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

¹ University of California, Los Angeles
² Jagiellonian University, Poland
³ Vanderbilt University, Nashville

^* To whom correspondence should be addressed. E-mail: hubbellw{at}jsei.ucla.edu.

Submitted on January 5, 2005
Revised on March 9, 2005
Accepted on 17 June 2005

	Abstract

In Site-Directed Spin Labeling, the relative solvent accessibility of spin labeled side chains is taken to be proportional to the Heisenberg exchange rate (Wex) of the nitroxide with a paramagnetic reagent in solution. In turn, relative values of Wex are determined by CW 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 wex 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 SDSL data, and hence comparing crystal and solution structures.

Key Words: EPR-Spectroscopy, T4 Lysozyme, saturation recovery EPR, spin labeling

This article has been cited by other articles:

				M. Doebber, E. Bordignon, J. P. Klare, J. Holterhues, S. Martell, N. Mennes, L. Li, M. Engelhard, and H.-J. Steinhoff Salt-driven Equilibrium between Two Conformations in the HAMP Domain from Natronomonas pharaonis: THE LANGUAGE OF SIGNAL TRANSFER? J. Biol. Chem., October 17, 2008; 283(42): 28691 - 28701. [Abstract] [Full Text] [PDF]

				M. Sarewicz, A. Borek, F. Daldal, W. Froncisz, and A. Osyczka Demonstration of Short-lived Complexes of Cytochrome c with Cytochrome bc1 by EPR Spectroscopy: IMPLICATIONS FOR THE MECHANISM OF INTERPROTEIN ELECTRON TRANSFER J. Biol. Chem., September 5, 2008; 283(36): 24826 - 24836. [Abstract] [Full Text] [PDF]

				J. C. Klein, A. R. Burr, B. Svensson, D. J. Kennedy, J. Allingham, M. A. Titus, I. Rayment, and D. D. Thomas Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR PNAS, September 2, 2008; 105(35): 12867 - 12872. [Abstract] [Full Text] [PDF]

				T. I. Smirnova, T. G. Chadwick, R. MacArthur, O. Poluektov, L. Song, M. M. Ryan, G. Schaaf, and V. A. Bankaitis The Chemistry of Phospholipid Binding by the Saccharomyces cerevisiae Phosphatidylinositol Transfer Protein Sec14p as Determined by EPR Spectroscopy J. Biol. Chem., November 17, 2006; 281(46): 34897 - 34908. [Abstract] [Full Text] [PDF]

				J. Pyka, J. Ilnicki, C. Altenbach, W. L. Hubbell, and W. Froncisz Accessibility and Dynamics of Nitroxide Side Chains in T4 Lysozyme Measured by Saturation Recovery EPR Biophys. J., September 1, 2005; 89(3): 2059 - 2068. [Abstract] [Full Text] [PDF]