Principal Investigator
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P13 Harald Schwalbe
Professor
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Institute of Organic Chemistry
and Chemical Biology /
Center of Biomolecular
Magnetic Resonance
Goethe-University Frankfurt a.M.
Max-von-Laue-Str. 7
60438 Frankfurt am Main, Germany
Phone +49 (0)69 798-29737
Fax +49 (0)69 798-29515
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P13 NMR investigations of mammalian rhodopsin
Rhodopsin is the major membrane protein of the rod outer segment (ROS), a highly ordered array of hundreds of discs that are enclosed by a separate plasma membrane, in vertebrate retina. As a visual photoreceptor it contains a photoreactive retinal chromophore that is covalently attached to the protein and is converted into a receptor agonist by light. Rhodopsin is the best studied GPCR; structural studies, however, yielding information about its activated state shall now be initiated using light-triggered time-resolved NMR spectroscopy. We have obtained both selectively and uniformly isotope-labeled samples of rhodopsin in a high level mammalian expression system (HEK293) of the bovine opsin gene yielding mg quantities of functional protein with all posttranslational modifications for NMR studies. We will now prepare opsin using the cell-free expression system in collaboration with P2 (Dötsch / Bernhard) and compare functionality and NMR spectra. We will also study the interaction of rhodopsin with arrestin and peptides derived from transducin to obtain information about protein-protein interactions involved in the signalling cascade. Light-triggered time-resolved NMR spectroscopy will be extended to study ATP turnover and substrate stoichiometry for a full length ABC transporter together with P6 (Glaubitz).
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Publications
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Dastvan, R., Bode, B., Karuppiaj, M.P.R., Marko, A., Lyubenova, S., Schwalbe, H. and Prisner, T. (2010) Optimization of transversal relaxation of nitroxides for PELDOR spectroscopy in phospholipid membranes. J Phys Chem B 114, 13507-13516.
Saxena, K., Schieborr, U., Anderka, O., Duchardt-Ferner, E., Elshorst, B., Gande, S.L., Janzon, J., Kudlinzki, D., Sreeramulu, S., Dreyer, M.K., Wendt, K.U., Herbert, C., Duchaussoy, P., Bianciotto, M., Dirguez, P.-A., Lassalle, G., Savi, P., Mohammadi, M., Bono, F. and Schwalbe, H. (2010) Influence of heparin mimetics on the assembly of the FGF - FGFR4 signaling complex. J Biol Chem 283, 26628-26640.
Juhnke, H.D., Hiltscher, H., Nasiri, H.R., Schwalbe, H. and Lancaster, C.R.D. (2009) Production, characterization, and cetermination of the real catalytic properties of the putative ‘Succinate Dehydrogenase’ from Wolinella succinogenes. Mol Microbiol 71, 1088-1101.
Nasiri, H.R., Panisch, R., Madej, M.G., Bats, J.W., Lancaster, C.R.D. and Schwalbe, H. (2009) The Correlation of cathodic peak potentials of vitamin K3 derivatives and their calculated electron affinities. The role of hydrogen bonding and conformational changes. Biochim Biophys Acta 1787, 601-608.
Gieldon, A., Lopez, J.J., Glaubitz, C. and Schwalbe, H. (2008) Theoretical study of the human bradykinin-bradykinin B2 receptor complex. ChemBioChem 9, 2487–2497.
Lopez J.J., Shukla, A.K., Reinhart, C., Schwalbe, H., Michel, H. and Glaubitz, C. (2008) The structure of the neuropeptide bradykinin bound to the human G-protein coupled receptor bradykinin B2 as determined by solid-state NMR. Angew Chem Intl Ed Engl 47, 1668-1671.