Sequential domain unfolding in phosphoglycerate kinase: fluorescence intensity and anisotropy stopped-flow kinetics of several tryptophan mutants.
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.
Beechem
J M
JM
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, USA.
Sherman
M A
MA
Mas
M T
MT
D005454:Fluorescence Polarization; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics; D015003:Yeasts / Q000201:enzymology*
D006146:Guanidines; D014364:Tryptophan; D010735:Phosphoglycerate Kinase; D019791:Guanidine