Structural intermediates in folding of yeast iso-2 cytochrome c.
The kinetic properties of the folding reactions of iso-2 cytochrome c from Saccharomyces cerevisiae have been investigated by stopped-flow and temperature-jump methods. Three different structural probes are compared: (1) absorbance changes in the visible reflecting changes in heme environment, (2) ultraviolet absorbance changes due to the exposure of aromatic groups to solvent, and (3) tryptophan fluorescence attributable principally to the average distance between the tryptophan residue (donor) and the heme (quencher). In addition, two probes either indicative of or correlated with function, ascorbic acid reducibility and the 695-nm absorbance band, have been used to monitor specifically the rate of formation of the native protein on refolding. The fastest phase observed (tau 3) has a measurable relative amplitude only when monitored by visible absorbance changes, suggesting that this reaction involves changes in heme environment in the absence of significant changes in the heme to tryptophan distance or in the extent to which aromatic groups are exposed to solvent. Different slow phases are observed when complete refolding is monitored by visible or ultraviolet absorbance (tau 1a) as opposed to tryptophan fluorescence (tau 1b), the fluorescence changes being complete on a time scale 4-8-fold faster than for absorbance. A mid-range kinetic phase (tau 2) is detected by all three structural probes. When ascorbic acid reducibility or 695-nm absorbance changes are used to monitor the rate of formation of the native protein, two phases are detected: tau 2 and tau 1a. Taken together these results demonstrate that kinetic phase tau 1b results in the formation of a structural intermediate in folding with fluorescence close to that of the native protein but with distinct absorbance properties.
D003574:Cytochrome c Group / Q000031:analogs & derivatives*; D045304:Cytochromes c; D006146:Guanidines; D006418:Heme; D007700:Kinetics; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013053:Spectrophotometry; D013696:Temperature
D003574:Cytochrome c Group; D006146:Guanidines; C037958:iso-1-AM-cytochrome C; C032139:iso-2-cytochrome C; D006418:Heme; D045304:Cytochromes c