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 den aturation experiments, The wild-type protein and the global suppressor variant, N52I (isoleucine replaces asparagine 52), were examined. Den aturation was induced by guanidinium chloride (GdmCl) and monitored by circular dichroism (CD) spectropolarimetry without stopped-flow devic es, 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 rational ized as a requirement to bury the oxidation-induced positive charge an d remain folded under physiological conditions. Kinetic data are monoe xponential and permit characterization of the rate-limiting transition state for unfolding as a function of [GdmCl]. For the oxidized wild-t ype 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 positi on 52 in the oxidized protein, change the free energy and solvent acce ssibility of the transition state. In contrast, substituting c positio n 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 positio n 52 side chain is in a denatured environment, even though transition state solvent accessibility is only one-third that of the denatured st ate (relative to the native state).