Kinetic studies of the refolding of yeast phosphoglycerate kinase: comparison with the isolated engineered domains.
Unfolding and refolding kinetics of yeast phosphoglycerate kinase were studied by following the time-dependent changes of two signals: the ellipticity at 218 nm and 222 nm, and the fluorescence emission at 330 nm (following excitation at 295 nm). The protein is composed of two similar-sized structural domains. Each domain has been produced by recombinant DNA techniques. It has been previously demonstrated that the engineered isolated domains are able to fold into a quasinative structure (Minard, P., et al., 1989b, Protein Eng. 3, 55-60; Missiakas, D., Betton, J.M., Minard, P., & Yon, J.M., 1990, Biochemistry 29, 8683-8689). The behavior of the isolated domains was studied using the same two conformational probes as for the whole enzyme. We found that the refolding kinetics of each domain are multiphasic. In the whole protein, domain folding and pairing appeared to be simultaneous events. However, it was found that some refolding steps occurring during the refolding of the isolated C-domain are masked during the refolding of yeast phosphoglycerate kinase. The N-domain was also found to refold faster when it was isolated than when integrated.
Missiakas
D
D
Laboratoire d'Enzymologie Physicochimique et Moléculaire, Unité de Recherche du Centre National de la Recherche Scientifique, Université de Paris-Sud, Orsay, France.
Betton
J M
JM
Chaffotte
A
A
Minard
P
P
Yon
J M
JM
D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors
D006146:Guanidines; D011994:Recombinant Proteins; D010735:Phosphoglycerate Kinase; D019791:Guanidine