Refolding kinetics of cytochrome c(551) reveals a mechansitic difference between urea and guanidine

The energetic parameters for the folding of small globular proteins can be very different if derived from guanidine hydrochloride (GdnHCl) or urea den aturation experiments. A study of the equilibrium and kinetics of the refol ding of wild-type (wt) cytochrome c(551) (cyt c(551)) from Pseudomonas aeru ginosa and of two site-directed mutants (E70Q and E70V) shows that the noni onic nature of urea reveals the role of a salt bridge between residues E70 and K10 on the transition state, which is otherwise completely masked in Gd nHCl experiments. Mixed denaturant refolding experiments allow us to conclu de that the masking effect of GdnHCl is complete at fairly low GdnHCl conce ntrations (congruent to0.1 M). The fact that potassium chloride is unable t o reproduce this quenching effect, together with the results obtained on th e mutants, suggests a specific binding of the Gdn(+) cation, which involves the E70-K10 ion pair in wt cyt c(551).We propose, therefore, a simple kine tic test to obtain a mechanistic interpretation of nonlinear dependences of DeltaG(w) on GdnHCl concentration on the basis of kinetic refolding experi ments in the presence of both denaturants.