Dependence of the size of the initially collapsed form during the refolding of barstar on denaturant concentration: evidence for a continuous transition.

Two-site fluorescence resonance energy transfer (FRET) measurements have been made to determine how two intra-molecular distances contract in the sub-millisecond collapse reaction that occurs initially during the refolding of the small protein barstar. FRET measurements were made on two, single-Cys and single-Trp-containing mutant forms of barstar, Cys25 and Cys62, in each of which a thionitrobenzoate (TNB) adduct was attached to the cysteine thiol. In each protein, the core tryptophan, Trp53, acted as the FRET donor, and the TNB adduct, located either at C25 or at C62, acted as the FRET acceptor. The stabilities as well as observable folding kinetics of the Cys25 and Cys62 mutant proteins were found to be identical. The presence of the TNB adduct on the cysteine did not alter the stability or folding kinetics of either protein. Thus, the FRET-monitored changes in the two labeled mutant proteins, Cys25-TNB and Cys62-TNB, could be compared directly. Refolding was commenced from unfolded protein in 8M urea, and both the Trp53 to C25-TNB distance and the Trp53 to C62-TNB distance were found to contract upon dilution of urea. The extent of contraction of each distance, which was measured at a few milliseconds of refolding, was dependent continuously on the concentration of urea present during refolding, and was different for the two distances. For either FRET pair, the gradual contraction of distance with a decrease in the concentration of urea in which refolding occurs, was continuous with the contraction of the polypeptide chain that is seen with a decrease in the concentration of urea in the range in which the protein remains completely unfolded. It therefore appears that the products of the initial sub-millisecond refolding reaction of barstar are collapsed forms, whose dimensions do not change cooperatively in an all-or-none manner, but instead, change gradually with a change in concentration of urea. Thus, the sub-millisecond polypeptide chain collapse reaction of barstar upon denaturant dilution, appears to be a continuous structural transition.