Human immunodeficiency virus type 1 (HIV-1) protease plays a fundamental role in the maturation and life cycle of the retrovirus HIV-1, as it functions in regulating post-translational processing of the viral polyproteins gag and gag-pol; thus, is a key target of AIDS antiviral therapy. Accessibility of substrate to the active site is mediated by two flaps, which must undergo a large conformational change from an open to a closed conformation during substrate binding and catalysis. The electron paramagnetic resonance (EPR) method of site-directed spin labeling (SDSL) with double electron-electron resonance (DEER) spectroscopy was utilized to monitor the conformations of the flaps in apo HIV-1 protease (HIV-1PR), subtypes B, C, and F, CRF01_A/E, and patient isolates V6 and MDR 769. The distance distribution profiles obtained from analysis of the dipolar modulated echo curves were reconstructed to yield a set of Gaussian-shaped populations, which provide an analysis of the flap conformations sampled. The relative percentages of each conformer population described as “tucked/curled”, “closed”, “semi-open”, and “wide-open” were determined and compared for various constructs. The results and analyses show that sequence variations among subtypes, CRFs and patient isolates of apo HIV-1PR alter the average flap conformation in a way that can be understood as inducing shifts in the relative populations, or conformational sampling, of the previously described four conformations for HIV-1PR.
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