PhD projects


Structural Biology of Membrane Proteins

PhD project:

GPCRs & their G proteins reconstituted in planar membranes: A generic platform to investigate receptor mediated signalling

Principal Investigator:

H. Vogel, Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL); horst.vogel|

In collaboration with:

F. Bernhard/V. Doetsch, Institute of Biophysical Chemistry, Johann Wolfgang Goethe-University of Frankfurt, D-60438 Frankfurt/Main; fbern|, vdoetsch|

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|

The PhD project will focus on the in vitro expression of several GPCRs and their G proteins, labeling of the proteins with suitable fluorescent probes, the reconstitution of the receptors and their G proteins into planar membranes and finally the monitoring of the activation of the receptors and the subsequent interaction with their G proteins by single molecule imaging in order to quantify the details of these central signalling reactions. We will establish the planar membrane system as a generic platform for investigating GPCR mediated cellular signalling. The project concentrates on the following GPCRs: (i) Vasopressin receptors, VR1 &VR2, a system on which the Frankfurt group has already gained experience; (ii) OR17-40, a prototypical human odorant receptor, which has been expressed recombinantly in HEK cells and investigated in live cells by the EPFL group. Odorant receptors are still very difficult to express efficiently in recombinant systems for yet unknown reasons. Therefore, the combination of in vitro expression and reconstitution in planar membranes will be of central importance not only for elucidating molecular details of OR activation but also eventually to create artificial sensor systems based on native olfactory receptors.

The group of H. Vogel has long-standing experience to investigate the structural dynamics of membrane proteins in general and GPCRs in particular using different biophysical techniques. Of central importance in the present project are the novel techniques to label membrane proteins site-selectively suitable for single molecule spectroscopies and fluorescence microscopies (Guignet 2004; Lill 2005; Meyer 2006a, b; Jacquier 2006; Prummer 2006).

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 (Pocanschi 2006; Zoonens 2005; Popot 2003).

The group of F. Bernhard/V. Doetsch has achieved a breakthrough in expressing GPCRs in a cell free system suitable for investigating the function of these proteins by biophysical and structural approaches (Klammt 2005, 2006, 2007).

- Cell-free expression of wt and tagged GPCRs and their G proteins (V. Dötsch/F. Bernhard, 12 months)

V1BR, V2R and OR17-40 comprising polyhistidine sequences and fused to AGT or ACP tags as well as their G proteins, again with polyhistidine sequences will be expressed in cell free systems. Efficient expression protocols for the V1BR and V2R receptors have already been established. Optimal reaction conditions will be determined by robotic throughput screens. Different cell-free expression modes in presence of a variety of detergents or lipids will be analysed in order to result in highest protein quality. The structural and functional folding of the synthesized GPCRs will be evaluated by ligand binding assays or by a diverse number of complementary biophysical methods. Fusion proteins of the GPCRs with their cognate Galpha proteins will further be constructed and cell-free produced for the characterization of signal transfer in planar membranes.

- Solubilization of OR17-40 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 odorant receptors will be analysed. Suitability of amphipols for the cell-free production of odorant receptors will be analysed. The functionality of the OR will be tested by their potency to activate their G proteins.

- Imaging GPCR-mediated signaling in planar membranes by single molecule microscopy (H. Vogel’s group, 18 months)

At the EPFL planar membranes comprising reconstituted GPCR will be transferred to different solid supports, preferentially to silicon chips which carry one or multiple (sub)micrometer-sitzed holes and which are covered on one surface by a 100 nm film of Al-oxide (Schmidt 2000; Danelon 2006). In this configuration the chip functions as a mono-mode wave guide: Light waves addressed from one side towards the chip will create a high-intensity evanescent wave, concentrated in the holes and suited to excite fluorophores in the planar membrane assembled across these holes; fluorescent probes might arise from fluorescent ligands bound to the GPCR or from probes directly integrated in the receptor e.g. in form of GFP-fusion proteins, or labelling an ACP-tag as pioneered by the EPFL group for imaging membrane proteins, especially GPCRs (Meyer 2006 a, b; Jacquier 2006), or by labelling a GPCR mutant protein selectively and reversibly on polyhistidine sequences (Guignet 2004). Membranes, assembled on one side of the chip will be freely accessible from both sides and thus allow to integrate from the cytoplasmic receptor side a (fluorescently labelled) G protein and from the extracellular receptor side a fluorescent ligand which would be able to activate the GPCR and in turn the G protein. We will use this generic chip platform to measure simultaneously fluorescence signals arising from the GPCR, its G protein and its activating ligand in order to get information about the state of oligomerization of the signalling proteins involved and to quantify the thermodynamics and kinetics of the molecular interactions of GPCR mediated signaling by single molecule microscopy (Stanasila 2003; Lill 2005; Meyer 2006a; Perez 2006; Prummer 2006). The different GPCRs and their G proteins obtained from the Frankfurt group will be equipped with orthogonal fluorescent probes and reconstituted in the planar membrane systems. Single molecule fluorescence sectrosopy and microscopy will be used to investigate the monomer/oligomer state of the proteins as well as their mutual interactions during agonist-mediated activation of the receptors.

