wolfgang Goethe-Universität Frankfurt, Germany

Haseloff RE (2003)Alterations in the protein expression of rat brain capillaryendothelial cells induced by oxidative stress6th Symposium Signal Transduction of the Blood-BrainBarriers. Szeged, Ungarn

Keller S (2004)Can Cell-Penetrating Peptides Cross Lipid Membranes?Institut für Physikalische Chemie. Martin-Luther-Universi-tät Halle, Germany

Klussmann E (2004)Anchored cAMP signalling in the vasopressin-inducedaquaporin-2 shuttle in renal principal cellsThe Biotechnology Centre of Oslo. University of Oslo, Nor-way

Knobeloch KP (2003)Zytokin-Rezeptoren und Zytokin-abhängige Signalwege:the role of Interferon Consensus Sequence Binding Pro-tein in hematopoiesisInternational Hannover Workshop on Cytokine Receptorsand Cytokine Signaling. Hannover, Germany

Krause E (2004)Massenspektrometrie in der ProteomforschungBundesanstalt für Materialforschung. Berlin-Adlershof,Germany

Krause G (2004)Gemeinsamkeiten von Struktur und Funktion des TightJunction Proteins Occludin und des Adherens JunctionProteins alpha-CateninInstitut für Chemie und Pathobiochemie der Freien Univer-sität Berlin, Germany

Krause G (2004)The tight junction protein occludin and the adherensjunction protein alpha-catenin share a common interacti-on mechanism with ZO-17th International Symposium Signal Transduction in theblood-brain barriers. Potsdam, Germany

Krause G (2004)Identifizierung von Wechselwirkungsepitopen zwischenZellkontaktproteinen der tight junctionsWorkshop Neue Peptidtechnologien. Max Brünger Zen-trum, Universität Leipzig, Germany

Krause G (2004)Structural determinants for the activation of glycoproteinhormon receptorsInstitut für Reproduktionsmedizin, Universität Münster,Germany

von Kries JP (2004)Screening for beta-Catenin AntagonistsMax-Planck-Institute of Molecular Cell Biology and Gene-tics Dresden, Germany

von Kries JP (2004)Screening in an academic setupEMBL Hamburg, Germany

Leitner D (2004)PASTE und PAPST for biomoleculesSpanish NMR User’s meeting. Madrid, Spain

Maul B (2003)Neuropeptidasen und AlkoholsuchtZentralinstitut für Seelische Gesundheit. Mannheim, Ger-many

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Oehme P (2004)Theoretische und klinische Aspekte der SuchtZentrum für Human- und Gesundheitswissenschaften, FBHumanmedizin der Freien Universität Berlin, Germany

Oehme P (2004)Toleranz als essentieller Schutzmechanismus - Reflektio-nen aus pharmakologischer SichtWissenschaftliche Konferenz der Leibniz-Sozietät e.V.Berlin, Germany

Oschkinat H (2003). From Gene to Structure as viewed by NMR. Structures ofmembrane proteins by solid-state NMR. NMR in Molecu-lar BiologyEuroconference on Structural Genomics. Obernai, France

Oschkinat H (2003) NMR-Spectroscopy of membrane proteins5th International Conference on Molecular Structural Bio-logy. Vienna, Austria

Oschkinat H (2003) A concept for structure determination of small membraneproteins by 3D magic angle spinning NMR and its applica-tion to the x spectrin SH3 domain45th Rocky Mountain Conference on Analytical Chemistry.Denver, USA

Oschkinat H (2003)NMR-Spectroscopy of membrane proteins8th International Dahlem Symposium on Cellular SignalRecognition and Transduction. Berlin, Germany

Oschkinat H (2003)NMR-Spectroscopy of membrane proteins4th Colloquium on Transport. Rauischholzhausen, Germa-ny

Oschkinat H (2003)The solid-state MAS-NMR structure of the 62-residuealpha-spectrin SH3 domain and the potential of NMR forthe investigation of membrane proteins44th ENC Experimental Nuclear Magnetic Resonance Con-ference. Savannah, USA

Oschkinat H (2003) The solid-state MAS-NMR structure of the 62-residuealpha-spectrin SH3 domain and the potential of NMR forthe investigation of membrane proteinsThe 3rd Alpine Conference on Solid-State Nuclear Magne-tic Resonance. Chamonix-Mont, France.

Oschkinat H (2004)Protein-protein interaction investigated by solution andSolid-state NMREMBL Heidelberg, Germany

Oschkinat H (2004)Structure determination of proteins by magic angle spin-ning MAS NMRBarossa Valley, Australia

Oschkinat H (2004)Protein-protein interactions viewed by solution and solid-state NMRParis, France

Oschkinat H (2004)Protein-Protein Interactions and Membrane Proteins Inve-stigated by Solution and Solid-State NMRUniversity of Florence, Italy

Oschkinat H (2004) Molecular Profiles - Target search in molecular pharma-cologyMax-Delbrück-Center Berlin-Buch, Germany

Oschkinat H (2004)Protein-protein interactions viewed by solution and solid-state NMRTechnische Universität München, Germany

Oschkinat H (2004)Structural genomics, membrane proteins and automationVentura, USA

Oschkinat H (2004)Viewing protein-protein interactions and membrane pro-teins by solution and solid-state NMRBiozentrum der Universität Basel, Switzerland

Oschkinat H (2004)NMR analysis of protein modulesNew York, USA

Oschkinat H (2004)Structures of membrane proteins by solution an solid andsolid-state NMRNew Jersey, USA

Oschkinat H (2004)Zelluläre Protein-Interaktion und pharmakologische Inter-ferenzCharité-Universitätsmedizin Berlin, Germany

Oschkinat H (2004)Structure determination of proteins by magic angle spin-ning MAS NMRMartin-Luther-Universität Halle-Wittenberg, Germany

Oschkinat H (2004)NMR approaches to structures of membrane proteinsAscona, Schweiz

Oschkinat H (2004)An apoE-derived peptide mediates uptake of PEG-liposo-mes onto brain capillary endothelial cellsBad-Herrenalb, Germany

Oschkinat H (2004)NMR-Spectroscopy of membrane proteins5th International Conference on Molecular Structural Bio-logy. Vienna, Austria

Oschkinat H (2004)Quality control of the vasopressin V2 receptor in the ERand ER/Golgi intermediate compartment4th Action Meeting on new Drugs and Treatment. Berlin,Germany

Oschkinat H (2004)Aufbau der Technologieplattform NMR-Messtechnik fürdie ProteomforschungBraunschweig, Germany

Oschkinat H (2004)Magic-angel-spinning solid-state NMR of proteinsIFIA-BioNMR. Karlsruhe, Germany

Oschkinat H (2004)Structure determination of proteins by magic angle spin-ning MAS NMRUniversity of Osaka, Japan

Oschkinat H (2004)Design, Synthesis and Characterization of ß-Sheet-For-ming peptides: The FBP 28 WW DomainAwaji, Japan

Oschkinat H (2004)Protein structure determination by magic-angle spinningsolid state NMRLille, France

Oschkinat H (2004)Protein Structure Determination by Solid State MAS NMRFrauenchiemsee, Germany

Pankow K (2004) Influence of the natriuretic peptide structure on degrada-tion by NEPPeptidase-Workshop, EMC Rotterdam, The Netherlands

Pohl P (2004)Fluid transport through membrane channels and epitheli-al cellsTagung der Gesellschaft für Biochemie und Molekularbio-logie. Münster, Germany

Pohl P (2004)Fluid transport through membrane channels and epitheli-al cellsJohannes Keppler Universität, Linzer Winter Workshop.Linz, Austria

Pohl P (2004)Fluid transport through membrane channels and epitheli-al cells: Calcium and Transport ProcessesUniversität des Saarlandes in Homburg, Germany

Rademann J (2004)Polymer-unterstützte C-Acylierungen gegen MalariaNovabiochem Seminar, Technische Universität Berlin,Germany

Rademann J (2004)P1-Variation in Peptidisosteren: Molekulare Diversitätdurch die Kombination von C- and N-AcylierungenMax-Bergmann Kreis. Munster, France

Rademann J (2004)Molekulare Werkzeuge für die chemische IndustrieBioclub der Freien Universität Berlin. Berlin, Germany

Rademann J (2004)P1-Site Diversity in Peptide Isoster Libraries via SmoothCC-Couplings on Linker Reagents.ACS National Meeting. Philadelphia, USA

Rademann J (2004)New linkers, resins and reagents - some problems of poly-mer-supported synthesis and attemps to solve them. ABC Technologies Symposium. Basel, Switzerland

Rademann J (2004)Molekulare Werkzeuge für die Chemische Biologie(Antrittsvorlesung) Freie Universität Berlin, Germany

Rademann J (2004)New roads to Chemical Diversity: From P1-Site Mutants inMalaria Inhibitors to Combinatorial Synthesis of ComplexModified DendrimersQuaid-iAzzam University. Islamabad, Pakistan

Rademann J (2004)Diversitäts-orientierte Synthese von Glycolipiden durchhydrophob unterstützte Synthese von Glycopeptiden Forschungszentrum Borstel, Germany

Rademann J (2004)Reversibly cross-linked dendrimers - A strategy for thefacile synthesis and combinatorial variation of complex,multivalent protein-mimics7th International Symposium on Biomolecular Chemistry -ISBOC-7. Sheffield, United Kingdom

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Reif B (2003)Molecular Interactions between the yeast prion proteinSup35 and the molecular chaperone Hsp104Euresco Conference on NMR in Molecular Biology. Ober-nai, France

Reif B (2003)Aggregation behaviour of misfolding peptides and proteinsmodulated by molecular chaperones: solution and solid-state NMR experimentsIMB-Symposium „Protein Folding and Aggregation – FromPrinciples to Pathological Implications”. Jena, Germany

Reif B (2003)Use of Protons in MAS Solid-State NMR in perdeuteratedpeptides and proteinsEMBO-ILL workshop on Deuterium labeling techniqes forbiomolecular NMR and neutron scattering. Grenoble,France

Reif B (2004)Molecular Interactions between Sup35 and Hsp104 cha-racterized by Solution State NMRBaltimore, USA

Reif B (2004)Use of Protons in MAS Solid-State NMR in perdeuteratedpeptides and proteinsAsilomar Conference Center, Pacific Grove, California,USA

Reif B (2004)Protons in MAS Solid-State NMRForschungszentrum Karlsruhe, Germany

Reif B (2004)Use of perdeuteration in MAS Solid State NMRDenver, Colorado, USA

Reif B (2004)Multidimensional NMR in Structural BiologyIl Ciocco, Italy

Rosenthal W (2003) Cyclic AMP-triggered exocytosisVIIIth International Dahlem Symposium on Cellular SignalRecognition and Transduction. Berlin, Germany

Rosenthal W (2003)Biomedical research and biotechnology in Berlin-Buch:Bioprofile – wohin führt der Weg? Von der Heilpflanze zumDrug DesignBioTechnica - Workshop: International Biotechnology -New Concepts in Molecular Medicine from Finland andGermany. Berlin, Germany

Rosenthal W (2003)Leuchttürme der Berliner Wissenschaft. Biotechnologie inBerlin-BrandenburgVeranstaltung der Initiative „An Morgen denken“. Berlin,Germany

Rosenthal W (2003)Mechanismen der AntidiureseSymposium Fortschritt und Praxis der Endokrinologie zurEröffnung des ambulanten Hochschulzentrums für Endo-krinologie, Diabetologie und Ernährung. Berlin / Potsdam,Germany

Rosenthal W (2004)The human V2 vasopressin receptor: gene, the protein, themutation and rescue strategies of mutant receptorsAnnual congress of the European Renal Association –European Dialysis and Transplant Association. Lisbon, Por-tugal

Schmieder P (2004)Towards the structure of the chromophore in bacterialphytochromesHerbsttagung des Hahn-Meitner-Instituts Berlin, Germany

Schmieder P (2004)Aspekte der Strukturbestimmung von Proteinen mittels derNMR-SpektroskopieHahn-Meitner-Institut Berlin, Germany

Schülein R (2004)Compartmentalization of NDI-casing vasopressin V2receptor mutants in the early secretory pathway5th Global NDI Conference. Phoenix, USA

Schülein R (2004)Functional significance of the cleavable signal peptides ofcorticotropin-releasing factor receptorsSymposium on Inflammation and Pain. Berlin, Germany

Schülein R (2004)Intracellular transport of G protein-coupled receptorsalong the secretory pathwaySymposium on Neuro-Immune Interaction in Pain. Berlin,Germany

Siems, WEACE and NEP - relations to alcohol preferencePeptidase-Workshop, EMC-Rotterdam, The Netherlands

Sun X (2004)Development and Properties of domain-selective murineACE formsPeptidase-Workshop, EMC-Rotterdam, The Netherlands

EXTERNAL FUNDING 2003 / 2004DRITTMITTEL

Distribution of annual expenditure shown for the sources of income since 1998Verteilung der jährlichen Ausgaben über die Drittmittelgeber seit 1998(total expenses per year in italics)(kursiv: Gesamtausgaben pro Jahr)

