Zusammenspiel von molekularen Konformationen und biologischer Funktion / Interplay between Molecular Conformations and Biological Function

Bewilligungen / Grants 2006  

Synthetic selectivity filters for porin-like ion channels

Bewilligung: 05.04.2006 Laufzeit: 2 Jahre

The project aims to turn the broad pore of a porin-like ion channel (OmpF) into a selective ion channel by incorporation of synthetic selectivity filters. Towards this end, OmpF hybrids with artificial selectivity filters being linked to one or two attachment sites of the protein portion will be synthesized by a combination of solid-phase synthesis, recombinant methods, chemical ligation and protein splicing. The azide-alkyne [3+2] cycloaddition and sulfide linkages will be used for the covalent attachment and positioning of the selectivity filter in the pore interior. The orientation and local conformtion of the selectivity filter, which is crucial for its function, will be studied by X-ray crystallography.

Universität Marburg
Fachbereich Chemie
Bioorganische Chemie
Professor Dr. Ulrich Koert

Universität Marburg
Fachbereich Chemie
Biochemie
Professor Dr. Lars-Oliver Essen

Universität Dortmund
Fachbereich Chemie
Chemische Biologie
Professor Dr. Henning D. Mootz

Ansprechpartner:
Universität Marburg
Fachbereich Chemie
Bioorganische Chemie
Professor Dr. Ulrich Koert
Postfach
35032 Marburg
Tel.: 06421 282 6970
Fax: 06421 282 5677
Homepage: http://www.chemie.uni-marburg.de

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Conformation-activity relationship of the archazolids: Development of a novel class of highly potent V-ATPase inhibitors

Bewilligung: 05.04.2006 Laufzeit: 1 Jahr

Das Vorhaben wurde am  19.11.2007 von Göttingen nach Heidelberg umgesetzt.

Vacuolar type ATPases (V-ATPases) are heteromultimeric, proton translocating proteins which are localized in a multitude of eukaryotic membranes and energize many different transport processes. Their malfunction is correlated with various diseases such as renal acidosis or cancer. The polyketide natural products archazolid A and B constitute novel types of particularly efficient (IC50 in the low nanomolar range) and specific inhibitors of V-ATPases, both in vitro and in vivo. Their unique structures have not been synthetically attained, nor has the conformation of the archazolids been studied.
This project aims to analyse in detail the interdependence of conformation and biological function of these potent natural enzyme inhibitors. Of particular interest will be the elucidation of the 3D-structure in solution and of the bioactive conformation, the energetic and structural correlation between these two states and the interplay between conformation, configuration and activity. In an initial pilot project, a proof of principle shall be obtained by which the conformation of the archazolids and the time-scale of ligand binding are resolved.

EMBL - Europäisches Laboratorium
für Molekularbiologie, Heidelberg
Biomolecular NMR Spectroscopy
Dr. Teresa Carlomagno
Meyerhoferstraße 1
69117 Heidelberg

Helmholtz-Zentrum für
Infektionsforschung GmbH, Braunschweig
Abt. Medizinische Chemie
Dr. Dirk Menche
Inhoffenstraße 7
38124 Braunschweig
Tel.: 0531 6181 9407
Fax: 0531 6181 795

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Elucidation of the conformational dynamics of the spliceosome using small molecule inhibitors

Bewilligung: 03.04.2006 Laufzeit: 2 Jahre

Pre-mRNA splicing is carried out by an elaborate ribonucleoprotein (RNP) complex, the spliceosome, which is assembled anew on each pre-mRNA intron by the stepwise interaction of snRNPs and multiple non-snRNP splicing factors. During its maturation into a catalytically active RNP, the spliceosome is repeatedly remodeled, undergoing multiple compositional and conformational changes. At present, only a limited number of spliceosome assembly/maturation intermediates can be distinguished, and they appear to represent a mixture of RNP conformational states. The specific inhibition of spliceosomal proteins that facilitate conformational changes in RNA and/or protein (e.g., spliceosomal helicases, PPIases or kinases) may enable the isolation of novel, conformationally homogeneous maturation intermediates of the spliceosome. It is thus planned to exploit small molecule inhibitors targeted at a particular enzyme to stall human spliceosomes at defined maturation states. Inhibitors will be identified using medium-throughput splicing or enzymatic assays, and rationally improved via crystal structure analysis of enzymes alone or complexed with inhibitors. 3D-structure probing of purified novel intermediates will be performed with pre-mRNA or snRNAs site-specifically labeled with chemical or fluorescent probes.

