Trp42 rotamers report reduced flexibility when the inhibitor acetyl-pepstatin is bound to HIV-1 protease

The Q7K/L33I/L63I HIV-1 protease mutant was expressed in Escherichia coli a nd the effect of binding a substrate-analog inhibitor, acetyl-pepstatin, wa s investigated by fluorescence spectroscopy and molecular dynamics. The dim eric enzyme has four intrinsic tryptophans, located at positions 6 and 42 i n each monomer. Fluorescence spectra and acrylamide quenching experiments s how two differently accessible Trp populations in the apoenzyme with k(q1) = 6.85 x 10(9) M-1 s(-1) and k(q2) = 1.88 x 10(9) M-1 s(-1), that merge int o one in the complex with k(q) = 1.78 x 10(9) M-1 s(-1) 500 ps trajectory a nalysis of Trp chi (1)/chi (2) rotameric interconversions suggest a model t o account for the observed Trp fluorescence. In the simulations, Trp6/Trp6B rotameric interconversions do not occur on this timescale for both HIV for ms. In the apoenzyme simulations, however, both Trp42s and Trp42Bs are flip ping between chi (1)/chi (2) states; in the complexed form, no such interco nverions occur. A detailed investigation of the local Trp environments samp led during the molecular dynamics simulation suggests that one of the apoen zyme Trp42B rotameric interconversions would allow indole-quencher contact, such as with nearby Tyr59. This could account for the short lifetime compo nent. The model thus interprets the experimental data on the basis of the c onformational fluctuations of Trp42s alone. It suggests that the rotameric interconversions of these Trps, located relatively far from the active site and at the very start of the flap region, becomes restrained when the apoe nzyme binds the inhibitor. The model is thus consistent with associating co mponents of the fluorescence decay in HIV-1 protease to ground state confor mational heterogeneity.