7000

6000

5000

4000

3000

2000

1000

0

1998 1999 2000 2001 2002 2003 2004

Others 11 3 6 3 5 37 28

Foundations 216 174 112 2 231 158 116

Industry 92 53 29 51 134 197 110

EU 43 74 217 122 710 414 1984

Berlin 52 54 51 0 0 0 0

BMBF 514 696 773 5422 1045 974 886

DFG 656 1050 992 1267 1073 1383 1349

1583

2105 2178

29893163

4473

6867

Exte

rnal

Fund

ing

129

1500

1250

1000

750

500

250

0

T€

1383

DFG BMBF Berlin EU Industry Foundations Others

974

0 37

2003

Expenses during the period reported: contribution of the sources of incomeDrittmittelausgaben im Berichtszeitraum: Verteilung über die Drittmittelgeber

414

197 158

2000

1500

1000

500

0

T€

DFG BMBF Berlin EU Industry Foundations Others

0 28

2004

1984

110 116

1349

886

PARTICIPATION IN RESEARCH NETWORKS2003 / 2004BETEILIGUNG AN NETZWERKEN UNDVERBUNDPROJEKTEN 2003 / 2004

Sonderforschungsbereiche der DeutschenForschungsgemeinschaft

Sonderforschungsbereich 449: Struktur und Funktionmembranständiger RezeptorenTeilprojekt A3: Struktur und Funktion von Transportsigna-len des Vasopressin V2-RezeptorsRalf Schülein, Walter RosenthalLaufzeit: 01.99-12.04

Sonderforschungsbereich 449: Struktur und Funktionmembranständiger RezeptorenTeilprojekt B1: Bestimmung der Raumstrukturen vonRezeptor-gebundenen Agonisten und Antagonisten mittelsFestkörper-NMR-SpektroskopieHartmut OschkinatLaufzeit: 01.02-12.04

Sonderforschungsbereich 449: Struktur und Funktionmembranständiger RezeptorenTeilprojekt A6: Untersuchungen von CRF- und CRF-Rezep-tor-Mutanten zur Entwicklung eines Modells für dieLiganderkennung von G-Protein-gekoppelten Rezeptorender Familie 2Michael Bienert, Michael Beyermann, Walter RosenthalLaufzeit: 01.99-12.04

Sonderforschungsbereich 498: Protein-Kofaktor-Wech-selwirkungen in biologischen Prozessen Teilprojekt C1: Schlüsselreaktionen der biologischen Was-serstoffaktivierung am Beispiel der [NiFe]-Hydrogenasen Hartmut Oschkinat, Bärbel Friedrich (HU)Laufzeit: 01.03-12.05

Sonderforschungsbereich 498: Protein-Kofaktor-Wech-selwirkungen in biologischen Prozessen Teilprojekt B6: NMR-spektroskopische Untersuchungenvon lichtinduzierten Strukturveränderungen in Protein-Chromophorkomplexen Peter SchmiederLaufzeit: 01.03-12.05

Sonderforschungsbereich 366: Signalerkennung und –UmsetzungTeilprojekt Z3: Herstellung und Haltung genetisch verän-derter Mäuse Elvira Rohde, Ivan Horak Laufzeit: 01.09-12.05

Sonderforschungsbereich 366: Signalerkennung und –UmsetzungTeilprojekt A11: Verbreitung und Bedeutung N-terminalerSignalpeptide bei G-Protein-gekoppelten Rezeptoren Ralf Schülein, Walter RosenthalLaufzeit: 01.97-12.05

Sonderforschungsbereich 594: Molekulare Maschinen inProteinfaltung und ProteintransportTeilprojekt A3: Biochemische und NMR-Strukturuntersu-chungen an Sup35p im Komplex mit Hsp104, Hsp40 undHsp70Bernd ReifLaufzeit 05.01-04.04

Forschergruppen der DeutschenForschungsgemeinschaft

Forschergruppe 299: Optimierte molekulare Bibliothekenzum Studium biologischer ErkennungsprozesseTeilprojekt 2/2-1: Struktur, Stabilität und Spezifikation vonnichtkatalytischen Proteindomänen und deren Verwen-dung als Werkzeuge für das Design einer stabilen minima-len ß-Faltblattstruktur und das Verständnis von pathologi-schen ProzessenHartmut Oschkinat, Michael BienertLaufzeit: 06.01-12.05

Forschergruppe 299: Optimierte molekulare Bibliothekenzum Studium biologischer ErkennungsprozesseTeilprojekt 2/2-2: Theoriegestützte NMR-spektroskopischeAnalyse von Protein-Ligand-Wechselwirkungen unter Ver-wendung von Peptid-Bibliotheken Hartmut OschkinatLaufzeit: 06.01-12.05

Forschergruppe 299: Optimierte molekulare Bibliothekenzum Studium biologischer ErkennungsprozesseTeilprojekt 7/2-1: Studium der Ligand-Erkennung von CRH-Rezeptoren mit Peptid- und nichtpeptidischen Biblio-thekenMichael Bienert, Jens Schneider-MergenerLaufzeit: 06.99-12.05

Forschergruppe 463: Innovative Arzneistoffe und Träger-systeme – Integrative Optimierung zur Behandlung ent-zündlicher und hyperproliferativer Erkrankungen Teilprojekt 7B: Hirntargeting mittels oberflächenmodifi-zierter Nanosuspensionen und Apolipoprotein E-Peptidbeladener TrägersystemeMargitta DatheLaufzeit: 11.01-12.07

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Graduiertenkollegs der Deutschen Forschungs-gemeinschaft

GK 238/3: Schadensmechanismen im Nervensystem – Ein-satz von bildgebenden Verfahren Ingolf BlasigLaufzeit: 01.03-12.05

GK 276: Signalerkennung und -umsetzung Alexander Oksche, Walter RosenthalLaufzeit: 10.99-09.05

GK 865: Vaskuläre Regulationsmechanismen Alexander Oksche, Walter Rosenthal Laufzeit: 04.03-03.07

GK 441: Chemie in InterphasenJörg RademannLaufzeit: 10.99-09.07

Leitprojekte des Bundesministeriums fürBildung und Forschung

Proteinstrukturfabrik 01 GG 9812: Strukturanalyse mithohem Durchsatz für medizinisch relevante Proteine Teilprojekt 9: NMR-SpectroscopyHartmut Oschkinat, Peter Schmieder, Dietmar LeitnerLaufzeit: 10.98-09.04

Proteinstrukturfabrik 01 GG 9812: Strukturanalyse mithohem Durchsatz für medizinisch relevante Proteine Teilprojekt 10: NMR-structure determinationHartmut Oschkinat, Dirk Labudde, Ilia PoliakovLaufzeit: 10.98-09.04

Verbundprojekte des Bundesministeriums fürBildung und Forschung

Verbundprojekt 0312992J: Proteomische Methoden fürmolekulare Strukturen von Zielproteinen aus dem Myco-bacterium tuberculosis Genom und ihrer Ligandkomplexezur Suche von WirkstoffenHartmut Oschkinat, Jens von KriesLaufzeit: 04.04-06.06

Verbundprojekt 0312890G: Proteomweite Analyse mem-brangebundener ProteineHartmut OschkinatLaufzeit: 04.03-03.06

EU-Projekte

EU-Projekt QLK3-2000-00924: Exploiting synthetic SH2-scaffolded repertoire libraries to profile cancer cells andinterfere with cancer-related phenotypesHartmut OschkinatLaufzeit: 12.00-30.11.03

EU-Projekt QLRT-2000-00987: Antidiuretics using short-action vasopressin – V2-receptor agonists as a new thera-peutic strategy of urinary incontinence and voiting disor-dersWalter RosenthalLaufzeit: 09.01-08.04

EU-Projekt QLK3-CT-2002-02149: Anchored cAMP signal-ling – Implications for treatment of human diseaseEnno Klussmann, Walter RosenthalLaufzeit: 11.02-20.05

EU-Projekt QLK3-CT-2002-01989: Target specific deliverysystems for gene therapy based on cell-penetrating pep-tidesMichael Bienert, Johannes Oehlke, Margitta DatheLaufzeit: 03.03-02.06

COOPERATIONS WITH CONTRACT2003 / 2004VERTRAGLICHE KOOPERATIONEN 2003 / 2004

Vereinbarung über die Zusammenarbeit zwischen derFreien Universität Berlin und dem ForschungsverbundBerlin e. V. für das FMP- Freie Universität Berlin

Vereinbarung über die Zusammenarbeit zwischen derCharité – Universitätsmedizin Berlin und dem For-schungsverbund Berlin e. V. für das FMP- Charité – Universitätsmedizin Berlin

Kooperationsvereinbarung: Gemeinsamer Neubau undNutzung des Genomzentrums- Max-Delbrück-Centrum für Molekulare Medizin, Berlin,

Germany

Kooperationsverbarung: Gemeinsame Nutzung des Her-mann-von-Helmholtz-Hauses- Max-Delbrück-Centrum für Molekulare Medizin, Berlin,

Germany

Kooperationsvereinbarung für das Verbundprojekt „Pro-teomweite Analyse membrangebundener Proteine (Pro-Amp)- Johann Wolfgang Goethe-Universität Frankfurt, Ger-

many- GSF-Forschungszentrum für Umwelt und Gesundheit

GmbH, Germany- Max-Planck-Gesellschaft zur Förderung der Wissen-

schaften e. V., Germany

Kooperationsvereinbarung: Ausbau und Intensivierungder wissenschaftlichen Zusammenarbeit- Deutsches Primatenzentrum GmbH, Göttingen, Germany- Bernhard-Nocht-Institut für Tropenmedizin, Hamburg,

Germany- Heinrich-Pette-Institut für experimentelle Virologie und

Immunologie, Hamburg, Germany- Forschungszentrum Borstel, Zentrum für Medizin und

Biowissenschaften, Borstel, Germany- Institut für Molekulare Biotechnologie e.V., Jena

Kooperationsvereinbarung: Automatisierte Methoden fürmolekulare Strukturen von biologischen Makromolekü-len aus dem Mycobakterium-tuberculosis-Genom undihrer Ligandkomplexe zur Suche von Wirkstoffen mitHochdurchsatz Chiptechnologien (Strukturgenomprojekt)- European Molecular Biology Laboratory (EMBL), Ham-

burg und Heidelberg, Germany

- Max-Planck-Gesellschaft zur Förderung der Wissen-schaften e. V., München, Germany

- Max-Planck-Institut für Infektionsbiologie, Berlin, Ger-many

- Technische Universität München, Wissenschaftszen-trum Weihenstefan, Freising, Germany

- Combinature Biopharm AG, Berlin, Germany- Marresearch GmbH, Norderstedt, Germany- Biomax Informatics AG, Martinsried, Germany

Forschungs- und Entwicklungsvereinbarung- IKOSATEC GmbH, Garching, Germany- Combinature Biopharm AG, Berlin-Buch, Germany

Kooperationsvereinbarung: Strukturanalyse mit hohemDruck für medizinisch relevante Proteine- Proteinstrukturfabrik: Projekt des Bundesministeriums

für Bildung und Forschung, Germany

Kooperationsvereinbarung zur Erbringung von Verwer-tungsleistungen- Ascenion GmbH, München, Germany

Kooperationsvereinbarung über die Fortsetzung derÖffentlichkeitsarbeit auf dem Forschungscampus Berlin-Buch- BBB Management GmbH, Berlin, Germany

Kooperationsvereinbarung: (Forschungs- und Entwick-lungsvereinbarung) Identifizierung von 2-D-separiertenProteinen über In-Gel-Verdau, Massenspektrometrie undDatenbankanalyse- Bundesinstitut für Risikobewertung, Berlin, Germany- Bundesinstitut für Verbraucherschutz und Veterinärme-

dizin, Germany

Kooperationsvereinbarung: MassenspektrometrischeCharakterisierung von Fluoreszenz-markierten Glyko- undPhosphopeptiden- Biosyntan GmbH, Berlin-Buch, Germany

Forschungs- und Entwicklungsvereinbarung: Develop-ment of isotope labelled media for protein expression inbacteria, yeast, insect cells and higher organisms- Cambridge Isotope Laboratories, Massachusetts, USA

Nutzungs- und Dienstleistungsvereinbarung: Gemein-same Nutzung von Forschungsgeräten und technologi-schen Einrichtungen- Charité-Universitätsmedizin Berlin, Germany

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Kooperationsvereinbarung zur Nutzung von Übertra-gungsstrecken als Zugangsleitungen zum Gigabit-Wis-senschaftsnetz G.WiN- Verein zur Förderung eines Deutschen Forschungsnet-

zes e. V., Berlin, Germany

Kooperationsvereinbarung über die Zusammenarbeit imRahmen von Projekten- Freie Universität Berlin, Germany

Kooperationsvereinbarung: Transfer von Naturstoffen,Derivaten und Analoga- Hans-Knöll-Institut für Naturstoff-Forschung e. V. , Ger-

many

Collaboration Agreement mit der AG Oschkinat- Max-Delbrück-Centrum für Molekulare Medizin, Berlin

Kooperationsvereinbarung: Molecular Fingerprinting ofthe Blood-Brain Barrier in Hypoxia-Targeting Brain Ves-sels to Treat Stroke- National Research Council of Canada, Montreal

Infrastrukturnutzungsvereinbarung- PFS biotech AG, Berlin, Germany

Kooperationsvereinbarung: Nutzung der PrimärdatenbankPD Dr. IE Blasig- Ressourcenzentrum für das Deutsche Humangenom-

projekt, Germany

Kooperationsvereinbarung für Auftragsmessungen Prof. Dr. H. Oschkinat- Schering AG, Berlin, Germany

Kooperationsvereinbarung: Structure determination byNMR- University of Oxford, UK

