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

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: 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 (K₂₁) and the values of the rate constants of the U_S→U_F and U_F→U_S 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, 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.