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Accessibility of Nitroxide Side Chains: Absolute Heisenberg Exchange Rates from Power Saturation EPR

Christian Altenbach ^*, Wojciech Froncisz , Roy Hemker ^*, Hassane Mchaourab ^* and Wayne L. Hubbell ^*

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

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.

In site-directed spin labeling, the relative solvent accessibility of spin-labeled side chains is taken to be proportional to the Heisenberg exchange rate (W_ex) of the nitroxide with a paramagnetic reagent in solution. In turn, relative values of W_ex are determined by continuous wave power saturation methods and expressed as a proportional and dimensionless parameter . In the experiments presented here, NiEDDA is characterized as a paramagnetic reagent for solvent accessibility studies, and it is shown that absolute values of W_ex can be determined from , and that the proportionality constant relating them is independent of the paramagnetic reagent and mobility of the nitroxide. Based on absolute exchange rates, an accessibility factor is defined (0 < < 1) that serves as a quantitative measure of side-chain solvent accessibility. The accessibility factors for a nitroxide side chain at 14 different sites in T4 lysozyme are shown to correlate with a structure-based accessibility parameter derived from the crystal structure of the protein. These results provide a useful means for relating crystallographic and site-directed spin labeling data, and hence comparing crystal and solution structures.

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