Kooperationsvereinbarung- The University Wisconsin, Madison, USA

InfrastrukturnutzungsvereinbarungCombinature Biopharm AG, Berlin, Germany

Kooperationsvereinbarung China-Konsortium- Deutsches Diabetes Zentrum (DDZ) an der Heinrich-

Heine-Universität, Düsseldorf, Germany- Deutsches Primatenzentrum GmbH (DPZ), Göttingen- Forschungszentrum Borstel (FZB) – Zentrum für Medi-

zin und Biowissenschaften, Borstel, Germany- Hans-Knöll-Institut für Naturstoff-Forschung (HKI),

Jena, Germany- Heinrich-Pette-Institut für Experimentelle Virologie und

Immunologie (HPI) , Germany - Leibniz-Institut für Neurobiologie IfN, Magdeburg, Ger-

many- Leibniz-Institut für Molekulare Biotechnologie (IMB),

Jena, Germany- Institut für Pflanzenbiochemie (IPB), Halle a.d. Saale,

Germany- Institut für Pflanzengenetik und Kulturpflanzenfor-

schung (IPK), Gatersleben, Germany

MEETINGS, WORKSHOPS, SYMPOSIA2003 / 2004WISSENSCHAFTLICHE VERANSTALTUNGEN

2003

6th Symposium Signal Transduction in the Blood-BrainBarriersBlasig IE, Haseloff RFSzeged/UngarnSeptember 2003

Berlin Magnetic Resonance Seminar and High-Field NMRFacility Users MeetingOschkinat HForschungsinstitut für Molekulare Pharmakologie, Berlin-Buch, GermanyDecember 2003

4th Progress report meeting, EU-Project: Anti-diuresisusing short-acting vasopressin-V2-receptor agonists as anew therapeutic strategy of urinary incontinence and voi-ding disordersKlussmann E, Rosenthal W, Lauterjung UMagnus-Haus, Berlin, GermanyJune 2003

1st Progress report meeting, EU-Project: Anchored cAMPsignalling / RTD grant - Implication for treatment of humandiseasesKlussmann E, Rosenthal W, Lauterjung UMagnus-Haus, Berlin, GermanyJune 2003

6. Deutsches Peptidsymposium (mit internationaler Betei-ligung)Bienert M, Dreissigacker M, Dathe MHumboldt-Universität zu Berlin, GermanyMarch 2003

8th International Dahlem Symposium on Cellular SignalRecognition and Transduction, BerlinRosenthal WCharite - Universitätsmedizin Berlin, GermanyJune 2003

2004

34. Jahrestagung der Deutschen Gesellschaft für Immu-nologieHorak IBerlin, Henry Ford Building, GermanySeptember 2004

Festsymposium zur Einweihung des 900-MHz-Spektrome-ters am FMPOschkinat H, Steuer A, Maul BMax-Delbrück-Communications Center, Berlin-Buch, Ger-manyJuly 2004

7th International Symposium on Signal Transduction in theBlood-Brain BarriersBlasig IE, Haseloff RFPotsdam, GermanySeptember 2004

EMBO Practical Course "Multidimensional NMR in Struc-tural Biology II” Oschkinat H (Co-Organisator)Ciocco, ItalySeptember 2004

25. Tagung des Max-Bergmann-KreisesBienert MMunster, FranceOctober 2004

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WORK IN PANELS 2003 / 2004GREMIENARBEIT 2003 / 2004

Walter Rosenthal

- Mitglied im Kuratorium des Deutschen Instituts fürErnährungsforschung, Potsdam-Rehbrücke

- Stellvertretender Sprecher der Sektion C Lebenswis-senschaften der Leibniz Gemeinschaft

- Mitglied des Wissenschaftlichen Beirats des Hans-Knöll-Insituts, Jena

- Kuratoriumsmitglied der Berlin-BrandenburgischenFortbildungsakademie (BBFA)

- Leiter des Instituts für Pharmakologie, Charité-Univer-sitätsmedizin Berlin

- Schatzmeister im Verbund biowissenschaftlicher undbiomedizinischer Gesellschaften (vbbm)

Hartmut Oschkinat

- Mitglied des Aufsichtsrats der Firma PSF Bio AG, Berlin

Ivan Horak

- Wissenschaftlicher Direktor der Forschungseinrichtungfür Experimentelle Medizin, FU Berlin

- Ombudsmann für Gute Wissenschaftliche Praxis desForschungsverbundes Berlin e.V.

- Sprecher der Arbeitsgruppe Tierhaltung der Gemein-samen Kommission nach § 3 (3) UniMedG

- Mitglied des Beirats in der Transgenic Core Facility derRCC Gen bio tec GmbH, Campus Berlin-Buch

Michael Bienert

- Wissenschaftlicher Sekretär des Max-Bergmann-Krei-ses

REVIEW ACTIVITIES 2003 / 2004GUTACHTERTÄTIGKEIT 2003 / 2004

Bienert, MichaelOrganisations- Deutsche Forschungsgemeinschaft

Research Institutions- Universität Halle- Humboldt-Universität zu Berlin

Journals- Org Lett- Drug Design Review-Online- Tetrahedron Lett- J Mass Spec- Amino Acids- J Pept Sci- J Am Chem Soc- J Mol Recognit- Pept Res

Blasig, IngolfOrganisations- Deutsche Forschungsgemeinschaft- Research Grant Council Hong Kong- Jubiläumsfond Österreichische Nationalbank- Assoziazone Italiana per la Ricerca Cancro

Research Institutions- Philipps-Universität Magdeburg- Universität Potsdam- Charité-Universitätsmedizin Berlin

Journals- Neurochemistry- Glia- J Neurosci- J Pharm Pharmacol- J Neurochem- Neurochemical Research

Carstanjen, DirkJournals- Bone marrow transplantation

Dathe, MargittaOrganizations- The Israel Science Foundation

Journals- J Pep Res- Eur J Biochem- Biochim Biophysica Acta

- Comb Chem - Biophys J- J Biol Chem- New J Chem- Biochem J- Regul Peptides- Org Biomol Chem

Freund, ChristianOrganisations- University of Oxford

Journals- Regul Peptides

Hagen, VolkerResearch Institutions- Bayrische-Maximilians Universität Würzburg

Journals- Angew Chemie- ChemBioChem- J Am Chem Soc- Blood- Eur J Med Chem- J Mol Struct

Haseloff, RainerJournals- J Photochemistry and Photobiology B-Biology- Free Radicals Research

Horak, IvanOrganisations- Deutsche Forschungsgemeinschaft- Deutsche Krebshilfe- Boehringer Ingelheim Fonds

Research Institutions- Yorkshire Cancer Research, UK- Universität zu Lübeck- Taussig Cancer Center, The Cleveland Clinic Foundation

Journals- J Biol Chem- Immunity- Eur J Immunol- J Mol Cell Biol- Blood- Nucleic Acids Research- Journal Leukemia

Klussmann, EnnoOrganisations- Health Research Board, Ireland

Journals- JCI- Glia- Biol of the Cell- Biol Cell- Neuroscience Letters- J Cell Sci- Kidney Int- J Biol Chem- Eur J Cell Biol

Krause, EberhardJournals- J Anal Chem- J Pept Sci- J Mass Spec- Biochemistry

Krause, GerdJournals- QSAR Journal- J Comput Aid Mol Des

Meyer, ThomasResearch Institutions- Georg-August-Universität Göttingen

Journals- Circulation

Oehlke, JohannesJournals- Biochim Biophys Acta- Eur J Biochem

Oschkinat, HartmutOrganisations- Deutsche Forschungsgemeinschaft - Österreichische Akademie der Wissenschaften- Schering AG- Swiss Federal Institute of Technology Zürich- Studienstiftung Bonn- Schweizerischer Nationalfond zur Förderung von

Wissenschaftlicher Forschung- FWF Der Wissenschaftsfond

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Research Institutions- Freie Universität Berlin- Ludwig-Maximilians-Universität München- Bayerische-Maximilians Universität Würzburg- Humboldt-Universität zu Berlin- Ecole Normale Supérieure de Lyon- Charité-Universitätsmedizin Berlin- Eidgenössische Technische Hochschule Zürich

Journals- J Mol Biol- J Magn Reson- J Biol NMR- J Am Chem Soc- Nat Struct Biol- Science- Biochemistry- Proc Natl Acad Sci U.S.A.

Piontek, JörgJournals- GLIA

Pohl, PeterOrganisations- Wellcome Trust

Research Institutions- Christian-Albrechts-Universität zu Kiel- Humboldt-Universität zu Berlin

Journals- Biochemistry- Biophys J- Eur J Biochem- J Bioelectrochemical Society- BioMed Central- TIPS- Proc Natl Acad Sci U.S.A.

Richter, ReginaJournals- Vasc Pharmacol- Eur J Pharmacol- J Cardiovasc Pharma- Psychopharmacology

Rosenthal, WalterOrganisations- Deutsche Forschungsgemeinschaft - Ärztekammer Berlin- Fakulte de Science Chile- Boehringer Ingelheim- Österreichischer Wissenschaftsfond- Alexander von Humboldt Stiftung- Ministerio Dell`Istruzione, Dell ùniversita` E Della Ricera

Research Institutions- Freie Universität Berlin- Technische Universität Berlin/DRFZ- Philipps-Universität Marburg- Eidgenössische Technische Hochschule Zürich- Eberhard-Karls-Universität Tübingen- Institut für Zoo- und Wildtierforschung- Université Montpellier- Bayerische Maximilians-Universität München- Christian-Albrechts-Universität zu Kiel- Universität des Saarlandes - Oregon Health and Science University Portland- Fachhochschule Lausitz

Journals- Neuroscience Letters- Nephrology Dialysis Transplantation- Human Molecular Genetics- Biology of the cell- Pharmacol & Toxicol- Hormone Research- Encyclopedia of Biological Chemistry- Eur J of Cell Biol- EJB- Eur J Biochem- Eur Biophys J Biophys- FEBS Lett- The Lancet- Kidney Int

Schmieder, PeterOrganizations- Studienstiftung des deutschen Volkes

Journals- ChemBioChem- J Magn Reson- Chemistry (Wiley VCH)

Schülein, RalfJournals- FEBS Lett- Biotechniques

Siems, Wolf-EberhardJournals- J Mol Med

Utebergenov, DarkhanJournals- J Neurochem

Vinkemeier, UweOrganizations- European Molecular Biology Organizations- Boehringer Ingelheim Fonds- Österreichische Akademie der Wissenschaften- DAAD

Journals- Biochim Biophys Acta- Biochem Biophys Res Comm- Dev Cell- EMBO J- Eur J Biochem- FEBS Left- J Cell Biol- Mol Cell Biol- Nucleic Acids Res- Proc Natl Acad Sci U.S.A.

ACADEMIC TEACHING 2003 / 2004LEHRE 2003 / 2004

Beyermann, MichaelBiophysik-Praktikum zum Biacore-Gerät Freie Universität Berlin

Bienert, MichaelVorlesung Proteine und PeptideHumboldt-Universität zu Berlin

Vorlesung zum Biophysikpraktikum (Biacore-Gerät)Freie Universität Berlin

Blasig, IngolfVorlesung Funktionelle BiochemieUniversität Potsdam

Mastercourse „Medical Neurosciense“. Reguläre Vorle-sung, Seminare und PraktikumUniversität Potsdam

Mastercourse „Medical Neurosciense“. Fakultative Vor-lesung und SeminarUniversität Potsdam

Dathe, MargittaBiophysik für Studenten der Biochemie: CD-Spektrosko-pieFreie Universität Berlin

Freund, ChristianVorlesung Protein EngineeringFreie Universität Berlin

Vorlesung Molekulare ImmunologieFreie Universität Berlin

Horak, IvanVorlesung Knock-Out-MäuseFreie Universität Berlin

Keller, SandroVorlesung Biophysik für Studenten der Biochemie: Iso-therm Titration CalorimetryFreie Universität Berlin

Klussmann, EnnoKursus der Allgemeinen Pharmakologie und ToxikologieCharite-Universitätsmedizin Berlin

Vorlesung Molekulare Pharmakologie und zelluläre Signal-transduktion: A-Kinase-AnkerproteineCharite-Universitätsmedizin Berlin

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Vorlesung Molekulare Pharmakologie und zelluläre Signal-transduktion: AffinitätspräzipationstechnikenCharite-Universitätsmedizin Berlin

Vorlesung Molekulare Pharmakologie und zelluläre Signal-transduktion für Biochemiker, Biologen, Mediziner undPharmazeutenCharite-Universitätsmedizin Berlin

Kursus der Allgemeinen Pharmakologie und Toxikologiefür HumanmedizinerCharite-Universitätsmedizin Berlin

Krause, EberhardVorlesung Molekulare Pharmakologie und zelluläre Signal-transduktion – Massenspektrometrie Proteinanalytik (Pro-teomics)Charite-Universitätsmedizin Berlin

Krause, GerdVorlesung Grundlagen Molecular ModellingTechnical University of Applied Sciences, Berlin

Vorlesung Grundlagen Molecular ModellingTechnical University of Applied Sciences, Berlin, FB Bio-informatics

Krause, WSpezialkurs SuchtCharite-Universitätsmedizin Berlin

Psychopharmakologie im Praktikum Molekulare undzelluläre SignaltransduktionCharite-Universitätsmedizin Berlin

Lorenz, DorotheaKursus Mikroskopische Techniken (Intrazelluläre Ca2+-Messungen)Humboldt-Universität zu Berlin

