Trp42 rotamers report reduced flexibility when the inhibitor acetyl-pepstatin is bound to HIV-1 protease
| Autor: | Monique Laberge, Ferenc Tölgyesi, László Polgár, Judit Fidy, Beáta Ullrich, Zoltán Szeltner |
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| Rok vydání: | 2000 |
| Předmět: |
Models
Molecular Time Factors Protein Conformation Stereochemistry Biochemistry Fluorescence spectroscopy chemistry.chemical_compound Molecular dynamics Protein structure HIV Protease HIV-1 protease Pepstatins Escherichia coli Computer Simulation Molecular Biology Conformational isomerism Acrylamide biology Chemistry Tryptophan Active site Kinetics Crystallography Spectrometry Fluorescence Mutagenesis biology.protein Pepstatin Research Article Protein Binding |
| Zdroj: | Protein Science. 9:2232-2245 |
| ISSN: | 1469-896X 0961-8368 |
| DOI: | 10.1110/ps.9.11.2232 |
| Popis: | The Q7K/L331/L631 HIV-1 protease mutant was expressed in Escherichia coli and the effect of binding a substrate-analog 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) = 6.85 x 10(9) M(-1) s(-1) and k(q2) = 1.88 x 10(9) M(-1) s(-1), that merge into one in the complex with k(q) = 1.78 x 10(9) M(-1) s(-1). 500 ps trajectory analysis of Trp X1/X2 rotameric interconversions suggest a model to account for the observed Trp fluorescence. In the simulations, Trp6/Trp6B rotameric interconversions do not occur on this timescale for both HIV forms. In the apoenzyme simulations, however, both Trp42s and Trp42Bs are flipping between X1/X2 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. |
| Databáze: | OpenAIRE |
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