PhD projects


Structural Biology of Membrane Proteins

PhD project:

Functional and molecular characterization of GPCRs:

Synergies of biochemical and structural techniques

Principal Investigator:

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

In collaboration with:

A. Engel, M.E. Müller Institute for Microscopy, Biozentrum, University of Basel, 4056 Basel, Switzerland; andreas.engel|

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

The hormone melatonin modulates a diverse number of physiological processes including circadian entrainment, blood pressure regulation, oncogenesis, retinal physiology, seasonal reproduction and osteoblast differentiation. Two melatonin receptors, MT1 and MT2, belonging to the superfamily of G-protein coupled receptors (GPCRs) have been identified in eukaryotic cells. Both receptors can couple to multiple and distinct signal transduction cascades, revealing a highly complex regulation system. A variety of G-proteins including Gi and Gα subtypes have been identified as coupling partners. A number of amino acid residues located in the MT1 and MT2 transmembrane regions are proposed to be involved in melatonin binding. Parts of the transmembrane helices 3, 4, 6 and 7 are suspected to form a melatonin binding pocket while molecular or structural details are still unclear. We will address the molecular characterization of the MT1 and MT2 receptors by new in vitro approaches based on the efficient synthesis of the full-length receptors and modified derivatives. Ligand binding and complex formation of the two receptors will be studied in defined in vitro systems by a variety of complementary biochemical approaches. The binding mechanisms of melatonin as well as of antagonists and of MT2 selective ligands will be characterized. Signal transduction and coupling to specific G-proteins will be analysed by a recently developed translational fusion approach. Characterization of oligomeric association of the two receptors will be complemented by single particle analysis in the group of A. Engel. Crystallization and structural evaluation of the two receptors by X-ray analysis will be addressed in the group of E. Pebay-Peyroula.

The institute of Biophysical Chemistry at the University of Frankfurt has established highly efficient cell-free expression systems for the rapid production of even very large membrane proteins. Different modes of expression are possible and the proteins can be produced in soluble form in presence of detergents. Protocols for the high level synthesis of the two human melatonin receptors MT1 and MT2 have already been developed. Both GPCRs can be produced in amounts of 2-4 mg per one ml of reaction in less than 24 hours. A diverse variety of biochemical approaches in order to evaluate quality and functional folding of the cell-free produced proteins has been developed. Furthermore, ligand interactions of GPCRs can be characterized by leading technologies like surface plasmon resonance, scintillation proximity or NMR titration assays.

The group of A. Engel has outstanding expertise in the efficient reconstitution of membrane proteins into lipid bilayers. Furthermore, the group has accumulated exceptional knowledge in the structural analysis of membrane proteins in different hydrophobic environments by electron microscopy (EM).

The group of E. Pebay-Peyroula is highly experienced in the crystallization and X-ray analysis of membrane proteins. Their knowledge will provide clear guidelines for sample preparation at optimal qualities and in suitable environments.

Central topics of this PhD project will be the detailed analysis of molecular mechanisms in signal perception and transduction within the melatonin system. Interaction of the individual receptors with different natural and artificial agonists and antagonists and mapping of the ligand binding sites will be a major issue. In addition, topologies and oligomeric association of the receptors in homo-and heteromeric complexes will be studied. Impact and effects of ligand interaction and variations in the hydrophobic environments on the functional conformation of the receptors will be of special interest. Receptor oligomer formation and identification of protein interfaces will be characterized by single particle analysis and biochemical approaches. The structural analysis of the melatonin receptors will focus on crystallization and X-ray analysis. An interactive research and training program will be emphasized in this project.

- Molecular biology – protein biochemistry (F. Bernhard/V. Dötsch, 24 months)

Based on the rapid production and modification of protein samples by our cell-free systems, the interaction, ligand binding properties and G-protein coupling of the MT1 and MT2 receptors will be studied. Functional domains of the receptors will be confined and analysed by genetic engineering techniques involving directed mutagenesis, fragmentation and construction of chimeric receptors. Cell-free production protocols will further be optimized by established robotic screening processes in order to produce GPCR samples at highest possible qualities for crystallization purposes.

- EM characterisation of melatonin receptors in membranes (A. Engel, 6 months)

Receptors will be efficiently reconstituted in lipid bilayers of different compositions. Oligomeric associations of the receptors in different detergent/lipid environments will be analysed by electron microscopy. The effect of ligand interaction on the formation of GPCR complexes will be analysed.

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

Crystallization screens of purified receptors samples will be set up at different conditions. Stability and ligand bind activity of the proteins in different detergent environments will be analysed and crystallization screens will be modified accordingly. Growth conditions of initial crystals will be refined for optimal diffraction properties.

Selected Publications:

  1. Schwarz, D., Klammt, C., Koglin, A., Löhr, F., Schneider, B., Dötsch, V., and Bernhard F. (2007).
    Preparative scale cell-free expression systems: New tools for the large scale preparation of integral membrane proteins for functional and structural studies. Methods, 41, 355-369.
  2. Klammt, C., Schwarz, D., Eifler, N., Engel, A., Piehler, J., Haase, W., Hahn, S., Dötsch, V., and Bernhard, F. (2007)
    Cell-free production of G-protein coupled receptors for functional and structural studies. J. Struct. Biol., 158, 482-493.
  3. Klammt, C., Schwarz, D., Dötsch, V., and Bernhard, F. (2006).
    Cell-free production of integral membrane proteins on a preparative scale. Meth. Mol. Biol., 375, 57-78.
  4. Klammt, C., Löhr, F., Schäfer, B., Haase, W., Dötsch, V., Rüterjans, H., Glaubitz, C., and Bernhard, F. (2004).
    High level cell-free expression and specific labeling of integral membrane proteins. Eur. J. Biochem., 271, 568-580.
  5. Nury, H., Dahout-Gonzalez, C., Trézéguet, V., Lauquin, G., Brandolin, G., Pebay-Peyroula, E. (2006).
  6. Relations between structure and function of the mitochondrial ADP/ATP carrier. Annual Review of Biochemistry, 75, 713-741.
  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. 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.