Characterization of an intermediate in the folding pathway of phosphoglycerate kinase: chemical reactivity of genetically introduced cysteinyl residues during the folding process.
The unfolding-refolding kinetics of yeast phosphoglycerate kinase were studied using the chemical reactivity of genetically introduced cysteinyl residues as conformational probes and far-ultraviolet circular dichroism. A unique internal cysteinyl residue was introduced in several mutants at selected positions in the N- and C-domains. The cysteinyl residues were at positions 97 (the unique cysteinyl residue of the wild-type enzyme), 183 in the N-domain, 285 and 324 in the C-domain. A similar strategy has been used to study the unfolding-refolding transition under equilibrium conditions [Ballery et al. (1990) Protein Eng. 3, 199-204]. Except for the mutant C97A,A183C, whose cysteinyl residue is located at the domain interface, three labeling phases were observed during the refolding process, indicating the presence of three species, the unfolded, intermediate, and folded proteins. The comparison of the data obtained following the accessibility of the thiol group to 5,5'-dithiobis(2-nitrobenzoate) and ellipticity at 218 nm indicated that all mutants have the same folding pathway and allowed us to characterize the intermediate. In this species, each domain appeared to have a high content of secondary structure but a flexible tertiary structure; this intermediate, which had the characteristics of a molten globule, remained in fluctuating equilibrium with a widely unfolded form. The same folding intermediate was detected for mutant C97A,A183C; however, the cysteinyl residue being totally accessible to the reagent, it is likely that in this intermediate the interdomain interactions are not established. Domain pairing and formation of the native tertiary structure occur simultaneously in the slow phase of refolding. The validity and limitations of the methodology are discussed.
Ballery
N
N
Laboratoire d'Enzymologie Physicochimique et Moléculaire, Centre National de la Recherche Scientifique, Université de Paris-Sud, Orsay, France.
Desmadril
M
M
Minard
P
P
Yon
J M
JM
D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013438:Sulfhydryl Compounds / Q000737:chemistry
D013438:Sulfhydryl Compounds; D010735:Phosphoglycerate Kinase; D003545:Cysteine