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

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• 作者：Christian Altenbach ; Wojciech Froncisz ; Roy Hemker ; Hassane Mchaourab ; Wayne L. Hubbell
• 刊名：Biophysical Journal
• 出版年：2005
• 出版时间：September 2005
• 年：2005
• 卷：89
• 期：3
• 页码：2103-2112
• 全文大小：265 K

文摘

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 Π. 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 Π, 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 < ρ < 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.