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Pure Appl. Chem., 2001, Vol. 73, No. 3, pp. 415-419

Tryptophan rotamers that report the conformational dynamics of proteins

Judit Fidy1*, Monique Laberge1, Beata Ullrich1, Laszlo Polgar2, Zoltan Szeltner2, Jacques Gallay3 and Michel Vincent3

1 Institute of Biophysics and Radiation Biology, Faculty of General Medicine, Semmelweis University, Budapest, H-1444, P.O.B. 263, Hungary
2 Institute of Enzymology, Biological Research Center, HAS, Budapest, H-1518, P.O.B. 7, Hungary
3 LURE, Université Paris-Sud, Bâtiment 209D, Orsay, F-91405, France

Abstract: 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.