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Monitoring Inhibitor-Induced Conformational Population Shifts in HIV-1 Protease by Pulsed EPR Spectroscopy

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Department of Chemistry, P.O. Box 117200, University of Florida, Gainesville, Florida 32611-7200
*To whom correspondence should be addressed. E-mail: [email protected]. Telephone: (352) 392-2345. Fax: (352) 392-0872.
Cite this: Biochemistry 2009, 48, 37, 8765–8767
Publication Date (Web):August 19, 2009
https://doi.org/10.1021/bi901201q
Copyright © 2009 American Chemical Society

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    Abstract

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    Double electron−electron resonance (DEER), a pulsed electron paramagnetic resonance (EPR) spectroscopy technique, was utilized to characterize conformational population shifts in HIV-1 protease (HIV-1PR) upon interaction with various inhibitors. Distances between spin-labeled sites in the flap region of HIV-1PR were determined, and detailed analyses provide population percentages for the ensemble flap conformations upon interaction with inhibitor or substrate. Comparisons are made between the percentage of the closed conformer seen with DEER and enzymatic inhibition constants, thermodynamic dissociation constants, and the number of hydrogen bonds identified in crystallographic complexes.

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    Experimental details, sample preparation, time domain echo curves, and data analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

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