Interflap Distances in HIV-1 Protease Determined by Pulsed EPR Measurements

Citations Over TimeTop 10% of 2007 papers

Abstract

Human immunodeficiency virus type 1 (HIV-1) protease, being responsible for the cleavage of the viral polyproteins gag and gag-pol, is an essential component in the processing of viral proteins encoded in the HIV viral genome. The present work utilizes site-directed spin labeling (SDSL) and double electron−electron resonance (DEER) spectroscopy to characterize flap conformations in HIV-1 protease in an inhibited and uninhibited form. Because HIV protease is a homodimer, incorporation of the labeling probe at K55C yields a spin label pair within the protease, suitable for distance measurements by pulsed techniques. DEER experiments were performed with and without protease inhibitors to determine distances between residues 55−55' within the flap region of the protease. Analysis of the EPR results demonstrates that the flap region spans a continuous, rather than segmental, range of motion in its uninhibited state. Upon addition of inhibitor, the distance between spin labels shortens by ∼3−4 Å (regardless of spin label choice), indicating a closing of the flaps. More importantly, these results characterize the range of flap opening in an uninhibited form, showing distances that span 26−48 Å, indicative of a broad range of possible conformations.

Related Papers

• → Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence(1991)480 cited
• → Cleavage site analysis in picornaviral polyproteins: Discovering cellular targets by neural networks(1996)230 cited
• → HIV-1 protease specificity of peptide cleavage is sufficient for processing of gag and pol polyproteins(1988)269 cited
• → Comparison of the HIV‐1 and HIV‐2 proteinases using oligopeptide substrates representing cleavage sites in Gag and Gag‐Pol polyproteins(1991)161 cited
• → Effect of two novel inhibitors of the human immunodeficiency virus protease on the maturation of the HIV gag and gag-pol polyproteins(1990)29 cited