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Accessibility and Dynamics of Nitroxide Side Chains in T4 Lysozyme Measured by Saturation Recovery EPR

Janusz Pyka ^*, Jan Ilnicki ^*, Christian Altenbach , Wayne L. Hubbell  and Wojciech Froncisz ^*

^* Department of Biophysics, Faculty of Biotechnology, Jagiellonian University, 30-387 Krakow, Poland; and Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095-7008

Correspondence: Address reprint requests to Wayne L. Hubbell, Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA 90095-7008. Tel.: 310-206-8839; Fax: 310-794-2144; E-mail: hubbellw{at}jsei.ucla.edu.

Long pulse saturation recovery electron paramagnetic resonance spectroscopy is applied to the investigation of spin-labeled side chains placed along a regular helix extending from 128 to 135 in T4 lysozyme. Under an argon atmosphere, analysis of the exponential saturation recovery curves gives the spin-lattice relaxation rates of the nitroxides, which depend on the nitroxide side-chain dynamics. In the presence of the fast-relaxing paramagnetic reagents O₂ or NiEDDA, global analysis of the saturation recovery provides the spin-lattice relaxation rates as well as the Heisenberg exchange rates of the nitroxide with the reagents. As previously shown with power saturation methods, such exchange rates are direct measures of the solvent accessibility of the nitroxide side chains in the protein structure. The periodic dependence of the spin-lattice relaxation rates and the exchange rates along the 128–135 sequence reveal the presence of the helical structure, demonstrating the use of these parameters in structure determination. In general, multiple exponentials are required to fit the saturation recovery data, thus identifying multiple states of the side chain. In one case, multiple conformations detected in the spectrum are not evident in the saturation recovery, suggesting rapid exchange on the timescale of spin-lattice relaxation.

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