Meyer, ThomasVorlesung Innere Medizin für ZahmedizinerUniversität Göttingen

Praktikum Innere MedizinUniversität Göttingen

Vorlesung Pathologie und Klinik internistischer und chirur-gischer ErkrankungenUniversität Göttingen

Oschkinat, HartmutVorlesung Biologische NMR-SpektroskopieFreie Universität Berlin

Vorlesung Biophysikalische MethodenFreie Universität Berlin

Vorlesung Grundlagen und neue Techniken derbiologischen NMR-SpektroskopieFreie Universität Berlin

Vorlesung Grundlagen der biologischen NMR-Spektro-skopieFreie Universität Berlin

Pohl, PeterVorlesung MikroskopieHumboldt-Universität zu Berlin

Vorlesung Experimentelle BiophysikHumboldt-Universität zu Berlin

Vorlesung BiomechanikHumboldt-Universität zu Berlin

Vorlesung Biophysik IIIJohannes-Kepler Universität Linz

Vorlesung Biophysik IJohannes-Kepler Universität Linz

Übungen zur PhysikJohannes-Kepler Universität Linz

Rademann, JörgVorlesung HeterocyclenUniversität Tübingen

Vorlesung StereochemistryFreie Universität Berlin

Vorlesung Combinatorial ChemistryQuaid-i-Azzam University, Islamabad

Vorlesung Chemie für BiologenUniversität Tübingen

Vorlesung Aktuelle Methoden der BioorganischenSyntheseFreie Universität Berlin

Vorlesung Bioorganic and Natural Product ChemistryFreie Universität Berlin

Richter ReginaVorlesung Anwendung der Mikrodialysetechnik in derNeuropharmakologieCharite-Universitätsmedizin Berlin

Rosenthal, WalterVorlesung Gentherapie ICharite-Universitätsmedizin Berlin

Vorlesung Weitere alternative TherapieformenCharite-Universitätsmedizin

Vorlesung PharmakokinetikCharite-Universitätsmedizin Berlin

Vorlesung Arzneimittel-Metabolismus/PharmakogenetikCharite-Universitätsmedizin Berlin

Vorlesung Gentherapie II (Mechanismen)Charite-Universitätsmedizin Berlin

Vorlesung StammzellenCharite-Universitätsmedizin Berlin

Vorlesung HomöopathieCharite-Universitätsmedizin Berlin

Vorlesung PlaceboCharite-Universitätsmedizin Berlin

Vorlesung Antivirale Therapie, Antigenese, GentherapieCharite-Unviersitätsmedizin Berlin

Schmieder, PeterVorlesung Molekulare Pharmakologie und zelluläre Signal-transduktionFreie Universität Berlin

Vorlesung Multidimensionale NMR-Spektroskopie-Grund-lagen und Anwendungen in der StrukturaufklärungTechnische Universität Berlin

Vorlesung Molekulare Pharmakologie und zelluläre Signal-transduktionTechnische Universität Berlin

Grundlagen und Anwendungen der MehrdimensionalenNMR-SpektroskopieTechnische Universität Berlin

Schülein, RalfKursus der Allgemeinen Pharmakologie und ToxikologieCharite-Universitätsmedizin Berlin

Vorlesung Molekulare Pharmakologie und zelluläre Signal-transduktion für Biochemiker, Biologen, Mediziner undPharmazeutenChartie-Universitätsmedizin Berlin

Kursus der allgemeinen PharmakologieCharité-Universitätsmedizin Berlin

Vorlesung Molekulare Pharmakologie und zelluläre Signal-transduktionCharité-Universitätsmedizin Berlin

Vinkemeier, UweVorlesung Aktuelle Themen der zellulären Siganltrans-duktionFreie Universität Berlin

Vorlesung Mechanismen der molekularen Signalverarbei-tungFreie Universität Berlin

Praktikum Mechanismen der SignalverarbeitungFreie Universität Berlin

Wiesner, BurkhardLaser Scanning Mikroskopie: Möglichkeiten und Grenzenoptischer Methoden zur Untersuchung subzellulärer Pro-zesseFreie Universität Berlin

Konfokale Mikroskopie (Molekulare Pharmakologie undzelluläre Signaltransduktion)FU/FMP

Molekulare Pharmakologie und zelluläre Signaltransduk-tionFU/FMP

Mikroskopische Techniken (FRAP, FREt, Reflexionsmes-sungen)HU/FMP

Laser Scanning Mikroskopie: Möglichkeiten und Grenzenoptischer Methoden zur Untersuchung subzellulärer Pro-zesseFreie Universität Berlin

CALLS FOR APPOINTMENTS 2003 / 2004RUFE 2003 / 2004

Hermosilla, Ricardo (2003) Juniorprofessur für Pathologie der Signaltransduktion am Institut für Pharmakologie der Charite-Universitäts-medizin Berlin, Germany

Pohl, Peter (2004)Lehrstuhl für BiophysikTechnisch-Naturwissenschaftliche Fakultät der Johannes-Kepler-Universität Linz

Pires, Ricardo (2002)Associated Professor for Biochemistry and Structural Bio-logyInstituto De Ciencas Biomédicas, Universidade Federal doRio de Janeiro, Brasil

POSTDOCTORAL LECTURE QUALIFICATIONS2003 / 2004HABILITATIONEN 2003 / 2004

Oksche, Alexander (2003) Molekulare Grundlagen ange-borener und erworbener polyurischer StörungenCharité-Universitätsmedizin Berlin

Schülein, Ralf (2003) The early secretory pathway of mem-brane proteins: clinical and pharmacological implicationsCharité-Universitätsmedizin Berlin

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GRADUATIONS 2003 / 2004PROMOTIONEN 2003 / 2004

Alken, Martina (2004) Functional significance of N-termi-nal signal peptides of G protein-coupled receptorsFreie Universität Berlin

Andreeva, Anna (2004) Protein kinase C isoform anta-gonism controls occludin phosphorylisation and tightjunction assemblyFreie Universität Berlin

Barth, Michael (2004) Entwicklung neuer, hochbeladenerTrägermaterialien für die organische Festphasensyntheseauf Basis von vernetztem Polyethylenimin – Anwendung imBereich der Peptidsynthese, für Polymerreagenzien undzur Synthese von peptidfunktionalisierten DendrimerenEberhard Karls Universität Tübingen

Begitt, Andreas (2004) Nukleocytoplasmatischer Transportund Geninduktion durch den Transkriptionsfaktor STAT1Freie Universität Berlin

Boisguerin, Prisca (2004) Characterization of PDZ Domain/Ligand SpecifityFreie Universität Berlin

Castellani, Federica (2004) Structure determination ofimmobilized proteins by solid-state NMR spectroscopy Freie Universität Berlin

Eckhardt, Torsten (2004) Design, Synthese, Photochemieund biologische Anwendung von ausgewählten „caged“cyclischen Adenosin-3, 5´-monophosphatenHumboldt-Universität zu Berlin

Edemir, Bayram (2004) Klonierung und Charakterisierungeiner neuen AKAP18-Isoform, AKAP18δ, und ihre möglicheBeteiligung an der AVP-vermittelten AQP2-TranslokationFreie Universität Berlin

Meyer, Thomas (2004) Nukleäre Akkumulation und Ziel-generkennung von STAT1-TranskriptionsfaktorenFreie Universität Berlin

Osiak, Anna (2004) Die Rolle der ubiquitinähnlichen GeneUBL 14 und ISG15 in vivoFreie Universität Berlin

Sauer, Ines (2004) Apolopoprotein E - abgeleitete Peptideals Vektoren zur Überwindung der Blut-Hirn-SchrankeFreie Universität Berlin

Serowy, Steffen (2004) Migration von Protonen entlang derOberfläche ebener BilipidmembranenHumboldt-Universität zu Berlin

Thielen, Anja (2004) Identifizierung transportrelevanterAminosäurereste im proximalen C-Terminus des humanenVasopressin-V2-RezeptorsFreie Universität Berlin

Storm Robert (2004) Extracellular osmolality and solutecomposition participate in the expressional regulation ofAquaporin-2 in renal inner medullary collecting duct cells.Evidence for an involvement of the TonE/TonEBP pathwayFreie Universität Berlin

Wessolowski, Axel (2004) Amphipathische Hexapeptide -Interaktion mit MembranenFreie Universität Berlin

Zimmermann, Jürgen (2004) Struktur und Funktion vonEVH1-DomänenFreie Universität Berlin

Zühlke, Kerstin (2003) Strukturelle und funktionelle Bedeu-tung der konservativen Disulfidbrücke des Vasopressin-V2-RezeptorsFreie Universität Berlin

DIPLOMA THESES 2003 / 2004DIPLOMARBEITEN 2003 / 2004

2003

Brandenburg, MartinKerntransportverhalten trunkierter STAT-PorteineTechnische Fachhochschule Berlin

Hahn, Janina Untersuchungen zu Chromophor-Protein-Interaktionen inortsspezifischen Mutanten von Phytochrom Cph1 aus Cya-nobakterium SynechocystisFreie Universität Berlin

Heine, Markus Wasserstoffperoxyd-induzierte Veränderungen der Aktivi-tät und Expression ausgewählter Proteine in EpithelzellenFreie Universität Berlin

Paschke, Carmen Erweiterte Peakformanalyse zur Verbesserung der auto-matischen Resonanzzuordnung von NMR-SpektrenFachhochschule Lausitz

Schröder, StephanDerivatisierung phosphorylierter Peptide zur Erhöhung derSignalintensität in der MassenspektrometrieTechnische Universität Berlin

Thurisch, BorisStrategien zur gezielten Expressions-Inhibition des muri-nen FMIP-GensTechnische Fachhochschule Berlin

Wendt, Norbert Strukturuntersuchungen der Disulfidstruktur an N-termi-nalen CRF-RezeptorenTechnische Universität Berlin

2004

Cubic, Ivona Charakterisierung von Proteinen mittels Kapillar-HPLC undMassenspektrometrieTechnische Fachhochschule Berlin

El-Dashan, AdeebMilde C-Acylierungen von polymergebundenen Carbox-ylatphosphoranen für Anwendungen in der MedizinischenChemieEberhard-Karls-Universität Tübingen

Kotzur, Nico Synthese und Photochemie von photolabilen cAMP-Deri-vatenHumboldt-Universität zu Berlin

Kubald, SybilleExtrazelluläre Domäne des Rezeptors für den Cortico-tropin-Releasing-Faktor: Klonierung in Expression eines N-Terminus-Schleife-Konstrukts als BindungsmodellTechnische Fachhochschule Berlin

Lassowski, BrigitteUntersuchungen zur Clonierung und Reinigung von Tightjunction-ProteinenUniversität Potsdam

Renner, Armin Funktionelle Bedeutung des Signalpeptides des Cortico-tropin-Releasing-Faktor Rezeptors 2aJulius-Maximilians-Universität Würzburg

Roswadowski, Inga Untersuchungen zur Struktur und Funktion von TightJunction-ProteinenTechnische Fachhochschule Berlin

Schröder, Stephan Derivatisierung phophorylierter Peptide zur Erhöhung derIonisierungen der MassenspektrometrieTechnische Universität Berlin

Zech,Tobias Functional studies of the adapter protein CD2BP2Freie Universität Berlin

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INTERNSHIPS 2003 / 2004PRAKTIKANTEN 2003 / 2004

Ballmer, Boris10.11.2003–05.12.2003Freie Universität Berlin – BiochemieSupervision: Dr. Berger

Behling, Katja01.06.2004–25.06.2004Freie Universität Berlin – BiochemieSupervision: Dr.Berger

Behnken, Swantje16.08.2004–08.10.2004Universität Potsdam – BiochemieSupervision : Dr. Donalies

Bibow, Stefan01.05.2004–31.07.2004Humboldt-Universität zu Berlin – BiophysikSupervision: Prof. Reif

Bieberstein, Andrea06.10.2003–20.02.2004Fachhochschule Lausitz – BiotechnologieSupervision: Dr. Donalies

Blick, Kristin12.05.2003–20.06.2003Freie Universität Berlin – BiochemieSupervision: Dr. Berger

Böthe, Matthias08.12.2003-30.01.2004Freie Universität Berlin – BiochemieSupervision: S. Keller

Böthe, Matthias01.07.2004–11.08.2004Freie Universität Berlin – BiochemieSupervision: Dr. Dathe

Brückner, Kathrin07.04.2003–09.05.2003Technische Universität Dresden – ChemieSupervision: Dr. Siems

Christian, Frank15.09.2003–24.10.2003Freie Universität Berlin – BiochemieSupervision: Dr. Klussmann

Cubic, Ivona01.09.2003–29.02.2004Technische Fachhochschule – BiochemieSupervision: Dr. E. Krause

Curth, Sebastian14.06.2004–10.09.2004Technische Fachhochschule Berlin – BiotechnologieSupervision: Dr. Donalies

Elß, Franka04.10.2004–25.03.2005Fachhochschule Magdeburg – PharmakologieSupervision: Dr. E. Krause

Erel, Funda01.02.2003–31.03.2003Vorpraktikum BiotechnologieSupervision: Dr. Klußmann

Ernst, Oliver20.10.2003–19.04.2004Technische Universität Berlin – BiotechnologieSupervision: Dr. Donalies

Femmer, Christian01.08.2004–15.10.2004Technische Universität Berlin – BiotechnologieSupervision: Dr. Klose