Max-Planck-Institut für biophysikalische Chemie
Abt. Zelluläre Biochemie
Professor Dr. Reinhard Lührmann

Max-Planck-Institut für biophysikalische Chemie
Abteilung Zelluläre Biochemie
Priv.-Doz. Dr. Markus Wahl

Universität Dortmund
FB Chemie
Chemische Biologie
Professor Dr. Herbert Waldmann

Ansprechpartner:
Max-Planck-Institut für biophysikalische Chemie
Abt. Zelluläre Biochemie
Professor Dr. Reinhard Lührmann
Am Faßberg 11
37077 Göttingen
Tel.: 0551 2011 405
Fax: 0551 2011 197

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TGF-beta signalling biosensors

Bewilligung: 30.03.2006 Laufzeit: 3 Jahre

Transforming growth factor beta (TGF-beta)-type signaling underlies most tumorigenesis events and regulates embryonic development by providing positional information to cells. In recent years much has been discovered about the molecular and biochemical features of the TGF-beta signal transduction machineries, the conformational changes of the components of that machinery during signaling, the role of TGF-beta signaling in many developmental contexts, and the involvement of anomalous TGF-beta signaling in many diseases, including cancer. In contrast, investigation of the cell biology of TGF-beta signaling events has started only lately. To study the cell biology of TGF-beta signaling during development with high spatial and temporal resolution, it is proposed to establish biosensors to monitor the state of activation of different components of the TGF-beta signaling cascade. It is planned to use synthetic chemistry, protein/lipid conformation and TGF-beta signaling in fly, frog and fish embryos and to apply BiFC and FRET which have been used previously to study other signaling pathways. The goal is to establish a panel of reagents that will allow one to follow the cascade of signaling events in real time. Finally, the results will be transferred to study TGF-beta signaling during development and in disease.

Max-Planck-Institut für molekulare Zellbiologie und Genetik
Dr. Marcos A. González-Gaitán

University of Cambridge
Wellcome Trust/Cancer Research UK
Gurdon Institute
Professor Dr. James

Smith EMBL - Europäisches Laboratorium für Molekularbiologie
Gene Expression Unit
Dr. Carsten Schultz

Ansprechpartner:
Max-Planck-Institut für molekulare Zellbiologie und Genetik
Dr. Marcos A. González-Gaitán
Pfotenhauerstraße 108
01307 Dresden
Tel.: 0351 210 2539
Fax: 0351 210 1389

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Information transmission pathways in an allosteric protein

Bewilligung: 30.03.2006 Laufzeit: 3 Jahre

Many cellular processes require allosteric proteins which contain at least two spatially separated substrate or effector binding sites with interdependent activities. Despite a large body of crystal structures there is no general knowledge about the mechanics and energetics of information transmission within such proteins. It is proposed to establish such principles from the molecular analysis of information transmission in Tet repressor (TetR), a well described tetracycline (tc) dependent regulator of transcription. TetR variants with different allostery and a peptide inducer, triggering a different structural change than tc, have already been isolated. It will be determined if there are more mechanisms of information transmission for TetR and how these depend on the chemical structure of the effector. Residues essential for transmitting information protein-internally between the binding sites will be identified by alanine scanning. The synthesis of peptidomimetics, hybrid compounds derived from the inducing peptide and tc and novel tetracycline derivates will be established and the compounds used to study their mechanism of induction. Crystal structures from all significant novel complexes will be solved to describe the propagation of information in molecular detail.

Universität Erlangen-Nürnberg
Institut für Biologie
Lehrstuhl für Mikrobiologie
Professor Dr. Wolfgang Hillen

Universität Erlangen-Nürnberg
Institut für Biologie
Lehrstuhl für Biotechnik
Professor Dr. Yves Muller
Universität Erlangen-Nürnberg
Institut für Pharmazie und Lebensmittelchemie
Lehrstuhl für Pharmazeutische Chemie
Professor Dr. Peter Gmeiner

Ansprechpartner: 
Universität Erlangen-Nürnberg
Institut für Biologie
Lehrstuhl für Mikrobiologie
Professor Dr. Wolfgang Hillen
Staudtstr. 5
91058 Erlangen
Tel.: 09131 8528 081
Fax: 09131 8528 082

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Substrate control of the active conformation of the respiratory complex I

Bewilligung: 30.03.2006 Laufzeit: 3 Jahre

The NADH:ubiquinone oxidoreductase, also called respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Its mechanism is largely unknown due to its complicated composition. Its dysfunction is connected to many neurodegenerative diseases such as Parkinson's disease. NADH- but not NADPH-binding induces long-range conformational changes extending to more than 190 Å. They lead to the opening of the ubiquinone binding site inducing the active conformation of the complex. In this project the aim is to determine the modulation of the active conformation by nucleotide-binding. New nucleotides will be synthesized with modifications of the base, the base sugar and the nicotinamide ring, as well as photoreactive derivates. The conformational changes induced by the binding of these derivatives will be determined by means of electron microscopy, CD and FTIR difference spectroscopy. Interactions between the substrate and its binding site leading to different conformations will be validated using site-directed mutants.

