PhD projects

ITN FP7 - SBMPs

Structural Biology of Membrane Proteins

PhD project:

Developing new approaches to decipher the interactions stabilizing membrane proteins

A joint SMFS application, nanotechnological application and bioinformatics approach




Principal Investigator:

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


In collaboration with:

Prof. S. Filipek, Professor of Biomodelling, International Institute of Molecular and Cell Biology, 4 Ks. Trojdena St, 02-109 Warsaw, Poland, sfilipek|iimcb.gov.pl

JPK Instruments AG, Bouchéstr. 12, 12435 Berlin, Germany; pelzer|jpk.com

Factors determining membrane protein folding and stability play key issues in biological, medical and biotechnological applications. Destabilization of membrane proteins can lead to their malfunction and misfolding (sanders, 2004), which build the origin of many diseases such as cystic fibrosis, type II diabetes, retinitis pigmentosa, deafness, Charcot-Marie tooth disease..., ... Because of their great functional importance membrane protein malfunction severely impairs the functional state of cells, tissues, and organs. However, destabilization of membrane proteins presents serious bottlenecks for their structural investigation as well. Structural determination of membrane proteins requires their overexpression, purification and crystallization for X-ray diffraction and electron microscopy. Each of these steps is continuously reported to pose potential traps, and in many cases, cause the membrane protein to destabilize, malfunction and misfolding. Establishing conditions that prevent membrane protein destabilization and malfunction is currently done in a predominantly empiric and intuitive manner. Thus, understanding the factors leading to destabilization of membrane proteins is not only important to answer biologically pertinent questions but also to provide a consortium with essential information of how to improve the handling of membrane proteins. The relatively new technique of single-molecule force spectroscopy (SMFS), which involves mechanical unfolding of single protein molecules, provides substantial insights into the interactions that stabilize membrane proteins.


The Muller group has recently developed new SMFS instrumentation (www.nambition.com) and efficient approaches to characterize the molecular interactions stabilizing and destabilizing the functional state of native membrane proteins by SMFS. At this stage it is possible to detect and to locate the interactions that stabilize native membrane proteins. So far the technique has been applied to detect and locate interactions stabilizing the tertiary and secondary structures of membrane proteins like human aquaporin-1, bacteriorhodopsin, halorhodopsin, bovine rhodopsin, and sodium/proton antiporters. It could be further shown how these interactions change with the environment (e.g., pH, electrolyte, temperature). At the moment SMFS can only detect the sum of all interactions stabilizing the secondary structures of the membrane proteins. To understand how environmental changes alter the interactions we must decipher their nature. Continuous development of SMFS allowed to dissect dissipative and conservative contributions of interactions stabilizing membrane proteins. Additionally, it was possible to reveal the shape of the energy barriers stabilizing secondary structures of membrane proteins and to reveal their kinetic stability and mechanical properties. In this consortium we intend to further develop the SMFS technique to go beyond this stage and to reveal insights into the nature of these interactions. The PhD thesis will thus include SMFS experiments, SMFS development and the development of new algorithms for SMFS data analysis and interpretation.


The Filipek group research is focused on theoretical investigations of membrane proteins – their structures and processes. The AFM images of rhodopsin dimers served as a basis for construction of a complex of oligomeric rhodopsin and its G protein (Rho4-Gtabg) and later for a theoretical model of arrestin bound to rhodopsin dimer. A specific mechanism of recognition of activation state of rhodopsin by arrestin was also proposed. Since the stability is the major factor characterizing the proteins we recently started research on simulated mechanical unfolding to get insight into molecular interactions generating stabilization of particular domains of membrane protein. Such an analysis leads also to understanding of folding/unfolding processes and of the influence of the membrane onto it. For technical reasons the minimal speed of simulated unfolding is orders of magnitude faster that used in SMFS experiments, however, the developments in oscillating SMFS will bring both methods to the same time scale of unfolding/folding processes.

JPK Instruments is the European market leader in biological AFM. The AFM (NanoWizardTM) has been designed to perform live cell imaging and cell adhesion experiments at the molecular level. It has been recently demonstrated that the NanoWizard can also be used to unfold individual membrane proteins and to detect molecular interactions established within the protein. In this project JPK Instruments intends to further develop new strategies to perform SMFS at even better force and distance resolution, throughput, temperature stability, and dynamic SMFS required to determine the dynamic energy landscape of membrane proteins.


- SMFS and DFS characterization of native membrane proteins (D. Muller’s group, 3x8 months)

SMFS provides a quantitative view of the interactions stabilizing membrane proteins. In a first step we will detect and map molecular interactions within membrane proteins under different conditions that lead to misfolding. Dynamic SMFS will then map details of the energy landscape and observe the energy minima, energy barriers and kinetic properties of the membrane proteins structure. In the next step, we will develop oscillating SMFS exhibiting an extremely fast sampling rate and being operated with an ultrashort cantilever. This ensures the detection of maximum range and times of interactions to reveal insights into the different energies and their ranges stabilizing the proteins. The developments will be made in a continuous feedback with the partners of this project. Future developments will focus on force-feedback SMFS experiments with small oscillating amplitudes in the sub-nanometer range.


- Development of new data analysis algorithms Filipek’s group, 3x4 months)

The analysis of new data from modified SMFS requires developing of new algorithms for recognizing and proper classification of SMFS force plots. This analysis will be conducted in connection to simulated mechanical unfolding using steered molecular dynamics (SMD) method.


- Instrumental development (JPK Instruments, 3x1 months)

The PhD student and project leaders will be in close contact and in continuous feedback with JPK Instruments to develop the next generation of SMFS and DFS technology required to decipher the interactions of membrane proteins.

References:

  1. Muller DJ, et al.,
    Single-molecule studies of membrane proteins. Curr Opin Struc Biol, (2006).
  2. Kedrov A, et al.,
    Deciphering molecular interactions of native membrane proteins by single-molecule force spectroscopy. Annu Rev Biophys Biomol Struct, (2007).
  3. 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, (2007) 282, 11377-11385.
  4. 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, (2006) 358, 255-269.
  5. 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, (2006) 16, 252-259.
  6. S. Filipek, K.A. Krzysko, D. Fotiadis, Y. Liang, D.A. Saperstein, A. Engel & K. Palczewski,
    A concept for G protein activation by G protein-coupled receptor dimers: the transducin/rhodopsin interface Photochemical and Photobiological Sciences, (2004) 3, 628-638.
  7. A. Modzelewska, S. Filipek, K. Palczewski & P.S.H. Park,
    Arrestin interaction with rhodopsin - Conceptual models Cell Biochemistry and Biophysics, (2006) 46, 1-15.