THERMODYNAMICS OF THE COMPLEX PROTEIN UNFOLDING REACTION OF BARSTAR

The complex unfolding reaction of barstar has been characterized by st udying the apparent rate of unfolding, monitored by intrinsic Trp fluo rescence, as a function of temperature and guanidine hydrochloride (Gd nHCl) concentration. The kinetics of unfolding and folding of wild-typ e (wt) barstar at 5 degrees C were first studied in detail. It is show n that when unfolding is carried out using concentrations of GdnHCl in the posttransition zone of unfolding, the change in fluorescence that accompanies unfolding occurs in two phases: 30% of the change occurs in a burst phase that is complete within 4 ms, and 70% of the change o ccurs in a fast phase that is complete within 2 s. In contrast, when t he protein is unfolded at 25 degrees C, no burst-phase change in fluor escence is observed. To confirm that a burst-phase change in fluoresce nce indeed accompanies unfolding at low temperature, unfolding studies were also carried out on a marginally destabilized mutant form of bar star for which the burst-phase change in fluorescence is shown to be a s high as 70%. These results confirm a previous report [Nath et al., ( 1996), Nat. Struct. Biol. 3, 920-923], in which the detection of a bur st-phase change in circular dichroism at 222 nm during unfolding at 25 degrees C led to the inclusion of a rapidly formed kinetic intermedia te, I-U, on the unfolding pathway. To characterize thermodynamically t he unfolding pathway, apparent unfolding rates were then measured at s ix different concentrations of GdnHCl in the range 2.6 to 5.0 M, at fi ve different temperatures from 5 to 46 degrees C. The subsequent analy sis was done on the basis of the observation that a preequilibrium bet ween the fully folded state (F) and I-U gets established rapidly befor e further unfolding to the completely unfolded state (U). The results indicate that I-U has a specific heat capacity similar to that of F an d therefore suggest that I-U is as compact as F, with practically no e xposure of the hydrophobic core. On the other hand, the transition sta te of unfolding has a 45% greater heat capacity than F, indicating tha t significant hydration of the hydrophobic core occurs only after the rate-limiting step of unfolding.