Gartmann, Marco20.10.2003–31.12.2003Universität Potsdam – BiochemieSupervision: Dr. Haseloff

Gasser, Carlos14.05.2004–16.07.2004Freie Universität Berlin – BiochemieSupervision: Dr. Pohl

Giebel, Sebastian01.06.2004-16.07.2004Freie Universität Berlin – BiochemieSupervision: S. Keller

Groß, Anika02.02.2004–12.03.2004Freie Universität Belin – PharmazieSupervision: Dr. Schmieder

Güngör, Volkan01.02.2004–30.04.2004BerufsanfängerSupervision: H.-J. Mevert

Guse, Katrin19.04.2004-04.06.2004Freie Universität Berlin – BiochemieSupervision: S. Keller

Han, Soeng-Ji19.07.2004-03.09.2004Freie Universität Berlin – BiochemieSupervision: S. Keller

Hamidzadek, Nadja27.01.2003–27.03.2003Weiterbildung (Arbeitsamt) – BiochemieSupervision: Dr. Labudde

Heinze, Mathias01.02.2004–31.03.20004Humboldt-Universität zu BerlinSupervision: T. Jahn

Heuberger, Julian08.11.2004–14.12.2004Freie Universität Berlin – BiochemieSupervision: Dr. Dathe

Hübner, Florian01.11.2004-18.02.2005Technische Fachhochschule BerlinSupervision: Dr. Pohl

Hühn, Stephan29.03.2004-07.05.2004Freie Universität Berlin – BiochemieSupervision: S. Keller

Jahnke, Nadine01.06.2004-16.07.2004Freie Universität Berlin – BiochemieSupervision: S. Keller

Jehle, Stefan11.08.2003–24.10.2003Freie Universität Berlin – BiochemieSupervision: Prof. Oschkinat

Kalmbach, Norman06.01.2003–31.01.2003Technische Fachhochschule Berlin – BiotechnologieSupervision: Dr. Vinkemeier

Kaltofen, Sabine21.07.2003–10.10.2003Fachhochschule ZittauSupervision: Dr. Krabben

Kamitz, Anne08.01.2004–02.04.2004AbiturientinSupervision: Dr. Vinkemeier

Kieseritzky, Gernot01.03.2004–09.04.2004Freie Universität Berlin – BiochemieSupervision: Dr. Pohl

Kirsch, Jenny13.04.2004–30.04.2005Universität Potsdam – Biochem.Supervision: Dr. Blasig

Kotzur, Nico01.03.2004–31.12.2004Humboldt-Universität Berlin – ChemieSupervision: Dr. Hagen

Kraemer, Florian01.08.2004–24.09.2004Universität Potsdam – BiochemieSupervision: Prof. Horak

Kron, Anja01.09.2003–30.01.2004Fachhochschule – BiotechnologieSupervision: Dr. Leitner

Kronsbein, Helena10.03.2003–28.03.2003Technische Universität München – BiochemieSupervision: Dr. Schmieder

Krüger, Kerstin15.04.2003–14.10.2003Rharmazie-PraktikantinSupervision: Dr. Siems

Krüger, Magnus01.11.2004–30.04.2005Freie Universität Berlin – PharmazieSupervision: Dr. Beyermann

Kuhn, Ramona04.08.2003–26.09.2003Technische Universität – Berlin – UmweltschutzSupervision: Dr. Wiesner

Lassowski, Birgit01.01.2003–31.03.2003Universität Potsdam – BiotechnologieSupervision: Dr. Blasig

145

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Leddin, Mathias01.08.2003–31.01.2004Freie Universität Berlin – BioSupervision: Dr. Knobeloch

Lisewski, Ulrike14.04.2004–18.05.2004Freie Universität Berlin – BiochemieSupervision: Dr. Pohl

Marter, Katrin05.05.2004–31.10.2004Freie Universität Berlin – BiologieSupervision: Dr. Haseloff

Mehmedi, Burhan24.11.2003–26.12.2003Freie Universität Berlin – BiochemieSupervision: Dr. Siems

Mitschke, Doreen02.06.2003–11.07.2003Freie Universität Berlin – BiochemieSupervision: Dr. Klußmann

Müller, Jeanette19.04.2004-04.06.2004Freie Universität Berlin – BiochemieSupervision: S. Keller

Narayanan, Raghav17.05.2004–07.08.2004Neu DelhiSupervision: Dr. Freund

Neumann, Christine23.02.2004–02.04.2004Universität Marburg – BiologieSupervision: Dr. Vinkemeier

Neumann, Marleen03.02.2003–08.03.2003Technische Fachhochschule Berlin – BiotechnologieSupervision: Dr. Wiesner

Niehage, Christian01.08.2004–31.10.2004Freie Univerisät Berlin – BiochemieSupervision: Dr. Blasig

Oehlke, Elisabeth12.07.2004–30.08.2004Freie Universität Berlin – ChemieSupervision: Dr. Hagen

Overath, Thorsten01.09.2003–26.10.2003Technische Universität Berlin – BiotechnologieSupervision: Dr. Siems

Overath, Thorsten23.02.2004–14.05.2004Technische Universität – BiotechnologieSupervision: Dr. Siems

Petrovska, Silvija15.11.2004–10.12.2004Skopje-MazedonienSupervision: Dr. Siems

Pietske, Matthias19.07.2004-03.09.2004Universität Potsdam – BiochemieSupervision: S. Keller

Prigge, Matthias24.09.2003–07.11.2003Freie Universität Berlin – BiochemieSupervision: Dr. Pohl

Reibitz, Franziska01.02.2004–31.07.2004PharmazeutinSupervision: Dr. Siems

Roswadowski, Inga01.06.2004–30.09.2004Technische Fachhochschule Berlin – Biotech.Supervision: Dr. Blasig

Santamaria, Katja19.02.2003–28.02.2003Freie Universität BerlinSupervision: Dr. Klussmann

Schäfer, Zasia06.09.2004–08.10.2004Universität Potsdam – BiochemieSupervision: Dr. Carstanjen

Schlede, Stephanie04.08.2003–29.08.2003Universität Potsdam – BiotechnologieSupervision: Dr. Siems

Schmidt, Marco08.12.2003–31.01.2004Freie Universität Berlin – BiochemieSupervision: Dr. Hagen

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Schröder, Kati24.02.2003–14.03.2003Technische Universität Berlin – BiotechnologieSupervision: M. Alken

Schulz, Katrin01.12.2004–28.02.2005Charitè-Universitätsmedizin BerlinSupervision: Dr. Blasig

Schumacher, Anne04.08.2003–12.09.2003Humboldt-Universität zu Berlin – BiologieSupervision: Dr. Krabben

Schumacher, Anne01.06.2004–31.07.2004Freie Universität Berlin – BiologieSupervision: Dr. Diehl

Schumann, Franziska15.09.2004–28.02.2005Technische Fachhochschule Berlin. – ChemietechnologieSupervision: Dr. Beyermann

Schuster, Ariane01.07.2004–30.04.2005Universität Potsdam – BiochemieSupervision: Dr. Blasig

Seifert, Christoph03.03.2003–28.03.2003Freie Universität Berlin – BiochemieSupervision: Dr. Siems

Socher, Elke17.02.2003–19.03.2003Humboldt-Universität zu Berlin – ChemieSupervision: Dr. Hagen

Stengel, Florian25.05.2004–06.07.2004Freie Universität Berlin – BiochemieSupervision: Dr. Siems

Stenzel, Denise01.09.2003–30.01.2004Fachhochschule Lausitz – BiotechnologieSupersision: Dr. Knobeloch

Strohschein, Susan07.01.2003–15.02.2003FU Berlin – BiochemieSupervision: Dr. Blasig

Teodorczyk, Marcin07.06.2004–27.08.2004Italien – BiotechnologieSupervision: Dr. Klussmann

Trippens, Jessica19.10.2004–11.01.2005Technische Hochschule Berlin – BiotechnologieSupervision: Dr. Carstanjen

Vorwinkel, Jakob08.03.2004–07.05.2004Freie Universität Berlin – BiochemieSupervision: Dr. Pohl

Wellmann, Anke29.03.2004–13.08.2004Freie Universität Berlin: BiotechnologieSupervision: Dr. Krabben

Winkler, Franziska26.07.2004–26.08.2004Technische Fachhochschule Berlin – BiochemieSupervision: Dr. Siems

Wolf, Constanze13.04.2004–12.07.2004Universität Potsdam – BiologieSupervision: Dr. Blasig

Wolkenhauer, Jan01.01.2003–30.06.2003Universität Potsdam – BiotechnologieSupervision: Dr. Wiesner

Zuleger, Nikolaj01.09.2003–30.01.2004Fachhochschule Lausitz – BiotechnologieSupervision: Dr. Blasig

Zschörnig, Barbara25.08.2003–26.09.2003Universität Regensburg – BiochemieSupervision: Dr. Vinkemeier

Zimmerling, Katrin25.10.2004–18.03.2005Hochschule Anhalt – Pharmazeutische TechnologieSupervision: Dr. Dathe

GUEST SCIENTISTS 2003 / 2004GASTWISSENSCHAFTLER 2003 / 2004

2003

Antonenko, Juri30.01.2003–27.02.200301.11.2003–30.11.2003Belozerski Insitut Moscow, Russia

Alexandrov, Alexej15.03.2003–15.09.200301.11.2003–31.05.2004Kazan University, Russia

Ayuyan, Artem13.08.2003–30.11.2003Academy of the Sciences, Moscow, Russia

Balla, Zsolt01.09.2003–14.09.2003University of Debrecen, Hungary

Barany-Wallje, Elsa08.09.2003–29.02.2004 University of Stockholm, Sweden

Lents, Alexander24.07.2003–28.09.2003Academy of the Sciences Moscow, Russia

Pechanova, Olga25.06.2003–20.07.2003Academy of the Sciences Slovakia, Bratislava

Paulis, Ludovit17.11.2003–21.11.2003 Academy of the Sciences Slovakia, Bratislava

Pires, Jose R.01.11.2003–31.01.2004 University of Rio de Janeiro, Brasil

Woineshet, Zenebe25.06.2003–20.07.2003Academy of the Sciences Bratislava, Slovakia

Sokolov, Valerij20.06.2003–18.07.200301.10.2003– 31.10.2003Academy of the Sciences Moscow, Russia

2004

Alexandrov, Alexej01.11.2003–15.03.2004Kazan University, Russia

Antonenko, Juri30.04.2004–30.05.200401.10.2004–31.10.2004Belozerski Institute, University of Moscow, Russia

Andreeva, Anna01.04.2004–31.05.2004Russia, Scholarship holder

Arbuzova, Anna19.04.2004–28.05.2004University of New York in Stony Brook, USA

Barany-Wallje, Elsa08.09.2003–29.02.2004University of Stockholm, Sweden

Coin, Irene01.10.2004–31.10.2004Italy, Scholarship holder

Khanfar, Monther08.07.2004–19.09.2004University of Zarga, Hashemite, Jordanien Uni

Kumari, Neha01.05.2004–31.12.2004India, Scholarship holder

Lents, Alexander11.08.2004–11.10.2004Frumkin Institute for Electrochemistry, University of Mos-cow, Russia

Magalhaes de Souza, Crista01.01.2004–30.11.2004Oswald Criz Institute, Rio de Janeiro, Brasilia

Pires, Richardo01.11.2003–31.01.200401.12.2004–28.02.2005University of Rio de Janeiro, Brasil

Schreibelt, Gerty03.05.2004–25.09.2004VU Medical centre Amsterdam, The Netherlands

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Sokolov, Valerij11.03.2004–11.04.200418.10.200417.11.2004Frumkin Institute for Electrochemistry, University of Mos-cow, Russia

Sokolenko, Elena11.08.2004–11.10.2004Frumkin Institute for Electrochemistry, University of Mos-cow, Russia

Sommer, Klaus15.11.2004–28.01.2005Universität of Linz, Austria

LECTURES AT THE FMP 2003KOLLOQUIEN UND SEMINARE AM FMP 2003

Bauer, Hans (Institut für Molekularbiologie, Salzburg,Austria)Tight junction proteins 26.02.2003Host: Blasig IE

Beitz, Eric (Institut für Pharmazeutische Chemie, Univer-sität Tübingen, Germany)Aquaporin mediated water permeability in the inner ear 07.10.2003Host: Klussmann E

Berger, Hartmut (Forschungsinstitut für Molekulare Phar-makologie, Berlin-Buch, Germany)GS/GI coupling of the CRF receptors type 1 in HEK cells 14.01.2003

Beyermann, Michael (Forschungsinstitut für MolekularePharmakologie, Berlin-Buch, Germany)Struktur- und Bindungsstudien extracellulärer Rezeptor-domänen11.03.2003

Blasig IE (Forschungsinstitut für Molekulare Pharmakolo-gie, Berlin-Buch, Germany)Structure, Function and Regulation of Tight Junction Pro-teins,25.11.2003

Blechert, Siegfried (Institut für Chemie der TechnischenUniversität Berlin, Germany)Metathese und Wirkstoffchemie – eine starke Kombina-tion17.07.2005Host: Oschkinat H

Carstanjen, Dirk (Forschungsinstitut für Molekulare Phar-makologie, Berlin-Buch, Germany)Die Rolle des Interferon induzierten TranskriptionsfaktorsICSBP in der Reifung und Funktion von Zellen des myelo-poetischen Systems09.12.2003

