Introduction of a disulfide bond into cytochrome c stabilizes a compact denatured state.
We introduced a novel disulfide bond, modeled on that of bullfrog cytochrome c, into yeast iso-1-cytochrome c. The disulfide spontaneously forms upon purification. A variety of techniques were used to examine the denaturation of this variant and several non-cross-linked controls. Denaturation is reversible and, with the exception of the protein in which the two cysteines are blocked, consistent with a two-state process. Comparison of the calorimetric and van't Hoff enthalpy changes indicates that denaturation is two-state at pH 4.6. Calorimetric and fluorescence-monitored guanidine hydrochloride (GdnHCl) denaturation data indicate that the free energy of denaturation for the cross-linked protein (delta Gd at 300 K) is decreased relative to non-cross-linked controls. The dependence of delta Gd on GdnHCl concentration, the GdnHCl concentration that denatures half the protein, as well as the enthalpy, entropy, and heat capacity changes (mGdnHCl, Cm, delta Hd, delta Sd, and delta Cp, respectively), all decrease in magnitude upon introduction of the cross-link. The decrease in delta Hd and delta Sd were confirmed by monitoring absorbance at several wavelengths as a function of temperature. The cross-link also decreases the pH dependence of these observables. Circular dichroism studies indicate the denatured state of the cross-linked protein possesses more structure than non-cross-linked proteins, and this structure is refractory to increases in temperature and chemical denaturant. We conclude that the diminished values of delta Gd, delta Hd, delta Sd, delta Cp, and mGdnHCl result from the denatured state of the cross-linked variant being more compact and possessing more structure than non-cross-linked controls.
Betz
S F
SF
Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290.
Pielak
G J
GJ
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D003574:Cytochrome c Group / Q000737:chemistry*; D045304:Cytochromes c; D004220:Disulfides / Q000737:chemistry*; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
C014059:CYC1 protein, S cerevisiae; D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D004220:Disulfides; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c