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Determination of the Orientation of T4 Lysozyme Vectorially Bound to a Planar-Supported Lipid Bilayer Using Site-Directed Spin Labeling

Kerstin Jacobsen ^*, Shirley Oga , Wayne L. Hubbell  and Thomas Risse ^*

^* Fritz Haber Institute of Max-Planck Society, Department of Chemical Physics, 14195 Berlin, Germany; and Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095-7008

Correspondence: Address reprint requests to Thomas Risse, Fritz Haber Institute of Max-Planck Society, Dept. of Chemical Physics, Faradayweg 4-6, 14195 Berlin, Germany. E-mail: risse{at}fhi-berlin.mpg.de.

Site-directed spin labeling is used to investigate the structure of adsorbed T4 lysozyme (T4L). A monolayer of T4L is prepared by tethering the protein selectively via a His-tag to the chelating headgroups (NTA Ni) of a planar quartz-supported lipid bilayer. This results in a vectorially oriented ensemble of proteins on the surface, which gives rise to angular-dependent electron paramagnetic resonance spectra. Similar measurements of spin-labeled lipid bilayers were used to characterize the structure and dynamics of the supports. Electron paramagnetic resonance line shape was analyzed using the stochastic Liouville equation approach developed by Freed and co-workers. The simulations reveal a conservation of the secondary and tertiary structure of T4L upon adsorption although slight conformational changes in the presence of the surface can be detected by probing tertiary contact sites. The orientation of the entire protein was deduced on the basis of an anisotropic motional model for the spin-labeled side chain. In addition, a polar order but azimuthal disorder of the molecules was assumed to fit the data. These results demonstrate the utility of site-directed spin labeling in combination with spectral simulation to study not only the secondary and tertiary structure of adsorbed proteins in monolayer coverage but also their orientation with respect to the surface.