Universität Freiburg
Institut für Organische Chemie und Biochemie
Professor Dr. Thorsten Friedrich

Universität Freiburg
Institut für Organische Chemie und Biochemie
Professor Dr. Bernhard Breit
Universität Louis Pasteur,
Strasbourg Faculté de chimie
Chimi physique moléculaire et spectroscopie
Professor Dr. Petra Hellwig

Ansprechpartner:
Universität Freiburg
Institut für Organische Chemie und Biochemie
Professor Dr. Thorsten Friedrich
Albertstraße 21
79014 Freiburg
Tel.: 0761 203 6060
Fax: 0761 203 6096

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Pleckstrin domains: from allosteric regulation of protein function towards novel tools for monitoring intracellular reactions (Weiterführung)

Bewilligung: 30.03.2006 Laufzeit: 2 Jahre

Pleckstrin homology (PH) domains play a major role in lipid-mediated protein translocation and signaling events in proteins. Pleckstrin itself becomes activated upon phosphorylation by protein kinase C (PKC). During the previous funding period a FRET-based sensor (KCP-1) was developed that was able to monitor PKC activity in living cells. However, the structural basis for the conformational alterations linked to the FRET changes still needs to be demonstrated. In order to optimize KCP-1 for NMR-studies and its perfomance in cells, a reduction in size and an improvement of the FRET change to above 100% needs to be achieved. The dual parameter FRET probe KCAP-1 that permits the independent recording of PKC- and PKA-induced phosphorylation in cells opens the way to an entire platform of reporters useful for the intracellular dissection of signaling pathways and for screening. Efforts shall be continued to prepare fluorescently labeled, membran-permeant phosphoinositide derivates to enable monitoring of phospholopid-PH domain interaction by FRET. Furthermore, photoactivatable derivates of phosphoinositide will be added to the toolbox. The interaction of fluorescently labeled phosphoinositide with cytoskeletal PH domains from the Rho-GEF obscurin will be studied by using NMR and FRET.

EMBL - Europäisches Laboratorium für Molekularbiologie
Gene Expression Unit
Dr. Carsten Schultz

EMBL - Europäisches Laboratorium für Molekularbiologie
Computational & Structural Biology Unit
Dr. Michael Sattler

King's College
GKT School of Medicine
Cardiovascular Division
Professor Dr. Mathias Gautel

Ansprechpartner: 
EMBL - Europäisches Laboratorium für Molekularbiologie
Gene Expression Unit
Dr. Carsten Schultz
Postfach 10 22 09
69012 Heidelberg
Tel.: 06221 387 210
Fax: 06221 387 206

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Modulation of the slow conformational dynamics in Ras and Ras-related proteins by drugs: development of a new type of specific Ras-inhibitors (Weiterführung)

Bewilligung: 30.03.2006 Laufzeit: 2 Jahre

In the first funding period, different metal cyclen complexes were characterized which can serve as lead compounds shifting the conformational equilibrium from the effector-binding state of Ras to its weak binding state. The influence of these compounds on the conformational equilibrium was studied by 31P-NMR spectroscopy on appropriate effector loop Ras-mutants. ITC experiments and fluorescence spectroscopy showed that these compounds are able to inhibit the binding of the effector Raf-kinase to activated Ras. The structure of the complex of metal-cyclen bound to activated Ras was solved by NMR-spectroscopy combined with MD. The new data provide the basis for the synthesis of new compounds with high affinity for oncogenic variants of Ras. Structure-based design and high-throughput screening combined with SAR-by-NMR will be used in parallel. The influence of these newly synthesized compounds on the slow and fast dynamics of Ras alone and in its complexes with the effector proteins Raf and RalGDS will be studied by heteronuclear NMR-relaxation experiments and high-pressure NMR spectroscopy. The local and global dynamics data on Ras will then be correlated with the local thermodynamical entropic contribution defined by ITC measurements combined with pairwise alanine screening methods.

Universität Regensburg
Institut für Biophysik und physikalische Biochemie
Professor Dr. Hans Kalbitzer

Universität Regensburg
Institut für Organische Chemie Professor
Dr. Burkhard König

Universität Bochum
Fakultät für Chemie
Physikalische Chemie 1
Professor Dr. Christian Herrmann

Ansprechpartner:   
Universität Regensburg
Institut für Biophysik und physikalische Biochemie
Professor Dr. Hans Kalbitzer
Universitätsstraße 31
93053 Regensburg
Tel.: 0941 943 2595
Fax: 0941 943 2479