Abstract：
:Stopped-flow total intensity and anisotropy experiments on single tryptophan containing mutants of yeast phosphoglycerate kinase (PGK) located in either the carboxy-terminal domain (W308 and W333), amino-terminal domain (W48 and W122), or "hinge" region (W194 and W399) were performed. The results obtained for single tryptophans in individual domains suggest that the unfolding of PGK by guanidinium hydrochloride is a sequential process in which unfolding of the carboxy-terminal domain is followed by the unfolding of the amino-terminal domain. A kinetic intermediate has been detected which consists of an unfolded carboxy-terminal domain and an altered amino-terminal domain, identical in hydrodynamic properties with the native state, but hyperfluorescent. In contrast to the C-terminal tryptophans, which exhibit concurrent total intensity and anisotropy changes in the entire denaturant concentration range (0-->2 M), the N-terminal tryptophans experience a large increase in fluorescence intensity and a constant anisotropic environment at low concentrations of denaturant, corresponding to the first transition region of the equilibrium unfolding profile. Anisotropy changes for the N-terminal probes are observed above 1 M Gdn-HCl, the region corresponding to the second equilibrium unfolding transition. Stopped-flow experiments performed on PGK mutants with two tryptophans (i.e., with a single tryptophan in each domain) confirm that each domain unfolds independently, and that the individual site-specific mutations do not significantly alter the unfolding pathway. Unfolding kinetics experiments with tryptophans situated in the hinge reveal that the region sensed by W399 unfolds before the carboxy-terminal domain, whereas W194 senses unfolding of both domains.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Beechem JM,Sherman MA,Mas MTdoi
10.1021/bi00042a028subject
Has Abstractpub_date
1995-10-24 00:00:00pages
13943-8issue
42eissn
0006-2960issn
1520-4995journal_volume
34pub_type
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