Structural Intermediates in Folding of Yeast Iso-2 Cytochrome c

Barry T. Nall

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

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 (τ3) 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 (τ1a) as opposed to tryptophan fluorescence (τ1b), the fluorescence changes being complete on a time scale 4–8-fold faster than for absorbance. A mid-range kinetic phase (τ2) 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: τ2 and τ1a Taken together these results demonstrate that kinetic phase τ1b results in the formation of a structural intermediate in folding with fluorescence close to that of the native protein but with distinct absorbance properties.

Original languageEnglish (US)
Pages (from-to)1423-1429
Number of pages7
JournalBiochemistry
Volume22
Issue number6
DOIs
StatePublished - 1983
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

Fingerprint

Dive into the research topics of 'Structural Intermediates in Folding of Yeast Iso-2 Cytochrome c'. Together they form a unique fingerprint.

Cite this