PhD projects


Structural Biology of Membrane Proteins

PhD project:

Determination of structure/function relation in vasopressin receptors

A combined approach of NMR and X-ray crystallography

Principal Investigator:

V. Dötsch/ F. Bernhard, Centre for Biomolecular Magnetic Resonance, Institute of Biophysical Chemistry, University of Frankfurt; vdoetsch|; fbern|

In collaboration with:

E. Pebay-Peyroula, Inst de Biol. Struct. UMR5075 CEA-CNRS-Univ. J.Fourier, 41, rue Jules Horowitz, 38027 Grenoble cedex 1; eva.pebay-peyroula|

J.L. Popot, C.N.R.S./Université Paris-7 UMR 7099, Institut de Biologie Physico-Chimique, 13, rue Pierre-et-Marie-Curie, F-75005 PARIS; Jean-Luc.Popot|

In mammals, vasopressin is an essential antidiuretic peptide hormone that regulates water excretion from the kidney by increasing the osmotic water permeability of the renal collecting duct. The hormone is also a key regulator of blood pressure, smooth muscle contraction, platelet aggregation, hepatic glycogenolysis and uterine motility. Furthermore, in brain vasopressin may act as neurotransmitter in various physiological responses like thermoregulation and cardiovascular homeostasis or in modulations of learning and memory. Those pleiotropic effects of vasopressin are mediated via activation of the three different vasopressin receptor subtypes V1AR, V1BR and V2R. The receptors belong to the superfamily of G-protein coupled receptors (GPCRs) having common structural elements and a seven transmembrane topology. The classification of the vasopressin receptors is based on differences in their coupling to distinct second messenger cascades and on their pharmacological profiles for a variety of vasopressin compounds. The vasopressin receptors are involved in a variety of human diseases like congestive heart failure, nephrogenic diabetes insipidus or liver cirrhosis.

Up to now, the functional characterization of the vasopressin receptors has only extensively been studied in vivo in several mammalian species. In vitro studies and structural approaches have mostly been prevented by the limited availability of the receptors in conventional overproduction systems based on living cells. The high-level production of GPCRs in our recently developed cell-free expression system opens completely new avenues for their functional and structural characterization. Functional activities like ligand binding and G-protein coupling can be analysed in vitro in different environments in well defined systems. Furthermore, the efficient and unique labeling strategies based on cell-free expression makes the GPCRs suitable for structural approaches by NMR and X-ray analysis. This project is intended as a multidisciplinary combined approach in order to explore function/structure relationships in the vasopressin system.

The two human vasopressin receptors V1BR and V2R can be produced at high amounts by using cell-free expression systems developed at the institute of Biophysical Chemistry in Frankfurt. Biochemical approaches and single particle analysis revealed homodimer formation and high quality of the samples. The proteins can be efficiently labeled with stable isotopes and specifically labeled samples suitable for NMR analysis can be obtained in less than two days. The V2R receptor can be concentrated up to millimolar concentrations sufficient for NMR analysis. The institute is equipped with a series of high end NMR spectrometers providing ideal facilities for the structural studies of membrane proteins. The group has already intensive experience with the NMR analysis of integral α-helical membrane proteins and in particular with GPCRs.

The group of E. Pebay-Peyroula in Grenoble is a leading expert in the three-dimensional crystallization of membrane proteins. A variety of self-developed screening systems for the production of high-quality crystals have been developed. Core projects of this group comprise the structural analysis of GPCRs and a number of complementary approaches for the growth and handling of high quality crystals have been developed.

The group of J.L.Popot has exceptional experience with the synthesis and characterization of new and modified detergents and derivatives suitable for membrane protein studies. A recently developed series of amphipols shows outstanding properties in the stabilization and solubilization of membrane proteins.

This PhD project will focus on structure/function relations of the two receptors of the vasopressin system. Functional domains involved in ligand interaction will be mapped and characterized on the molecular level by molecular and biochemical approaches. Exisiting libraries of peptidic and nonpeptidic selective vasopressin agonists and antagonists will be used. Stability of the isolated receptors in a variety of environments composed of detergents and lipids and mixtures thereof will de determined. Fragmentation approaches in combination with functional analysis will be implemented in order to define optimal conditions and targets suitable for NMR analysis. A variety of protein solubilization conditions including mixed micelles, bicelles and novel detergents like amphipols will be evaluated in order to improve the spectral dispersion of the GPCRs and their derivatives in NMR measurements. Ligand binding studies and complex formation of isolated transmembrane spanning fragments of the V1BR and V2R receptors will be studied by NMR titration assays and complemented by biochemical techniques. Crystallization screens of the purified receptors will include samples that have been cell-free synthesized in a variety of different conditions including coexpression with inhibitors and other ligands for stabilization. Approaches to improve potential crystal contacts by increasing the hydrophilic surface of the receptors will further be considered. The project is intended to provide an excessive multidisciplinary approach for the structural evaluation of GPCRs. In order to gain maximal expertise and technical know-how, the project will include three participating labs:

