Occurrence of transient multimeric species during the refolding of a monomeric protein.
A set of protein fragments from yeast phosphoglycerate kinase were produced by chemical cleavage at a unique cysteinyl residue previously introduced by site-directed mutagenesis. Cross-linking experiments showed that the fragments corresponding to incomplete N-terminal domain form stable oligomeric species. Transient oligomeric species were also observed by both cross-linking and light scattering experiments during the folding process of the whole protein. These transient oligomeric species are formed during the fast folding phase and dissociate during the slow folding phase to produce the monomeric active protein. The multimeric species are not required for the protein to fold correctly. Unexpectedly, the distribution of oligomeric species is not dependent on protein concentration during the folding process. A kinetic competition mechanism is proposed as a possible solution to this paradox. These results provide direct evidence that the polypeptide chain can explore nonnative interactions during the folding process.
Pecorari
F
F
Laboratoire d'Enzymologie Physicochimique et Moléculaire Unité de Recherches du CNRS, Université de Paris-Sud, Bat 430, 91405 Orsay, cedex France.
Minard
P
P
Desmadril
M
M
Yon
J M
JM
D000818:Animals; D003432:Cross-Linking Reagents; D006736:Horses; D007700:Kinetics; D046911:Macromolecular Substances; D009132:Muscles / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017354:Point Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012542:Scattering, Radiation; D014461:Ultracentrifugation
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010735:Phosphoglycerate Kinase