Tryptophan rotamers that report the conformational dynamics of proteins

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.

The red‐edge effects: 30 years of exploration

“…In the latter case, for the protein studied in a water-glycerol mixture, temperaturedependent shifts of spectra and decrease in t F are observed in the region 250-290 K. Measurements of lifetime distributions over the emission spectrum show a progressive change of short-living component from positive to negative with the increase of l em , which is characteristic of dielectric relaxations. Temperaturedependent REE were detected and characterized in the time domain for HIV protease (151). Studies on temperature-dependent spectral shifts as a function of l ex allowed determination of the range of dielectric relaxations in this protein.…”

Room-Temperature Ionic Liquids: Slow Dynamics, Viscosity, and the Red Edge Effect

“…An extreme example of this slow dynamics can be observed for example upon excitation of tryptophan , residues in denatured proteins. In this case, local environments provided by nearby amino acids are different for each tryptophan and do not get averaged.…”

A Study of the Time-Resolved Fluorescence Spectrum and Red Edge Effect of ANF in a Room-Temperature Ionic Liquid

“…This phenomenon is uncommon for typical solvents, but it commonly occurs in colloidal gels, micelles, and even proteins. In the case of proteins, the phenomenon can be observed for example when photoexciting tryptophan residues. , Each tryptophan residue in a protein is surrounded by a specific local environment provided by nearby amino acids. Because the protein sequence does not change with time, local environments remain different, and therefore, different tryptophan residues absorb and emit at different wavelengths.…”