  1. C. Schmidt, M. Mayer and H. Vogel.
    A chip-based biosensor for functional analysis of single ion channels. Angew. Chemie Int. Ed. 39, 3137-3140 (2000).
  2. L Stanasila, J-B Perez, H Vogel and S Cotecchia.
    Oligomerization on the alpha 1 and beta-adrenergic receptor subtypes:  Potential implications in receptor internalization.  J. Biol. Chem. 278, 40239-4025 (2003).
  3. E Guignet, R Hovius and H Vogel.
    Reversible site-selective labeling of membrane proteins in live cells. Nature Biotechnol. 22, 440-444 (2004).
  4. Y Lill, KL Martinez, MA Lill, BH Meyer, H Vogel and B Hecht. Kinetics of the initial steps of G-protein coupled receptor mediated cellular signaling revealed by single molecule imaging. ChemPhysChem 6, 1633-1640 (2005).
  5. BH Meyer, JM Segura, KL Martinez, R Hovius, N George, K Johnsson, H Vogel.
    FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells.  Proc Natl Acad USA 103, 2138-2143 (2006a).
  6. BH Meyer, KL Martinez, J-M Segura, R Hovius, N George, K Johnsson and H Vogel. Covalent labeling of cell-surface proteins for in-vivo FRET studies. FEBS Lett. 580, 1654-1658 (2006b).
  7. V Jacquier, M Prummer, JM Segura, H Pick, H Vogel. Visualizing odorant receptor trafficking in living cells down to the single-molecule level. Proc. Natl. Acad. Sci. USA 103, 14325-14330 (2006).
  8. J-B Perez, J-M Segura, KL Martinez, D Abankwa and H Vogel. Monitoring the diffusion of single heterotrimeric G proteins in supported cell-membrane sheets reveals their partitioning into microdomains. J. Mol. Biol., 363, 918-930 (2006).
  9. C Danelon, J-B Perez and H Vogel.
    Cell Membranes Suspended Across Nano-Aperture Arrays. Langmuir 22, 22-25 (2006).
  10. M Prummer, BH Meyer, R Francini, J-M Segura and H Vogel.
    Posttranslational covalent labeling reveals heterogeneous mobility of individual G-protein coupled receptors in living cells. ChemBioChem 7, 908-9011 (2006).
  11. Klammt, C., Srivastava, A., Eifler, N., Junge, F., Beyermann, M., Schwarz, D., Michel, H., Doetsch, V., Bernhard, F.,
    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 (2007).
  12. Klammt, C., Schwarz, D, Löhr, F., Schneider, B., Dötsch, V., Bernhard, F.,
    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 (2006).
  13. Klammt, C., Schwarz, D., Fendler, K., Haase, W., Dötsch, V., Bernhard, F.
    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 (2005).
  14. CL Pocanschi, T Dahmane, Y Gohon, F Rappoport, HJ Apell, JH Kleinschmidt, JL Popot.
    Amphipathic polymers: Tools to fold integral embrane proteins to their active form.  Biochemistry 45, 13954-13961 (2006).
  15. M Zoonens, LJ Catoici, JL Popot.
    NMR study of a membrane protein in detergent-free aqueous solution. Proc Natl Acad Sci USA 102, 8893-8898 (2005)
  16. JL Popot et al: Amphipols.
    Polymeric surfactants for membrane biology research. Cell Mol Life Sci 60, 1559-1574 (2003).