PhD projects


Structural Biology of Membrane Proteins

PhD project:

Exploring structure, assembly and function of the vasopressin receptors

A joint AFM – EM – SMFS approach

Principal Investigator:

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

In collaboration with:

D. Muller, Professor of Cellular Machines, Center of Biotechnology, University of Technology Tatzberg 47-51, D-01307 Dresden, Germany; mueller|

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

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. 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 will be structurally analyzed in vitro of the reconstituted receptor using EM, AFM and SMFS. With the reconstituted GPCR being set into its native environment the membrane bilayer, we will precisely tune the functional state of the GPCR, which will be structurally investigated. This project is intended as a multidisciplinary combined approach in order to explore function/structure relationships in the vasopressin system.

This PhD project will focus on structure, assembly and function relations of the two receptors of the vasopressin system being reconstituted into the native membrane. Functional domains involved in ligand interaction will be mapped and characterized on the molecular level by microscopic approaches. Stability of the isolated receptors in a variety of environments composed of detergents and lipids and mixtures thereof will de determined by SMFS. A variety of protein reconstitution conditions will be evaluated in order to improve the reconstitution yield and to maintain the full functionality of the GPCRs. Ligand binding and complex formation of the V1BR and V2R receptors will be structurally studied by EM and AFM, while SMFS will be used to locate the ligand binding and interaction strength, and to determined the dynamic energy landscape guiding the different functional states of the receptor. 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:

In summary this project implies various activities:

- Molecular biology – protein biochemistry (V. Dötsch, 4 months)

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. The V2R receptor can be concentrated up to millimolar concentrations sufficient for reconstitution into the lipid membrane and structural analysis by EM and AFM. Using our cell free expression system we will further provide samples of the full length human vasopressin receptors V1BR and V2R receptor as well as truncated forms of the receptor solubilized in different detergents. These samples will be used for reconstitution experiments into protein liposomes that are necessary for the EM, AFM and SMFS studies. Ligand binding of the individual fragments will be analyzed by surface plasmon resonance experiments.

- Reconstitution and EM of human vasopressin receptors V1BR and V2R in membranes (A. Engel’s group, 12 months)

The receptors will reconstituted in lipid bilayers (N-terminal domain and entire protein). Reconstitution will be achieved by reducing the detergent concentration below the CMC (either by dilution, adsorption to BioBeads or cyclodextrin, or by dialysis). To optimize the conditions, different lipid compositions, pH and salts will be explored. Reconstituted receptors will be analysed by electron microscopy of negatively stained solubilized receptors and by EM of freeze-fractured, metal shadowed samples. Even relatively low resolution structures should be sufficient to distinguish between oligomeric state of the receptors. The effect of ligand interaction on the formation of GPCR complexes will be assessed by measuring oligomerization under different conditions by blue native gel electrophoresis, and by EM of negatively stained samples. For conditions that prove to stabilize the receptor structure, 2D crystallization will be explored and assessed by EM of negatively stained samples and by electron diffraction.

- AFM and SMFS characterization of vasopressin receptors in membranes (D. Muller’s group, 20 months)

AFM will be applied to image the oligomeric state of densely packed vasopressin receptors. High-resolution AFM images will enable to resolve details of the receptors surface being set into different functional states. This will reveal first insights into the conformational changes being associated with the activation of the GPCR. SMFS provides a quantitative view of molecular interactions establishing the energetic landscape of membrane proteins.  Here, we will analyze the molecular interactions of the vasopressin receptor as a function of the physiological environment (lipids, pH, electrolyte) and the homo- and heterooligomeric states. Having mapped the vasopressin receptor’s primary structure with these interactions we will then investigate how they change upon binding to pharmacological compounds targeting the receptor. Using dynamic force spectroscopy (DFS) we will measure the interactions at different time scales, which will allow reconstructing the energy landscape of the receptor before and after binding to other physiologically relevant molecules. This will allow drawing a detailed picture of the functional mechanisms of the vasopressin receptor.

Selected Publications:

  1. 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).
  2. 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).
  3. 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).
  4. Trbovic, N., Klammt, C., Koglin, A., Löhr, F., Bernhard, F., and Dötsch, V.
    Efficient strategy for the rapid backbone assignment of membrane proteins. J. Am. Chem. Soc., 127, 13504-13505 (2005).
  5. Muller DJ, et al.,
    Single-molecule studies of membrane proteins. Curr Opin Struc Biol (2006).
  6. Kedrov A, et al.,
    Deciphering molecular interactions of native membrane proteins by single-molecule force spectroscopy. Annu Rev Biophys Biomol Struct (2007).
  7. P.S.-H. Park, K.T. Sapra, M. Koliński, S. Filipek, K. Palczewski & D.J. Müller,
    Stabilizing effect of Zn2+ in native bovine rhodopsin. Journal of Biological Chemistry 282, 11377-11385 (2007).
  8. T. Sapra, P.S.H. Park, A. Engel, S. Filipek, D.J. Müller & K. Palzcewski,
    Detecting molecular interactions that stabilize native bovine rhodopsin. Journal of Molecular Biology 358, 255-269 (2006).
  9. D. Fotiadis, B. Jastrzebska, A. Philippsen, D.J. Müller, K. Palczewski & A. Engel,
    Structure of the rhodopsin dimer: A working model for G-protein coupled receptors. Current Opinion in Structural Biology 16, 252-259 (2006).
  10. Fotiadis D, Liang Y, Filipek S, Saperstein DA, Engel A, Palczewski K.
    Atomic-force microscopy: Rhodopsin dimers in native disc membranes. Nature 421, 127 (2003).
  11. Murata K, Mitsuoka K, Hirai T, Walz T, Agre P, et al.
    Structural determinants of water permeation through aquaporin-1. Nature 407: 599 (2000).
  12. Remigy HW, Caujolle-Bert D, Suda K, Schenk A, Chami M, Engel A.
    Membrane protein reconstitution and crystallization by controlled dilution. FEBS Lett 555, 160 (2003).
  13. Signorell GA, Kaufmann TC, Kukulski W, Engel A, Remigy HW.
    Controlled 2D crystallization of membrane proteins using methyl-beta-cyclodextrin. J Struct Biol 157, 321 (2007).