Dynamics and Function of Spin Labelled Membrane Proteins Studied by Multi-Frequency EPR
Final Report Abstract
The goals of the present proposal comprise development and application of multifrequency EPR spectroscopy of site-directed spin labelled membrane proteins. A high-field pulse EPR spectrometer working at 95 GHz/3.4 T was set up in our laboratory according to the design of that developed in Prof. Möbius' group. Site-directed spin labelled variants of the channel forming colicin A and of sensory rhodopsin light receptors were used as model systems to study structural details and conformational dynamics during protein function. The EPR data characterize the molten globule properties of colicin A with a resolution of the side, chain locations and reveal a compact hydrophobic protein core. Inter-spin distance measurement of doubly spin labelled colicin A variants enabled us to suggest a model of the membrane bound closed channel state. In a joint effort together with our collaboration partners Prof. Mobius, Berlin, and Prof. Groenen, Leiden, we succeeded to explain the heterogeneity of the g-tensor component gxx resolved by high-field EPR spectroscopy. New synthesis and incorporation of artificial amino acids with nitroxide side chains has now made possible to introduce spin labels not only in the N-terminal part of the protein but also in its centre, thus allowing EPR studies with a new quality of spin labelled proteins.
Publications
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Incorporation of spin-labelled amino acids into proteins. Magn Reson. Chem 2005, 43 Spec no.. S34-S39
Becker, C. F.; Lausecker, K.; Balog, M.; Kalai, T.; Hideg, K.; Steinhoff, H. J.; Engelhard, M.
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(2008) Conformation of the closed channel state of colicin A in proteoliposomes: an umbrella model. J. Mol. Biol. 378, 204-214
Padmavathi, P.V.L., and H.-J. Steinhoff
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(2009) Topology of the amphipathic helices of the colicin A pore-forming domain in E. coli lipid membranes studied by pulse EPR. Phys. Chem. Chem. Phys. 11:6770-7
Böhme, S., P. V. Padmavathi, J. Holterhues, F. Ouchni, J.P. Klare, H.-J. Steinhoff
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(2010) Heterogeneity in the nitroxide micro-environment: polarity and proticity effects in spin labeled proteins studied by multifrequency EPR. Appl. Magn. Reson. 37:391-403
Bordignon, E., H. Brutlach, L. Urban, K. Hideg, A. Savitsky, A. Schnegg, P. Gast, M. Engelhard, E.J.J. Groenen, K. Mobius, and H.-J. Steinhoff
