Tryptophan rotamers that report the conformational dynamics of proteins

The binding of acetyl-pepstatin to the Q7K/L33I/L63I mutant of HIV-1 protea se was studied by fluorescence, phosphorescence, and 500-ps molecular dynam ics. The protease is a homodimer with two tryptophans per monomer. Maximum entropy method (MEM) analysis and acrylamide quenching results show two try ptophyl. tryptophan (Trp) populations in the apoenzyme that merge into one in the complex. These results are in agreement with molecular dynamics simu lations indicative of Trp asymmetry in the apoenzyme as revealed by the occ urrence of nonequivalent Trp42 indole rotamer interconversions, not observe d for the complex. Analysis of the local Trp42B environments of the apoenzy me with respect to possible quencher groups shows that the chi (2) intercon versions do not influence the lifetime, while the chi (1) interconversions do. Upon binding the inhibitor, Trp42B acquires a single conformation with the same lifetime and orientation as that of Trp342, and also with less que nching accessibility. Thus, protein conformational dynamics become constrai ned with inhibitor binding. This conclusion is supported by red-edge effect experiments and phosphorescence lifetime measurements. The low temperature tau (p) (similar to5.8 s) is quenched to similar to 200 mus as protein mot ions become activated around the glass transition temperature. In the case of the complex, the phosphorescence lifetime data show a more cooperative a ctivation of the quenching mechanisms.