Danilov, Sergei M (Anestesiology Research Center, Chi-cago, USA)Structure-function studies of angiotensin-converting enzy-me using monoclonal antibodies24.11.2003Host: Siems WE

Dathe, Margitta (Forschungsinstitut für Molekulare Phar-makologie, Berlin-Buch, Germany)ApoE-Peptid-Lipidkomplexe: Trägermodelle für eine Wirk-stoffaufnahme ins ZNS14.10.2003

Dieckmann, Torsten (University of California)Mechanism of action of Rab GTPases in intracellular vesi-cular protein transport02.07.2003Host: Oschkinat H

Diehl, Anne (Forschungsinstitut für Molekulare Pharma-kologie, Berlin-Buch, Germany)Protein production for structure determination by NMRand X-ray25.02.2003

Duffy, Heather S. (Albert-Einstein College of Medicine,New York, USA)pH dependent inter- and intramolecular interactions onconnexin43: Regulation of a junctional complex 02.09.2003Host: Blasig IE

Folkers, Gerd (Eidgenössische Technische HochschuleZürich, Switzerland)Designing the Locks and Creating new Keys: MolecularDesign Methodology for Genetic Switches06.05.2003Host: Bienert M

Freund, Christian (Forschungsinstitut für Molekulare Phar-makologie, Berlin-Buch, Germany)GYF and AH3 domain mediated interactions in eukayoticsignaling10.06.2003

Glockshuber, Rudi (Eidgenössische Technische Hoch-schule Zürich, Switzerland)Catalysis of disulfide bond formation in Escherichia coli21.01.2003Host: Bienert M

Goody, Roger (Max-Planck-Institut für Molekulare Physio-logie, Dortmund, Germany)Mechanisms of actions of Rab GTPases in intracellularvesicular protein transport01.07.2003Host: Oschkinat H

Gottschalk, Kay (Weizmann Institute of Science, Israel)Computational Approaches to Transmembrane ProteinStructure27.11.2003Host: Reif B

Hagen, Volker (Forschungsinstitut für Molekulare Pharma-kologie, Berlin-Buch, Germany)Caged compounds: Design and applications13.05.2003

Hauri, Hans-Peter (Universität Basel, Switzerland)Protein traffic early in the secretory pathways: dynamicsand signals23.09.2003Host: Hermosilla R

Heinz, Dirk (Department of Structural Biology, Braun-schweig, Germany)Bacterial invasion at atomic resolution16.09.2003Host: Rosenthal W

Heise, Bernd (Institut für Experimentelle Physik der Uni-versität Ulm, Germany)Structural investigations on peptaibols via liquid and solidstate NMR18.03.2003Host: Oschkinat H

Helm, Volkhard (MPI Frankfurt, Germany)Studying protein-protein interactions in silico11.02.2003Host: Freund C

Hennemann, Hanjo (Center of advanced european studiesand research, Bonn, Germany)Ras recruitment system: analysis of protein function andprotein networks and its high throughput application21.10.2003Host: Rosenthal W

Hermosilla, Ricardo (Forschungsinstitut für MolekularePharmakologie, Berlin-Buch, Germany)Intracellular degradation pathways of wild-type and trans-port-defective mutant vasopressin V2 receptors09.07.2003

Hougardy, Stefan (Institut für Informatik der Humboldt-Uni-versität zu Berlin, Germany)Three-Dimensional similarity of Small Molecules13.11.2003Host: Vinkemeier U

Johnson, Nils (Forschungszentrum Karlsruhe, Germany)Analysis of protein network in living cells02.12.2003Host: Vinkemeier U

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Jordt, Sven (Department of Cellular & Molecular Pharma-cology, UCSF, USA)The Pain Gate – Functional Domains in the CapsaicinReceptor16.12.2003Host: Rosenthal W

Klussmann, Enno (Forschungsinstitut für Molekulare Phar-makologie, Berlin-Buch, Germany)Protein kinase A anchoring proteins and Rho are involvedin the AVP-indused shuttling of aquaporin11.02.2003

Krabben, Ludwig (Forschungsinstitut für Molekulare Phar-makologie, Berlin-Buch, Germany)NMR am Membranprotein30.09.2003

Krause, Eberhard (Forschungsinstitut für Molekulare Phar-makologie, Berlin-Buch, Germany)Structural analysis of peptides and proteins by mass spec-trometry22.04.2003

Krause, Gerd (Forschungsinstitut für Molekulare Pharma-kologie, Berlin-Buch, Germany)Predictions of structure-function relationships: Advan-tages and limits of structural bioinformatics08.04.2003

Kühne, Ronald (Forschungsinstitut für Molekulare Phar-makologie, Berlin-Buch, Germany)Luteinizing Hormone Releasing Hormone Receptor: Mole-cular dynamics and model-based ligand design24.06.2003

Ladizhansky, Vladimir (Francis Bitter Magnet Laboratory,Cambridge, UK)Techniques for modern solid-state NMR10.01.2003Host: Oschkinat H

Lang, Christine (Institut für Mikrobiologie und Genetik derTechnischen Universität Berlin, Germany)Production of proteins for structural genomics using yeastas hosts30.04.2003Host: Schade M

Markley, John L. (University of Wisconsin-Madison, USA)Structural Genomics of the Eukaryote Arabidopsis thaliana23.09.2003Host: Oschkinat H

Marx, Dominik (Lehrstuhl für Theoretische Chemie, Ruhr-Universität Bochum, Germany)Proton Transfer along Hydrogen-Bonded Networks20.05.2003Host: Pohl P

Monsees, Thomas (Zentrum für Dermatologie und Andro-logie, Giessen, Germany)Expression von Kininrezeptoren im Rattentestis: Eine funk-tionelle Bedeutung für die Spermatogenese13.01.2003Host: Siems WE

Mourot, Alexandre (Laboratoire De Chimie Bioorganique,France)Investigation of the conformational transitions of the nico-tinic receptor by photoaffinity labeling, 12.12.2003Host: Pohl P

Müller, Jürgen (National Cancer Institute at Frederick,USA)C-TAK1 is a regulator of the MAPK scaffold protein KSRand various other signaling proteins20.03.2003Host: Rosenthal W

Multhaup, Gerd (Institut für Chemie und Biochemie, FreieUniversität Berlin, Germany)Implication of amyloid precursor protein ligands in amyloi-dogenesis26.08.2003Host: Reif B

Palczewski, Krysztof (Department of Ophtalmology of theUniversity of Washington, USA)Structure, function and membrane organisation of verte-brate rhodopsin10.04.2003Host: Krause G

Reiser, Oliver (Institut für Organische Chemie der Univer-sität Regensburg, Germany)Arglabin, ein vielversprechender Farnesyltransferase-Inhibitor13.02.2003Host: Bienert M

Scharnagl, Hubert (Universitätsklinikum Freiburg, Germa-ny)Apolipoprotein E, Lipidstoffwechsel und die AlzheimerscheKrankheit12.06.2005Host: Dathe M

Schmalz, Hans-Günther (Universität zu Köln, Germany)Mediated synthesis of biologycally relevant small mole-cules27.03.2003Host: Oschkinat H

Schmidt, Gundula (Albert-Ludwigs-Universität Freiburg,Germany)Mechanism and function of Rho targeting bacterial toxins19.08.2003Host: Rosenthal W

Schmieder, Peter (Forschungsinstitut für Molekulare Phar-makologie, Berlin-Buch, Germany)Strukturen von GPCRs mit Lösungs-NMR22.07.2003

Schubert, Mario (University of British Columbia in Vancou-ver, Canada)Insights into mRNA degradation in E. coli - The S1 domainof Rnase E19.06.2003Host: Oschkinat H

Schülein, Ralf (Forschungsinstitut für Molekulare Pharma-kologie, Berlin-Buch, Germany)The early secretory pathway of G protein-coupled recep-tors: clinical and pharmacological implications28.01.2003

Serrano, Luis (European Molecular Biology Laboratory,Heidelberg, Germany)Sequence determinants of amyloid formation20.10.2003Host: Oschkinat H

Siems, Wolf-Eberhard (Forschungsinstitut für MolekularePharmakologie, Berlin-Buch, Germany)NEP und ACE, zwei „alte“ Enzyme mit vielen neuen Chan-cen16.12.2003

Sinning, Irmgard (Biochemie-Zentrum Heidelberg,Germany)Expression of G-protein coupled receptors in the eye oftransgenic Drosophila04.03.2003Host: Rosenthal W

Stiles, Brad (Institut für Experimentelle und KlinischePharmakologie und Toxikologie, Freiburg, Germany)Biotoxins: Antrax to Venoms14.02.2003Host: Rosenthal W

Stubbs, Milton T. (Institut für Biotechnologie, Martin-Luther-Universität Halle, Germany)Understanding protein - ligand interactions: Effects of fle-xibility18.02.2003Host: Oschkinat H

Vinkemeier, Uwe (Forschungsinstitut für Molekulare Phar-makologie, Berlin-Buch, Germany)Regulation der subzellulären Verteilung von STAT127.05.2005

Zamora, Salvador Ventura (Departemente de Bioquimica iBiologia Molecular, University of Barcelona, Spain)Short amino acid sequences can trigger protein amyloidformation in globular proteins15.07.2003Host: Reif B

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LECTURES AT THE FMP 2004KOLLOQUIEN UND SEMINARE AM FMP 2004

Ahnert-Hilger, Gudrun (Institut für Anatomie der Charitè-Universitätsmedizin Berlin, Germany)Regulation of vesicular neurotransmitter transporters byheterotrimeric G-proteins 17.02.2004Host: Klussmann E

Appelt, Christian (Forschungsinstitut für Molekulare Phar-makologie, Berlin-Buch, Germany)Peptide-lipid interaction: structural investigations by NMRand Molecular Dynamics13.04.2004

Baeuerlein, Edmund (Max-Planck-Institut für Biochemie,Martinsried, Germany)BIOMINERALISATION. Von Biologie zu Materialwissen-schaften und Molekularbiologie15.11.2004Host: Oschkinat H

Banci, Lucia (Centro Risonanze Magnetiche, University ofFlorence, Italy)NMR on Metalloprotein in Structural Genomics03.05.2004Host: Oschkinat H

Boelens, Wilbert (University of Njimegen, Department ofBiochemistry, The Netherlands)Involvement of the small heat shock protein aB-crystallinin cellular stress regulation06.04.2004Host: Reif B

Cordes, Frank (Konrad-Zuse-Zentrum für Informations-technik, Berlin, Germany) Conformation Databases for Virtual Screening18.05.2004Host: Kühne R

Gast, Klaus (Max-Delbrück-Center, Berlin-Buch, Germa-ny)Critical oligomers in protein misfolding and aggregation10.02.2004Host: Bienert M

Gershengorn, Marvin (NIH/NIDDK Bethesda, USA)Pharmacology of Thyrotropin-Releasing Hormone Recep-tors: Similarities and Differences02.03.2004Host: Krause G

Gohla, Antje (Departments of Immunology and Cell Bio-logy, La Jolla, USA)Regulation of cytoskeletal dynamics by Chronophin, anovel unconventional cofilin phosphatase30.03.2004Host: Rosenthal W

Harteneck, Christian (Institut für Pharmakologie derCharité Universitätsmedizin Berlin, Germany)Kationenkanäle auf der Suche nach neuen Funktionen: Diemolekulare Vielfalt der TRP-Kanäle07.09.2004Host: Rosenthal W

Heise, Tilmann (Heinrich-Pette-Institut Hamburg,Germany) The La Protein, a multifunctional RNA binding proteinessential for cell cycle progression05.10.2004Host: VinkemeierU

Henning, Mirko (Department of Molecular Biology & TheSkaggs Institute of Chemical Biology, La Jolla, USA) Labeling Methodology and 19F NMR of Fluorinated RNA02.06.2004Host: Reif B

Horuk, Richard (Berlex AG, Richmont, Californien, USA)CCR1 receptor antagonists from the bench to the Clinic07.12.2004Host: Kühne R

Huber, Otmar (Charité Universitätsmedizin Berlin, Germa-ny)The fate of Cell-cell contacts in apoptotic epithelial cells15.06.2004Host: Blasig IE

Hundsrucker, Christian/Weber, Viola (Forschungsinstitutfür Molekulare Pharmakologie, Berlin-Buch, Germany)Peptide Disruptors of AKAP-PKA Interactions07.12.2004

John, Susan (King's College, London, UK)Regulating the transcriptional activity of STAT516.11.2004Host: Vinkemeier U

Kofler, Michael (Forschungsinstitut für Molekulare Phar-makologie, Berlin-Buch, Germany)FMP, NWG Protein EngineeringRecognition Code of GYF domains07.12.2004

Kungl, Andreas (Universität Graz, Switzerland)Biophysical Investigations of Chemokine-Receptor andCo-Receptor Interactions06.01.2004Host: Oschkinat H

Labudde, Dirk/Leitner, Dietmar (Forschungsinstitut fürMolekulare Pharmakologie, Berlin-Buch, Germany)Tools and concepts for automation of protein structuredetermination by NMR09.03.2004

Langer, Gernot (Schering AG, Berlin, Germany)Early Drug Discovery - Target identification, Assay deve-lopment & High throughput screening 16.11.2004Host: Kühne R

Livett, Bruce G (University of Melbourne, Australia)Beauty and the Beast: molecular Prospecting for NovelDrugs from the Sea: the discovery, properties and deve-lopment of the novel analgesic, conotoxin Vc1.1 (ACV1)26.01.2004Host: Oehme P

