Folding of yeast iso-1-AM cytochrome c.
We describe a specific modification of iso-1 cytochrome c which results in blocking a single free sulfhydryl group. The derivative differs from the unmodified protein by the introduction of a small, uncharged group, thus maintaining the same charge balance as the native protein. The modified protein, obtained by treatment of iso-1 cytochrome c with iodoacetamide, has an activity indistinguishable from that of the unmodified protein in the lactate dehydrogenase-cytochrome c reductase system from yeast and has the same stability toward denaturation by guanidine hydrochloride. The kinetics of fluorescence changes associated with the guanidine hydrochloride induced folding-unfolding transition for modified iso-1 cytochrome c (iso-1-AM) have been investigated throughout the transition zone by using stopped-flow mixing. The results are compared to those for the yeast isozyme, iso-2 cytochrome c. The main features of the fluorescence-detected folding kinetics are similar, as might be expected for homologous proteins; however, the limiting value of the fraction of fast refolding protein (alpha 2) below the transition zone is smaller for iso-1-AM (approximately 0.7) than for iso-2 (approximately 0.9).
D000596:Amino Acids / Q000032:analysis; D001345:Autoradiography; D055598:Chemical Phenomena; D002621:Chemistry; D003574:Cytochrome c Group / Q000031:analogs & derivatives; D045304:Cytochromes c; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000032:analysis
D000596:Amino Acids; C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D006146:Guanidines; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D013438:Sulfhydryl Compounds; D045304:Cytochromes c; D019791:Guanidine