- Solution NMR analysis – functional analysis (V. Dötsch/F. Bernhard, 24 months)

Ligand binding properties and oligomeric complex formation of the two receptors will be analysed by an array of biochemical techniques. Systematic terminal fragmentation in combination with functional analysis will provide a number of targets for NMR analysis. V1BR and V2R derivatives suitable for structural analysis will be selected based on their spectral dispersion properties. Established combinatorial labeling approaches will be employed for the rapid assignment of GPCR derivatives and isolated domains. The interaction of receptor fragments and domains with other partners will be analysed on the molecular level by NMR titration experiments. Efficient purification protocols for functionally folded and monodisperse samples will be established. Concentration and stability of the proteins in suitable hydrophobic environments will be assessed and samples at optimal conditions will be generated for crystallization screens.

- Solubilization of GPCRs in amphipols (J.L. Popot, 6 months)

Recently developed amphipols provide a new and unique environment for the solubilization of membrane proteins. The impact of amphipols on the functional folding and complex formation of the two vasopressin receptors will be analysed. Suitability of amphipols for the cell-free production of GPCRs will be analysed.

- Crystallization and X-ray analysis (E. Pebay-Peyroula, 6 months

Crystallization of the purified receptors will be analysed in a variety of different screens and conditions. Besides full-length proteins, truncated and modified derivatives will further be analysed for crystal formation. Sample quality will continuously be improved in order to obtain high quality crystals.

Selected Publications:

  1. Klammt, C., Srivastava, A., Eifler, N., Junge, F., Beyermann, M., Schwarz, D., Michel, H., Doetsch, V., Bernhard, F. (2007)
    Functional analysis of cell-free-produced human endothelin B receptor reveals transmembrane segment 1 as an essential area for ET-1 binding and homodimer formation. FEBS J., 274, 3257-69.
  2. Klammt, C., Schwarz, D, Löhr, F., Schneider, B., Dötsch, V., Bernhard, F. (2006)
    Preparative scale cell-free expression systems: New tools for the large scale preparation of integral membrane proteins for functional and structural studies. FEBS J., 273, 4141-53.
  3. Nury, H., Dahout-Gonzalez, C., Trézéguet, V., Lauquin, G., Brandolin, G., Pebay-Peyroula, E. (2006).
    Relations between structure and function of the mitochondrial ADP/ATP carrier. Annual Review of Biochemistry, 75, 713-741.
  4. Pocanschi, C. L., Dahmane, T., Gohon, Y., Rappaport, F., Apell, H.-J., Kleinschmidt, J. H., Popot, J.-L. (2006).
    Amphipathic polymers: tools to fold integral membrane proteins to their active form. Biochemistry, 45, 13954-13961.
  5. Klammt, C., Schwarz, D., Fendler, K., Haase, W., Dötsch, V., Bernhard, F. (2005)
    Evaluation of detergents for the soluble expression of α-helical and B-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system. FEBS J., 272, 6024-38.
  6. Trbovic, N., Klammt, C., Koglin, A., Löhr, F., Bernhard, F., and Dötsch, V. (2005)
    Efficient strategy for the rapid backbone assignment of membrane proteins. J. Am. Chem. Soc., 127, 13504-13505.
  7. Pebay-Peyroula, E., Dahout-Gonzalez, C., Kahn, R., Trézéguet, V., Lauquin, G.J.-M., Brandolin, G. (2003)
    Structure of mitochondrial ADP/AP carrier in complex with carboxyatractyloside. Nature, 426, 39-44.
  8. Popot, J.-L., Berry, E. A., Charvolin, D., Creuzenet, C., Ebel, C., Engelman, D. M., Flötenmeyer, M., Giusti, F., Gohon, Y., Hervé, P., Hong, Q., Lakey, J. H., Leonard, K., Shuman, H. A., Timmins, P., Warschawski, D. E., Zito, F., Zoonens, M., Pucci, B., Tribet, C. (2003)
    Amphipols: polymeric surfactants for membrane biology research. Cell. Mol. Life Sci., 60, 1559-1574.
  9. Royant, A., Nollert, P., Edman, K., Neutze, R., Landau, E.M., Pebay-Peyroula, E., Navarro, J. (2001)
    X-ray structure of sensory rhodopsin II at 2.1 Å resolution. Proc. Natl. Acad. Sci. USA, 98, 10131-10136.
  10. Tribet, C., Audebert, R., Popot, J.-L. (1996)
    Amphipols: polymers that keep membrane proteins soluble in aqueous solutions. Proc. Natl. Acad. Sci. USA, 93, 15047-15050.