Folding of tryptophan mutants of barstar: evidence for an initial hydrophobic collapse on the folding pathway

Abstract

The contributions of the three tryptophan residues of barstar to the spectroscopic properties, stability, and folding of the protein have been studied by mutating two of the tryptophans, Trp38 and Trp44, individually as well as together, to phenylalanines, Phe. The three mutant proteins studied are shown to be similar to wt barstar in structure by activity measurements as well as by spectroscopic characterization. Fluorescence energy transfer between the tryptophans as well as quenching by their local structural environments complicates the analysis of the contributions of the individual tryptophans to the fluorescence of the wt protein, but it is demonstrated that Trp53, which is completely buried within the hydrophobic core, makes the dominant contribution to the fluorescence, while the fluorescence of Trp38 is largely quenched in the fully folded protein. GdnHCl- as well as temperature-induced equilibrium unfolding studies, using three different structural probes, indicate that W38FW44F, where both Trp38 and Trp44 have been removed, follows a two-state unfolding transition and is less stable than the wt barstar. The fluorescence-monitored folding and unfolding kinetics of W38FW44F have been studied in detail. W38FW44F folds 2-fold faster and unfolds 3-fold faster than wt barstar. A large fraction of the total fluorescence change that occurs during folding occurs in a burst phase within 4 ms after commencement of folding. A similar burst phase change in fluorescence, although to a smaller extent, is shown to occur during the folding of wt barstar. The results suggest that a very early folding intermediate accumulates within 4 ms of folding, and that this kinetic intermediate is sufficiently compact that Trp53, which is completely sequestered from solvent in the fully folded protein, is also significantly sequestered from solvent in this intermediate.