Changing the transition state for protein (un)folding

(Un)folding transition states of Saccharomyces cerevisiae iso-1-ferri- and ferrocytochromes c were studied using equilibrium and kinetic denaturation experiments, The wild-type protein and the global suppressor variant, N52I (isoleucine replaces asparagine 52), were examined. Denaturation was induced by guanidinium chloride (GdmCl) and monitored by circular dichroism (CD) spectropolarimetry without stopped-flow devices, Soret CD spectra indicate that thermal and GdmCl denatured states are different, and heat is the more effective denaturant. Equilibrium data show that the high stability of ferrocytochrome c can be rationalized as a requirement to bury the oxidation-induced positive charge and remain folded under physiological conditions. Kinetic data are monoexponential and permit characterization of the rate-limiting transition state for unfolding as a function of [GdmCl]. For the oxidized wild-type protein, the transition state solvent accessibility is nearly the same as that of the denatured state. Three perturbations, reducing the wild-type protein, reducing the N52I variant, and substituting position 52 in the oxidized protein, change the free energy and solvent accessibility of the transition state. In contrast, substituting c position 52 in the reduced protein apparently does not change the transition state solvent accessibility: allowing more detailed characterization. In the reduced proteins' transition states at 4.3 M GdmCl, the position 52 side chain is in a denatured environment, even though transition state solvent accessibility is only one-third that of the denatured state (relative to the native state).