title: Phosphorescence reveals a continued slow annealing of the protein core following reactivation of Escherichia coli alkaline phosphatase.
abstract: When Escherichia coli alkaline phosphatase (AP) is refolded in vitro after extensive denaturation in 6.2 M guanidine hydrochloride, the enzymatic activity reaches its asymptotic value in 1 h at 24 degrees C. In contrast, the structural rigidity of the hydrophobic core of the protein, monitored by the recovery of the tryptophan phosphorescence lifetime, 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 longer 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 otherwise. In the context of the rugged energy landscape model [Frauenfelder, H., et al. (1991) Science 254, 1598-1603], the slow annealing of the hydrophobic core is consistent with the presence of high-energy barriers that separate fully active intermediates along the folding pathway. The data suggest that the core of the protein undergoes continued structural rearrangements affecting the rigidity of the protein environment surrounding the emitting tryptophan and the protein lability long after the return of enzyme activity.
authors: Institute of Gerontology, University of Michigan, Ann Arbor 48109.
authors: V
authors: V
authors: Subramaniam
authors: N C
authors: NC
authors: Bergenhem
authors: A
authors: A
authors: Gafni
authors: D G
authors: DG
authors: Steel
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D007700:Kinetics; D008163:Luminescent Measurements; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: D006146:Guanidines; D000469:Alkaline Phosphatase; D019791:Guanidine