Llinas, Miguel (Department of Chemistry of the CarnegieMellon University, Pittsburgh, USA)Is it NMR or is it crystallography? CLOUDS: a directmethod for protein structure elucidation via NMR protondensities15.06.2004Host: Oschkinat H

Lorenz, Dorothea (Forschungsinstitut für Molekulare Phar-makologie, Berlin-Buch, Germany)Electron microscopy in cell biology. Methods and perspec-tives24.02.2004

Luy, Burkhard (Institut für Organische Chemie und Bioche-mie II der Technischen Universität München, Germany)Structural Investigations on the Pulmonary SurfactantAssociated Lipopeptide SP-C and Novel NMR-TechniquesConcerning Small Molecules in Organic Solvents29.07.2004Host: Reif B

Marg, Andreas (Forschungsinstitut für Molekulare Phar-makologie, Berlin-Buch, Germany)STAT – Abwege25.05.2004

Mayer, Thomas (Max-Planck-Institute of Biochemistry,Department of Cell Biology, Martinsried, Germany)Small molecules: versatile probes to study mitotic kinesins05.10.2005Host: von Kries J

Oelgeschläger, Thomas (Marie Curie Research Institut,Transcription Laboratory, Oxted, UK)Core promoter-specific regulation of RNA polymerase IItranscription04.05.2004Host: Vinkemeier U

Overduin, Michael (University of Birmingham, UK)Membrane binding domains: the good, the bad, and theugly14.04.2004Host: Oschkinat H

Pardo, Leonardo (Universidad Autonoma de Barcelona,Spain)Bioinformatic Approaches Leading to an Understanding ofthe Structure and Activity of G-protein coupled receptors30.11.2004Host: Kühne R

Rapp, Wolfgang (Rapp Polymer GmbH, Tübingen,Germany)Ein universelles Linkerkonzept zur sequenziellen Abspal-tung von Peptiden und Erzeugung von geschützten Pepti-damiden06.05.2004Hosts: Bienert M, Beyermann M

Reif, Bernd (Forschungsinstitut für Molekulare Pharmako-logie, Berlin-Buch, Germany)Misfolding proteins and molecular chaperones by solutionand solid-state NMR10.11.2004

Rosen, Michael (Southwestern Medical Center Dallas,University of Texas, USA)Structural and Biochemical Mechanisms of Signal Inte-gration by the Wiskott-Aldrich Syndrome Protein25.03.2004Host: Oschkinat H

Salditt, Tim (Institut für Röntgenphysik, Universität Göttin-gen, Germany)Probing the Structure, and Interactions of Polypeptides inLipid Bilayers by X-ray and Neutron Scattering12.03.2004Host: Freund C

Scott, John D. (FRS, Oregon Health & Sciences University,Portland, USA)The Molecular Architecture of Signal Transduction com-plexes05.02.2004Host: Klussmann E

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Seelig, Joachim (Biozentrum Universität Basel, Switzer-land)Detergents, Peptides and Microdomains in Membranes04.02.2004Host: Bienert M

Seitz, Oliver (Humboldt-Universität zu Berlin, Germany)Erzwungene Interkalation – Wie man Basenlücken stopftund Mutationen in DNA nachweist 19.10.2004Host: Bienert M

Soderhäll, Arvid (Forschungsinstitut für Molekulare Phar-makologie, Berlin-Buch, Germany) Suggested function of the antimicrobial cyc-RW peptidefrom MD-simulations22.06.2004

Sommer, Klaus (Medizinische Universität Wien, Austria)Inhibitor of apoptosis protein survivin is upregulated byoncogenic c-H-Ras28.10.2004Host: Pohl P

Stefan, Eduard (Forschungsinstitut für Molekulare Phar-makologie, Berlin-Buch, Germany)Phosphodiesterase pde4 is involved in the vasopressin-mediated water reabsorbtion by regulating the localizati-on of the water channel aquaporin-211.05.2004

Strauss, Holger (Forschungsinstitut für Molekulare Phar-makologie, Berlin-Buch, Germany)Biophysical investigations on the N-terminus of cyanobac-terial phytochrome Cph1d223.03.2004

Tycko, Robert (Laboratory of Chemical Physics, NIDDK,Bethesda, USA)Structure of Unfolded Proteins and Amyloid Fibrils: Expe-rimental Constraints from Solid State NMR 13.10.2004Hosts: Reif B, Oschkinat H

Wüstner, Daniel (Max-Delbrück-Center, Berlin-Buch, Ger-many)Transport und function of cholesterol in epi01.12.2004Hosts: Rosenthal W, Bienert M

TECHNOLOGIETRANSFER 2003/2004

Auch in den Jahren 2003 und 2004 hat das FMP für wirt-schaftlich vielversprechende ForschungsergebnisseSchutzrechte angemeldet, Verwertungschancen geprüftund gegebenenfalls Maßnahmen zur kommerziellen Ver-wertung ergriffen. Durch die räumliche Nähe zu biotech-nologischen Firmen auf dem Campus Berlin-Buch und inder Region Berlin/Brandenburg sind optimale Vorausset-zungen für die Anwendung von Forschungsergebnissengegeben.

Um die Verwertungsaktivitäten weiter zu optimieren,wurde im August 2004 eine Zusammenarbeit mit derAscenion GmbH (München) vertraglich vereinbart. Seit-dem bewertet die Ascenion Erfindungen von FMP-Mitar-beitern hinsichtlich patent- und vor allem marktrelevanterGesichtspunkte.

Das FMP hält derzeit acht eingetragene oder erteilteSchutzrechte auf insgesamt vier Erfindungen. Für weitereErfindungen sind Anmeldeverfahren zur Erteilung vonSchutzrechten eingeleitet.

TECHNOLOGY TRANSFER 2003/2004

Also in 2003 and 2004, the FMP submitted patent applica-tions for economically promising research results, asses-sed the prospects for commercial success and, whereappropriate, initiated commercialization activities. Due tothe proximity to biotech companies on the Berlin-Buchcampus and in the Berlin-Brandenburg region, excellentconditions for the utilization of research results are pro-vided.

To further optimize commercialization activities, the FMPentered a contract agreement with Ascension GmbH inMunich. Since that time Ascension has been assessinginventions of FMP employees with regard to patents andespecially to market-relevant aspects.

The FMP currently holds eight registered or grantedpatents on a total of four inventions. Applications for addi-tional patents have been submitted.

Inventions and patentsErfindungen und Schutzrechte

Hagen V, Bauer PJAls Verknüpfungsreagenzien einsetzbare dimaleinimido-substituierte Dihydroxyalkane und Verfahren zu deren Her-stellungDE 195 33 867 C1 (filed 24.04.1997)EP 96938012.0 (filed 02.05.2003)PCT/DE96/01742US 09/043,263 (filed 15.06.1999)priority establishing patent application: 13.09.1995

Hagen V, Kaupp UBNeue photolabile 8-substituierte cyclische Nucleotidester,Verfahren zu ihrer Herstellung und VerwendungDE 195 29 025.9EP 96927617.9-1270WO 97/05155priority establishing patent application: 29.07.1995

FMP-inventors in bold.

157

Hagen V, Kaupp B, Bendig, Wiesner BNeue, Photolabile Cumarinylmethylester von cyclischenNucleotiden, Verfahren zu deren Herstellung und ihre Ver-wendungDE 100 21 256 A1 PCT/EP01/03512EP 01 929 464.4-2110JP 2001-578448US 01/03512priority establishing patent application: 20.04.2000

Labudde D, Leitner D, Schubert M, Winter R, Oschkinat H,Schmieder PVorrichtung und Verfahren zur Zuordnung der NMR-Sig-nale von PolypeptidenDE 10144661.6-33 (filed 14.08.2003) EP 02777009.8PCT/EP02/09959US 10/799,447priority establishing patent application: 11.09.2001

Labudde DVerfahren zur Ermittlung von Verschiebungen der Hirna-reale durch Tumorbildung und deren Darstellung aufeinem BildschirmDE 100 13 360.6 (filed 22.07.2004)PCT/DE01/00955 JP 002003527899 T2US 10/221,064EP 01916923.4priority establishing patent application: 09.03.2000

Maric KTablett für Feuchtkammer (Gebrauchsmuster)DE 2002109.5 (filed 05.04.2001)priority establishing application: 8.12.2000

Meyer T, Vinkemeier UVerwendung von dimer-spezifischen Nuclear-Lokalisati-ons-Signalpeptiden (dsNLS) abgeleitet aus der STAT-DNABindungsdomäneDE 102 01 791.3 PCT/EP2003/000240EP 03702426.2-2107priority establishing patent application: 17.01.2002

Rosenthal W, Klußmann E, Oksche ANeue Spleißvariante eines Proteinkinase A-Ankerproteinsund Verwendung dieserDE 103 06 085.5 PCT/EP2003/09892CA PCT/EP 2003/09892EP 03 750 490.9-2405JP 502327850US 10/526.768priority establishing patent application: 7.2.2003

Rosenthal W, Klußmann E, Hundsrucker CPeptide zur Inhibition der Interaktion von Proteinkinase Aund Proteinkinase A-AnkerproteinDE 10 2004 031 579.5priority establishing patent application: 29.06.2004

Soderhäll A, Kühne R, et al.Neue, peptidomimetische, oral verfügbare LHRH Antago-nisten mit Tetrahydrocarbazol-Grundkörperrights sold in 2004

Siems W, Walther T, Melzig MFVerwendung von NEP-assoziierten Molekülen zur Behand-lung von nichtimmunogenen-nichthypertensiven Zivili-sationskrankheitenDE 103 11 984.1-41 PCT/DE2004/000491priority establishing patent application: 12.3.2003

Vinkemeier UNeue Nucleus-Export-Signalpeptide (NES), sie enthalten-de Fusionsproteine sowie deren VerwendungDE 100 35 867 A1PCT/EP01/08065EP 01954035.0US 10/333,082priority establishing patent application: 14.07.2000

Vinkemeier U, Meyer TA single residue modulates Tyrosine Dephosphorylation,oligomerization, and nuclear accumulation of Stat Tran-scription factorsEP 04 09 0039.1PCT/EP2004/001462priority establishing patent application: 10.2.2004

Vinkemeier U, Meyer TVerfahren zur Detektion von nukleozytoplasmatischenTransportprozessenDE 10 2004 046 327.1priority establishing patent application: 20.09.2004

Vinkemeier U, Meyer T, Marg AHyperactive Stat molecules and their use in assaysemploying gene activationEP 04 077 629.6priority establishing patent application: 17.09.2004

App

endi

x

FMP-inventors in bold.

STRUCTURE OF THE FORSCHUNGSINSTITUT FÜR MOLEKULARE PHARMAKOLOGIE (FMP)

Staff CouncilBurkhard Wiesner

Structural Biology

Cellular Signalling /Molecular Genetics

Chemical Biology

Administration, Technical and

Scientific Services

Computer ServicesThomas Jahn

Technical ServicesHans-Jürgen Mevert

LibraryMichael Beyermann

Renate Peters

Animal HousingRegina Richter

Microdialysis Service

Regina Richter

DNA Sequencing Service

Erhard Klauschenz

Solution NMRPeter Schmieder


Gerd Krause

Molecular Modelling

Ronald Kühne

Solid State NMR Bernd Reif

Protein EngineeringChristian Freund

Protein TraffickingRalf Schülein

Anchored SignallingEnno Klußmann

Cellular ImagingBurkhard Wiesner


Ingolf E. Blasig


Wolf-Eberhard Siems

Cytokine SignalingKlaus P. Knobeloch

Molecular Myelopoiesis

Dirk Carstanjen


Uwe Vinkemeier

Peptide SynthesisMichael Beyermann

Peptide BiochemistryHartmut Berger

Mass SpectrometryEberhard Krause

Synthetic Organic BiochemistryVolker Hagen

Medicinal ChemistryJörg Rademann

Screening UnitJens Peter von Kries

DirectorWalter Rosenthal

Public Relations Björn Maul

Safety OfficerHans-Ulrich Heyne

ForschungsverbundBerlin e. V.

Peptide Lipid Interaction/Peptide Transport

Margitta DatheJohannes Oehlke

Protein StructureHartmut Oschkinat

Cellular SignallingWalter Rosenthal

Molecular GeneticsIvan Horak

Peptide Chemistry & BiochemistryMichael Bienert

AdministrationThomas Ellermann

Stru

ctur

eof

the

FMP

159

ORGANIGRAMM

BetriebsratBurkhard Wiesner

Strukturbiologie Zell-Signalling/Molekulare Genetik Chemische Biologie Verwaltung, technischeund wissenschaftliche

Dienste

Computer-ServiceThomas Jahn

Technischer ServiceHans-Jürgen Mevert

BibliothekMichael Beyermann

Renate Peters

TierhaltungRegina Richter

MikrodialyseserviceRegina Richter

DNA-Sequenzier-service

Erhard Klauschenz

Lösungs-NMRPeter Schmieder

StrukturelleBioinformatikGerd Krause

Molekulares ModellingRonald Kühne

Festkörper-NMR Bernd Reif

Protein EngineeringChristian Freund

Protein-TrafficingRalf Schülein

Anchored SignallingEnno Klußmann

Zell-ImagingBurkhard Wiesner

MolekulareZellphysiologieIngolf E. Blasig

BiochemischeNeurobiologie

Wolf-Eberhard Siems

Zytokin-SignallingKlaus P. Knobeloch

Molekulare Myelopoese

Dirk Carstanjen

ZelluläreSignalverarbeitung

Uwe Vinkemeier

PeptidsyntheseMichael Beyermann

PeptidbiochemieHartmut Berger

MassenspektrometrieEberhard Krause

Synthetische OrganischeBiochemie

Volker Hagen

Medizinische ChemieJörg Rademann

Screening-UnitJens Peter von Kries

DirektorWalter Rosenthal

ÖffentlichkeitsarbeitBjörn Maul

ArbeitssicherheitHans-Ulrich Heyne

ForschungsverbundBerlin e. V.

