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

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Abstract
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: US → UF →N. US and UF are slow-refolding and fast-refolding unfolded forms of barstar, and N is the native protein. US and UF 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 US:UF (K21) and the values of the rate constants of the US → UF and UF → US reactions, k12 and k21 respectively, are shown to be independent of the concentration of GdnHCl. K21 has a value of 2.1 ± 0.1, and k12 and k21 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 K21, k12, and k21 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, UF → N, and US → IN → N, and IN is a native-like intermediate. The rate constants of the UF → N and US → IN reactions are both similar, with values of 37 s−1 in water. The IN → N reaction, which involves the same trans-cis isomerization process as the US → UF 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.