FOLDING OF TRYPTOPHAN MUTANTS OF BARSTAR - EVIDENCE FOR AN INITIAL HYDROPHOBIC COLLAPSE ON THE FOLDING PATHWAY

The contributions of the three tryptophan residues of barstar to the s pectroscopic properties, stability, and folding of the protein have be en studied by mutating two of the tryptophans, Trp38 and Trp44, indivi dually as well as together, to phenylalanines, Phe. The three mutant p roteins studied are shown to be similar to wt barstar in structure by activity measurements as well as by spectroscopic characterization. Fl uorescence energy transfer between the tryptophans as well as quenchin g by their local structural environments complicates the analysis of t he 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 t o the fluorescence, while the fluorescence of Trp38 is largely quenche d 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 to tal fluorescence change that occurs during folding occurs in a burst p hase within 4 ms after commencement of folding. A similar burst phase change in fluorescence, although to a smaller extent, is shown to occu r during the folding of wt barstar. The results suggest that a very ea rly 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 a lso significantly sequestered from solvent in this intermediate.