Detection of intermediates in the unfolding transition of phosphoglycerate kinase using limited proteolysis.
The accessibility of peptide bonds to cleavage by Staphylococcus aureus V8 protease bound on a Sepharose matrix was used as a conformational probe in the study of the unfolding-folding transition of phosphoglycerate kinase induced by guanidine hydrochloride. It was shown that the protein is resistant to proteolysis below a denaturant concentration of 0.4 M. The transition curve, determined by susceptibility toward proteolysis, was similar to that obtained following the enzyme activity [Betton et al. (1984) Biochemistry 23, 6654-6661]. Proteolysis under conditions where the folding intermediates are more populated, i.e., 0.7 M Gdn.HCl, gave two major fragments of Mr 25K and 11K, respectively. The 25K polypeptide fragment was identified as the carboxy-terminal domain. Its conformation was similar to that of a folding intermediate trapped at a critical concentration of denaturant, and in this form, it was not able to bind nucleotide substrates [Mitraki et al. (1987) Eur. J. Biochem. 163, 29-34]. From the present data and those previously reported, we concluded that the intermediate detected on the folding pathway of phosphoglycerate kinase has a partially folded carboxy-terminal domain and an unfolded amino-terminal domain.
Laboratoire d'Enzymologie Physico-chimique et Moléculaire, Groupe de Recherche du Centre National de la Recherche, Scientifique associé à l'Université de Paris-Sud, Orsay, France.
J M
JM
Betton
M
M
Desmadril
J M
JM
Yon
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002942:Circular Dichroism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006736:Horses; D007700:Kinetics; D008958:Models, Molecular; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments / Q000302:isolation & purification; D010735:Phosphoglycerate Kinase / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D012697:Serine Endopeptidases / Q000378:metabolism
D000596:Amino Acids; D006146:Guanidines; D010446:Peptide Fragments; D010735:Phosphoglycerate Kinase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D019791:Guanidine