PhD projects


Structural Biology of Membrane Proteins

PhD project:

Exploring structure, assembly and function of the endothelin receptor:

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|

Rapid advances in the understanding of endothelin as a naturally occurring peptide with developmental and regulatory roles in normal physiology, along with a number of deleterious effects under pathologic conditions (including vasoconstriction, fibrosis, vascular hypertrophy, and inflammation) have led to the development of endothelin-receptor antagonists (ERAs). Endothelin binds to two receptor subtypes, endothelin receptor type A (EDNRA, also known as ETA) and endothelin receptor type B (EDNRB, also known as ETB), and stimulates the generation of local mediators of vascular tone, such as nitric oxide, prostacyclins, and platelet-activating factors. These factors modulate the effects of endothelin in the cardiovascular system because of their opposing vasorelaxant action. Oligomerization of receptors has received much attention over the past several years. The consequences arising from oligomeric arrangements of receptors are proposed to play a central role in signal transduction. Except for a few cases, many of the recent studies investigating receptor oligomerization have involved heterologous expression systems of native and modified receptors. Heterodimeric and homodimeric receptors show different functions and consequently the receptors are modulated differently by drugs. However, the functional significant structure and assembly of the endothelin receptors could not be studied so far. Thus, in this consortium we will focus on studying the structure and assembly of the endothelin receptors in physiological conditions. Furthermore we will characterize the molecular interactions between and within endothelin receptors and its ligands and how these interactions modulate their functional state. Novel experimental setup will reveal the energy landscape of the receptors being set into different functional states. Since the energy landscape describes the pathways of biochemical reactions we will characterize how this landscape changes dynamically upon hetero- or homooligomerization and upon drug binding.

This PhD project will focus on the structure, assembly and function of the Endothelin B receptor. The project is intended to provide an excessive multidisciplinary approach for the structural evaluation. In order to gain maximal expertise and technical know-how, the project will include three participating labs:

The group of V. Dötsch/F. Bernhard has recently established a cell-free expression system that is capable of producing large amounts of prokaryotic and eukaryotic membrane proteins either as a precipitate or in a micelle-solubilized form by adding detergent directly to the reaction mixture. In this system the Endothelin B receptor can routinely be expressed to levels of 2-3 mg per ml of reaction mixture. In cooperation with the research groups of A. Engel and D. Müller attempts to identify the transmembrane α-helices of Endothelin B that are responsible for receptor dimerization as well as ligand binding. The energetic coupling between the individual transmembrane α-helices and its modulation by dimerization and ligand binding will be at the center of the proposed three-way collaboration.

The group of A. Engel has developed different methods to study structure and function of membrane proteins. Highly ordered 2D crystals of AQP1 were produced that led to the first structure of a human membrane channel. The group has explored different approaches to improve 2D crystallization of membrane proteins, and has analyzed the crystals by electron microscopy (EM) and atomic force microscopy (AFM). The latter method was used to assess the organization of rhodopsin in the native disk membranes of the retinal rod cells.

The group of D. Muller has recently developed efficient approaches to detect and to map the molecular interactions of native membrane proteins by single-molecule force spectroscopy (SMFS).  The technique developed allows observing the dynamic change of these molecular interactions upon oligomerization and changes of the physiological environment. Furthermore it was shown, that SMFS can detect and locate precisely the binding of molecular compounds to the membrane protein structure. Extending this approach by dynamic force microscopy allows to record the energy landscape of the membrane protein and to understand in detail the reaction pathways of the membrane protein function.

In summary this project implies various activities:

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

Using our cell free expression system we will provide samples of the full length Endothelin 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.

- EM and AFM characterization of Endothelin receptors in membranes (A. Engel’s group, 20 months)

Receptors will be 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. EM of freeze-fractured, metal shadowed samples will be used to assess the quality of reconstituted proteoliposomes. Oligomeric associations of the receptors in different detergent/lipid environments will be analysed by electron microscopy of negatively stained solubilized receptors and by atomic force microscopy of reconstituted receptors densely packed into proteoliposomes. Stable preparations of receptor and/or receptor-ligand complexes will be submitted to 2D crystallization trials, exploring different conditions (lipids, pH, salts and co-factors) and different methods (dialysis, cyclodextrin). 2D crystals will be analysed by negative stain EM and by electron diffraction of unstained samples kept at low temperature.

- SMFS characterization of Endothelin receptors in membranes (D. Muller’s group, 12 months)

SMFS provides a quantitative view of molecular interactions establishing the energetic landscape of membrane proteins.  Here, we will analyze the molecular interactions of the Endothelin receptor as a function of the physiological environment (lipids, pH, electrolyte) and the homo- and heterooligomeric states. Having mapped the Endothelin 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 Endothelin receptor.

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. 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.
  4. 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.
  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 16252-259 (2006).
  10. Fotiadis D, Liang Y, Filipek S, Saperstein DA, Engel A, Palczewski K.
    The G protein-coupled receptor rhodopsin in the native membrane. FEBS Lett 564, 281 (2004).
  11. 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).
  12. 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).