Peptid-Lipid-Inter-aktion/Peptidtransport

Margitta DatheJohannes Oehlke

Proteinstruktur Hartmut Oschkinat

Zell-SignallingWalter Rosenthal

Molekulare GenetikIvan Horak

Peptidchemie &Biochemie

Michael Bienert

VerwaltungThomas Ellermann

INDEX

AAl-Gharabli, Samer ............................................................. 90Alken, Martina ..................................................................... 40Andreeva, Anna Y. ............................................................... 49Antonenko, Yuri ................................................................... 57Appelt, Christian ............................................................ 16, 18Ayuyan, Artem ..................................................................... 57

BBall, Linda ............................................................................. 13Balling, Rudolf ........................................................................ 2Bárány-Wallje, Elsa ............................................................ 76Barth, Michael ..................................................................... 90Bauer, Jörg ........................................................................... 90Becker, Matthias ................................................................. 55Beck-Sickinger, Annette G. ................................................. 2Begitt, Andreas .................................................................... 66Benedict, Melanie ............................................................... 63Ben-Slimane, Uta ................................................................ 33 Berger, Hartmut ............................................................. 70, 80Beyermann, Michael .................................................... 70, 72Bienert, Michael ................................................... 70, 75, 76Blasig, Ingolf ........................................................................ 49Blasig, Rosel ........................................................................ 64Blick, Helmut ...................................................................... 101Blum, Christopher ............................................................... 43 Bohne, Kerstin ..................................................................... 64 Boisguerin, Prisca ............................................................... 13Boldt, Liane .......................................................................... 60Brauße, Kerstin ................................................................. 107Bräutigam, Matthias ............................................................. 2Brockmann, Christoph ........................................... 13, 25, 26Buschner, Michael ............................................................ 101

CCarstanjen, Dirk ................................................................... 62Cartier, Regis ........................................................................ 66Castellani, Federica ............................................................ 13Chen, Zhongjing ................................................................... 29Chernogolov, Alex ............................................................... 29Chevelkov, Veniamin ........................................................... 29Coin, Irene ............................................................................ 74

DDasari, Muralidhar .............................................................. 29 Dathe, Margitta ....................................................... 70, 75, 76Dekowski, Brigitte ............................................................... 87Diehl, Anne ........................................................................... 13Djurica, Maja ....................................................................... 63Donalies, Ute ........................................................................ 40Dreissigacker, Marianne ................................................... 70

EEhrlich, Angelika ..................................................... 74, 75, 95Eichhorst, Jenny .................................................................. 48 Eilemann, Barbara .............................................................. 50Eisenmenger, Frank ...................................................... 24, 26 El-Dashan, Adeeb ............................................................... 90 Ellermann, Thomas ........................................................... 106Erdmann, Christoph ............................................................ 95 Evers, Heide ......................................................................... 14

FFälber, Katja ......................................................................... 13Fechner, Klaus ..................................................................... 82Fink, Uwe ...............................................................................29 Flinders, Jeremy .................................................................. 13Fossi, Michele ...................................................................... 13Freund, Christian ........................................................... 31, 32

GGeelhaar, Andrea ................................................................ 43Geißler, Daniel ..................................................................... 87Georgi, Monika .................................................................... 82Gomoll, Michael .................................................................. 43 Göritz, Petra ....................................................................... 101Griesinger, Christian ............................................................. 2

HHackel, Uwe ....................................................................... 101Hagen, Volker ................................................................. 70, 86 Hahn, Janina .................................................................. 16, 18Handel, Lilo ........................................................................... 14Hartmann, Gislinde ............................................................. 50 Haseloff, Reiner F. ............................................................... 49Hausbeck, Dana ................................................................ 107Heine, Markus ..................................................................... 50Heinrich, Nadja ................................................................... 82Heinze, Matthias ........................................................... 31, 33Henn, Volker ......................................................................... 43Hermann, Ingrid ................................................................. 108Heuer, Katja .................................................................... 32, 33Heyne, Alexander .............................................................. 108Heyne, Hans-Ulrich ........................................................... 101Hiller, Matthias ..................................................................... 13

161

Hologne, Maggy ...................................................................29Holtmann, Henrik ................................................................. 13Horak, Ivan ........................................................................... 36Hübel, Stefan ....................................................................... 26 Hundsrucker, Christian ....................................................... 43

JJahn, Reinhard ...................................................................... 2Jahn, Thomas .................................................................... 108Jehle, Stefan ........................................................................ 14Joost, Hans-Georg ................................................................ 2Joshi, Mangesh ................................................................... 13

KKallies, Axel .......................................................................... 63Kahlich, Bettina ................................................................... 55 Keller, Sandro ................................................................ 76, 78Kiesling, Alexandra ............................................................. 36Kirsch, Jenny ....................................................................... 50Kisser, Agnes ....................................................................... 60 Klauschenz, Erhard ........................................................... 101Kleinau, Gunnar ............................................................. 20, 23Klemm, Clementine .............................................................. 85Klemm, Janet ....................................................................... 64 Klose, Annerose .................................................................. 74Klose, Jana............................................................................ 74Klussmann, Enno ................................................................. 42Knobeloch, Klaus-Peter ..................................................... 59Köhler, Christian .................................................................. 13Königsmann, Jessica ...........................................................63 Kofler, Michael ..............................................................31, 33Kotzur, Nico ............................................................................87Krabben, Ludwig .................................................................. 13Krätke, Oliver .........................................................................74 Krause, Dagmar.....................................................................74 Krause, Eberhard ........................................................... 70, 83Krause, Gerd......................................................................... 20 Krause, Winfried .................................................................. 55 Kries, Jens-Peter von................................................ 3, 70, 92Krylova, Oxana O. ................................................................. 57Kühne, Ronald................................................................. 24, 25Kummerow, Mandy ............................................................. 66

LLabudde, Dirk ....................................................................... 13Lättig, Jens...................................................................... 20, 23 Lamer, Stephanie ................................................................. 85 Lange, Vivien ........................................................................ 14Lassowski, Birgit ................................................................. 50Lauterjung, Ulrike .............................................................. 104 Lechler, Ralf ......................................................................... 87 Leidert, Martina ................................................................... 14Lentz, Alexander................................................................... 57Leitner, Dietmar ................................................................... 13Lerch, Heidi .......................................................................... 85Liesenfeld, Oliver ................................................................. 76Lojek, Eva ............................................................................ 101Lödige, Inga .......................................................................... 66Lorberg, Dörte ...................................................................... 50 Lorenz, Dorothea ........................................................... 43, 48

MMac, Thien-Thi .................................................................... 14Manks, Silvia ...................................................................... 107Marg, Andreas ..................................................................... 66Margania, Valentina ........................................................... 57 Maul, Björn ................................................................. 102, 103McSorley, Theresa .............................................................. 43 Meier, Franziska .................................................................. 90 Meissner, Torsten ................................................................ 66Melchior, Frauke ................................................................... 2Messing, Claudia ............................................................... 107Mevert, Jürgen....................................................................101 Meyer, Stephanie ................................................................ 66 Meyer, Thomas .................................................................... 66Michl, Dagmar ..................................................................... 40 Mikoteit, Kerstin .................................................................. 50 Mohs, Barbara ................................................................... 101Mollajew, Rustam ................................................................ 57 Motzny, Katrin ................................................................ 31, 33 Müller, Sebastian L. ................................................ 20, 23, 50Muschter, Antje ................................................................. 100

NNarayanan, Saravanakumar ............................................. 29Nedvetsky, Pavel ................................................................. 43 Niehage, Christian .............................................................. 50Niendorf, Sandra ................................................................. 60 Nikolenko, Heike ................................................................. 78

Inde

x

OOczko, Brunhilde ................................................................. 48Oehlke, Johannes ......................................................... 70, 75Oschkinat, Hartmut ....................................................... 10, 12Osiak, Anna .......................................................................... 60Otto, Christel ...................................................................... 107Oueslati, Morad ................................................................... 40

PPahlke, Doreen .................................................................... 14Pankow, Kristin .................................................................... 55Pankow, Rüdiger ................................................................. 64 Panzer, Holger .................................................................... 101Passow, Josephine ........................................................... 107 Perepellichenko, Ludmila ................................................... 90 Peters, Renate ................................................................... 101Petschick, Heidemarie ....................................................... 36Piontek, Jörg ........................................................................ 49 Piotukh, Kirill .................................................................. 31, 33 Pires, Ricardo ...................................................................... 13Pisarz, Barbara .................................................................... 74Pisarz, Hans-Werner ........................................................ 108Pohl, Peter ............................................................................ 57Poliakov, Ilja ......................................................................... 14Prigge, Matthias .................................................................. 57Pritz, Stephan ....................................................................... 74

RRademann, Jörg ........................................................ 3, 70, 89Reif, Bernd ............................................................................ 28Rehbein, Kristina ................................................................. 14Richter, Regina M. ....................................................... 98, 101 Ringling, Martina ................................................................. 48 Rosenthal, Walter ............................................................ 6, 36Rossum, Barth van .............................................................. 13Roswadowski, Inga ............................................................. 50Rötzschke, Olaf .................................................................... 25Rückert , Christine ............................................................... 49 Rutz, Claudia ........................................................................ 40

SSantamaria, Katja ................................................................ 43 Saparov, Sapar M. .............................................................. 57 Sauer, Ines ..................................................................... 76, 78Scharnagel, Hubert ............................................................. 76Schlegel, Brigitte ........................................................... 16, 18Schmidt, Antje ..................................................................... 46Schmieder, Peter ................................................................. 16Schmikale, Bernhard .......................................................... 74 Schneeweiß, Ulrike ............................................................ 33 Schülein, Ralf ....................................................................... 33Schümann, Michael ............................................................ 85

Schumacher, Gabriele ...................................................... 107 Schumann, Björn ............................................................... 108Schrey, Anna .................................................................. 24, 26 Schröder, Nikolaj ................................................................. 14Schulz, Katrin ....................................................................... 50Schuster, Ariane .......................................................... 50, 152 Selfe, Joanna ....................................................................... 63 Serowy, Steffen ............................................................. 57, 76Shan, Ying ............................................................................ 66Siems, Wolf-Eberhard ........................................................ 52Singh Bal, Manjot ................................................................ 50 Söderhäll, Arvid ....................................................... 24, 25, 26 Sokolenko, Elena ................................................................. 57Sokolov, Valerij .................................................................... 57Soukenik, Michael .............................................................. 14Souza, Christina de ............................................................. 57Sperling, Birgit ................................................................... 107 Stefan, Eduard ..................................................................... 43 Steuer, Andrea ..................................................................... 10Strauss, Holger .............................................................. 16, 18 Sun, Xiaoou .......................................................................... 55Sylvester, Marc ............................................................. 32, 33

TTasadaque Ali Shah, Syed ................................................. 90 Tenz, Kareen ......................................................................... 85 Thielen, Anja ........................................................................ 40Thiemke, Katharina ................................................. 31, 32, 33 Thakur, Mina ........................................................................ 64 Tremmel, Sandra ................................................................. 74 Tsunoda, Satoshi ................................................................. 56

UUryga-Polowy, Viviane ....................................................... 90 Uschner, Michael .............................................................. 101 Utepbergenov, Darkhan ..................................................... 49

VVargas, Carolyn ................................................................... 14Venta, Nicola ........................................................................ 66Vinkemeier, Uwe .................................................................. 65 Vogelreiter, Gabriela ..................................................... 78, 82

WWaldmann, Herbert ............................................................... 2Walter, Juliane ..................................................................... 50 Weber, Viola ......................................................................... 43Weik, Steffen ........................................................................ 90Weisgraber, Karl .................................................................. 76Wendt, Stefanie ................................................................. 101Wessolowki, Axel ................................................................ 78Wichard, Jörg ...................................................................... 26

Inde

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Wiedemann, Urs ...................................................... 14, 20, 23Wiesner, Burkhard ........................................................ 46, 75Wietfeld, Doreen ................................................................. 74Wietstruck, Markus ............................................................ 60 Winkler, Lars ........................................................................ 50Wolf, Constanze ................................................................... 50Wolf, Yvonne .................................................................. 75, 78Wolff, Christian .................................................................. 100

ZZimmermann, Jürgen ................................................... 32, 33Zuleger, Nikolaj ............................................................ 50, 145

Forschungsinstitut für Molekulare Pharmakologie (FMP)Campus Berlin-BuchRobert-Rössle-Straße 1013125 BerlinTel.: +49-30-9489-2920Fax: +49-30-9489-2927www.fmp-berlin.de

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Res Report 2.Teil App - leibniz-fmp.de · duktion/Molekularer Genetik, Strukturbiologie und Chemi-scher Biologie geführt hat. Kennzeichnend für die wissenschaftliche Arbeit am FMP