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 (DELTAG(d) at 300 K) is decreased relative to non-cross-linked controls. The dependence of DELTAG(d) on GdnHCl concentration, the GdnHCl concentration that denatures half the protein, as well as the enthalpy, entropy, and heat capacity changes (m(GdnHCl), C(m), DELTAH(d), DELTAS(d), and DETLAC(p), respectively), all decrease in magnitude upon introduction of the cross-link. The decrease in DETLAH(d) and DETLAS(d) 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 DETLAG(d), DETLAH(d), DELTAS(d), DETLAC(p), and m(GdnHCl) result from the denatured state of the cross-linked variant being more compact and possessing more structure than non-cross-linked controls.