Quantitative analysis of the kinetics of denaturation and renaturation of barstar in the folding transition zone

The fluorescence‐monitored kinetics of folding and unfolding of barstar by guanidine hydrochloride (GdnHCl) in the folding transition zone, at pH 7, 25 °C, have been quantitatively analyzed using a 3‐state mechanism: U S → U F →N. U S and U F are slow‐refolding and fast‐refolding unfolded forms of barstar, and N is the native protein. U S and U F probably differ in possessing trans and cis conformations, respectively, of the Tyr 47‐Pro 48 bond. The 3‐state model could be used because the kinetics of folding and unfolding of barstar show 2 phases, a fast phase and a slow phase, and because the relative amplitudes of the 2 phases depend only on the final refolding conditions and not on the initial conditions. Analysis of the observed kinetics according to the 3‐state model yields the values of the 4 microscopic rate constants that describe the transitions between the 3 states at different concentrations of GdnHCl. The value of the equilibrium unfolded ratio U S :U F ( K 21 ) and the values of the rate constants of the U S → U F and U F → U S reactions, k 12 and k 21 respectively, are shown to be independent of the concentration of GdnHCl. K 21 has a value of 2.1 ± 0.1, and k 12 and k 21 have values of 5.3 × 10 −3 s −1 and 11.2 × 10 −3 s −1 , respectively. Double‐jump experiments that monitor reactions that are silent to fluorescence monitoring were used to confirm the values of K 21 , k 12 , and k 21 obtained from the 3‐state analysis and thereby the validity of the 3‐state model. The 3‐state model does not account for the kinetics of folding in the pretransition region, where folding occurs by 2 parallel pathways, U F → N, and U S → I N → N, and I N is a native‐like intermediate. The rate constants of the U F → N and U S → I N reactions are both similar, with values of 37 s −1 in water. The I N → N reaction, which involves the same trans‐cis isomerization process as the U S → U F reaction, occurs with a rate constant of 16 × 10 −3 s −1 and is independent of GdnHCl concentration. Thus, trans‐cis isomerization occurs 3 times faster in the folding intermediate than in the unfolded state.