PHOSPHORESCENCE REVEALS A CONTINUED SLOW ANNEALING OF THE PROTEIN CORE FOLLOWING REACTIVATION OF ESCHERICHIA-COLI ALKALINE-PHOSPHATASE

When Escherichia coli alkaline phosphatase (AP) is refolded in vitro a fter extensive denaturation in 6.2 M guanidine hydrochloride, the enzy matic activity reaches its asymptotic value in 1 h at 24 degrees C. In contrast, the structural rigidity of the hydrophobic core of the prot ein, monitored by the recovery of the tryptophan phosphorescence lifet ime, returns to its characteristic native-like value over several days . Moreover, the protein lability, measured by the rate of inactivation in 4.5 M guanidine hydrochloride, also changes on a time scale much l onger than the recovery of activity. These results clearly demonstrate that while the return of enzymatic activity, the traditional measure of the attainment of the native state, indicates that AP has refolded to its final, active conformation, the phosphorescence data indicate o therwise. In the context of the rugged energy landscape model [Frauenf elder, H., et al. (1991) Science 254, 1598-1603], the slow annealing o f the hydrophobic core is consistent with the presence of high-energy barriers that separate fully active intermediates along the folding pa thway. The data suggest that the core of the protein undergoes continu ed structural rearrangements affecting the rigidity of the protein env ironment surrounding the emitting tryptophan and the protein lability long after the return of enzyme activity.