REFOLDING RATE OF STABILITY-ENHANCED CYTOCHROME-C IS INDEPENDENT OF THERMODYNAMIC DRIVING-FORCE

N52I iso-2 cytochrome c is a variant of yeast iso-2 cytochrome c in wh ich asparagine substitutes for isoleucine 52 in an alpha helical segme nt composed of residues 49-56. The N52I substitution results in a sign ificant increase in both stability and cooperativity of equilibrium un folding, and acts as a ''global suppressor'' of destabilizing mutation s. The equilibrium m-value for denaturant-induced unfolding of N52I is o-2 increases by 30%, a surprisingly large amount for a single residue substitution. The folding/unfolding kinetics for N52I iso-2 have been measured by stopped-flow mixing and by manual mixing, and are compare d to the kinetics of folding/unfolding of wild-type protein, iso-2 cyt ochrome c. The results show that the observable folding rate and the g uanidine hydrochloride dependence of the folding rate are the same for iso-2 and N52I iso-2, despite the greater thermodynamic stability of N52I iso-2 Thus, there is no linear free-energy relationship between m utation-induced changes in stability and observable refolding rates. H owever, for N52I iso-2 the unfolding rate is slower and the guanidine hydrochloride dependence of the unfolding rate is smaller than for iso -2. The differences in the denaturant dependence of the unfolding rate s suggest that the N52I substitution decreases the change in the solve nt accessible hydrophobic surface between the native state and the tra nsition state. Two aspects of the results are inconsistent with a two- state folding/unfolding mechanism and imply the presence of folding in termediates: (1) observable refolding rate constants calculated from t he two-state-mechanism by combining equilibrium data and unfolding rat e measurements deviate from the observed refolding rate constants; (2) kinetically unresolved signal changes (''burst phase'') are observed for both N52I iso-2 and iso-2 refolding. The ''burst phase'' amplitude is larger for N52I iso-2 than for iso-2, suggesting that the intermed iates formed during the ''burst phase'' are stabilized by the N52I sub stitution.