PhD projects


Structural Biology of Membrane Proteins

PhD project:

Site-directed spin labeling & Solid-state NMR: An optimized approach to study protein structure and ligand binding in lipid bilayers

Principal Investigator:

Dr. M. Baldus, Solid-state NMR group, Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany, maba|

In collaboration with:

Dr. V. Gordeliy, Moscow Institute of Physics and Technology, Russia.

Prof. M. Engelhard (visiting scientist), Max-Planck Institute of Molecular Physiology, Dortmund, Germany.

Prof. G. Bueldt, Research Centre Juelich, Juelich, Germany.

Dr. G. Siegal, ZoBio inc., Leiden, The Netherlands

Solid-state NMR Nuclear Magnetic Resonance (ssNMR) methods become increasingly useful to study protein structure and dynamics in a lipid bilayer environment. Previously, we have shown that high-resolution ssNMR spectra and structural information can be obtained from membrane proteins in a lipid bilayer environment including GPCRs, ion channels, and seven-helix receptors(1-3). Current methods probe through-space distances between 13C, 15N and 1H spins in the range of 2-6 Å(4). From EPR and solution-state NMR, it is well known that the use of paramagnetic quenchers or site-directed spin labeling can potentially provide distance constraints over more than 10 Å. Thus far, the use of paramagnetic relaxation for ssNMR has been limited to studies involving lipid membranes(5), single-labeled membrane proteins(6) and multiply-labeled microcrystalline proteins(7, 8). In this Ph.D. project, the utility of spin labeling in the context of an integrated approach for 3D structure determination of membrane-embedded proteins by ssNMR shall be explored. This concept shall be applied to ligand-membrane protein complexes.

Stage 1: ssNMR Methodology and application to the sensory rhodopsin/transducer complex

Because previous EPR and NMR reports have shown that nitroxide spin labels may influence protein structure(9, 10), we want to compare in the first stage our spectroscopic findings to reference structures, in particular those available for the receptor-transducer complex of sensory rhodopsin from Natronomonas pharaonis. The groups of V. Gordeliy, M. Engelhard and G. Bueldt are the world leaders in the functional and structural characterization of this system(11, 12). In a preliminary set of experiments, we have analyzed ssNMR results of the L90C mutant of SRII which suggest that indeed paramagnetic quenching can be used to infer information about the overall fold of membrane embedded proteins but that local structural variations may be present for spin labels facing the protein interior (Figure 1).

Figure 1

Figure 1:
a) Overlay of 13C-13C ssNMR data of wild-type (black) and L90R1 (green) U[13C,15N]SRII/HtrII complex in lipid bilayers. Crosspeaks predicted for 3 residues within/outside 10Å of the paramagnetic tag are labeled in red/blue.
b) Residues highlighted in a) are shown on the 3D structure (1H2S.pdb). The orange sphere represents a radius of about 10 Å around the mutation side. Grey residues are not labeled.

These dependencies shall be further investigated using 2D ssNMR for variable sample temperatures using different spin-labeled protein variants. In parallel, we will develop ssNMR methodology that makes optimum use of the relaxation behavior induced upon 1H, 13C and 15N spin system parameters to probe protein structure in the distance range from 5 to 15 Å. Our data shall be compared to X-ray structures and MD calculations and we will apply these methods to study the signal transduction pathway of sensory rhodopsin in lipid bilayers by ssNMR. Here, our findings may resolve current discrepancies between X.ray crystallography and evidence based on other biophysical, in particular EPR, data regarding the structural alterations associated with receptor activation.

Stage 2: Application to a chimeric potassium channel

In the second stage, we want to use the combination of ssNMR and spin labelling to study protein function and ligand binding in a membrane-embedded ion channel (KcsA-Kv1.3). Earlier, we have demonstrated that ssNMR is a powerful instrument to probe the formation of a ligand-potassium channel complex in membranes(2). Natural and synthetic ligands not only play a central role in channel block but they also are known to modulate channel function(13). Using ssNMR studies on spin-labeled protein variants we want to further characterize which structural alterations are associated with channel activation and inactivation in a lipid bilayer environment for different ligands. The latter studies shall be carried out in combination with ligand screening using the proprietary TINS ligand screening hardware and compound collection from ZoBio headed by G. Siegal. A protocol for solubilizing the chimeric potassium channel in detergent micelles is already available.


  1. Luca, S., black, J. F., Sohal, A. K., Filippov, D. V., van Boom, J. H., Grisshammer, R. & Baldus, M. (2003)
    Proc. Natl. Acad. Sci. U. S. A. 100, 10706-10711.
  2. Lange, A., Giller, K., Hornig, S., Martin-Eauclaire, M.-F., Pongs, O., Becker, S. & Baldus, M. (2006).
    Nature 440, 959-962.
  3. Etzkorn, M., Martell, S., Andronesi, Ovidiu C., Seidel, K., Engelhard, M. & Baldus, M. (2007).
    Angewandte Chemie International Edition 46, 459-462.
  4. Baldus, M. (2006).
    Current Opinion in Structural Biology 16, 618-623.
  5. Villalain, J. (1996).
    Eur. J. Biochem. 241, 586-593.
  6. Tuzi, S., Hasegawa, J., Kawaminami, R., Naito, A. & Saito, H. (2001).
    Biophys. J. 81, 425-434.
  7. Nadaud, P. S., Helmus, J. J., Hofer, N. & Jaroniec, C. P. (2007).
    J. Am. Chem. Soc. 129, 7502-7503.
  8. Guido Pintacuda, Giraud, N., Pierattelli, R., Böckmann, A., Bertini, I. & Emsley, L. (2007).
    Angewandte Chemie International Edition 46, 1079-1082.
  9. McHaourab, H. S., Lietzow, M. A., Hideg, K. & Hubbell, W. L. (1996).
    Biochemistry 35, 7692-7704.
  10. Liang, B., Bushweller, J. H. & Tamm, L. K. (2006).
    J. Am. Chem. Soc. 128, 4389-4397.
  11. Gordeliy, V. I., Labahn, J., Moukhametzianov, R., Efremov, R., Granzin, J., Schlesinger, R., Buldt, G., Savopol, T., Scheidig, A. J., Klare, J. P. & Engelhard, M. (2002).
    Nature 419, 484-487.
  12. Moukhametzianov, R., Klare, J. P., Efremov, R., Baeken, C., Gäppner, A., Labahn, J. r., Engelhard, M., Büldt, G. & Gordeliy, V. I. (2006).
    Nature 440, 115-119.
  13. Kurata, H. T. & Fedida, D. (2006).
    Prog. Biophys. Mol. Biol. 92, 185-208.