The Q7K0L33I0L63I HIV-1 protease mutant was expressed in Escherichia coli and the effect of binding a substrateanalog inhibitor, acetyl-pepstatin, was investigated by fluorescence spectroscopy and molecular dynamics. The dimeric enzyme has four intrinsic tryptophans, located at positions 6 and 42 in each monomer. Fluorescence spectra and acrylamide quenching experiments show two differently accessible Trp populations in the apoenzyme with k q1 ϭ 6.85 ϫ 10 9 M Ϫ1 s Ϫ1 and k q2 ϭ 1.88 ϫ 10 9 M Ϫ1 s Ϫ1 , that merge into one in the complex with k q ϭ 1.78 ϫ 10 9 M Ϫ1 s Ϫ1 . 500 ps trajectory analysis of Trp x 1 0x 2 rotameric interconversions suggest a model to account for the observed Trp fluorescence. In the simulations, Trp60Trp6B rotameric interconversions do not occur on this timescale for both HIV forms. In the apoenzyme simulations, however, both Trp42s and Trp42Bs are flipping between x 1 0x 2 states; in the complexed form, no such interconverions occur. A detailed investigation of the local Trp environments sampled during the molecular dynamics simulation suggests that one of the apoenzyme Trp42B rotameric interconversions would allow indole-quencher contact, such as with nearby Tyr59. This could account for the short lifetime component. The model thus interprets the experimental data on the basis of the conformational 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 apoenzyme binds the inhibitor. The model is thus consistent with associating components of the fluorescence decay in HIV-1 protease to ground state conformational heterogeneity.
The binding of acetylpepstatin to the Q7K/L33I/L63I mutant of HIV-1 protease was studied by fluorescence, phosphorescence, and 500-ps molecular dynamics. The protease is a homodimer with two tryptophans per monomer. Maximum entropy method (MEM) analysis and acrylamide quenching results show two tryptophyl, tryptophan (Trp) populations in the apoenzyme that merge into one in the complex. These results are in agreement with molecular dynamics simulations indicative of Trp asymmetry in the apoenzyme as revealed by the occurrence of nonequivalent Trp42 indole rotamer interconversions, not observed for the complex. Analysis of the local Trp42B environments of the apoenzyme with respect to possible quencher groups shows that the c2 interconversions do not influence the lifetime, while the c1 interconversions do. Upon binding the inhibitor, Trp42B acquires a single conformation with the same lifetime and orientation as that of Trp42, and also with less quenching accessibility. Thus, protein conformational dynamics become constrained with inhibitor binding. This conclusion is supported by red-edge effect experiments and phosphorescence lifetime measurements. The low temperature tp (~5.8 s) is quenched to ~200 ms as protein motions become activated around the glass transition temperature. In the case of the complex, the phosphorescence lifetime data show a more cooperative activation of the quenching mechanisms.
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