KINETIC EVIDENCE OF MICROSCOPIC STATES IN PROTEIN FOLDING

Staphylococcal nuclease unfolds at acidic pHs and refolds at neutral pH. Previous kinetic analysis based on both the direct pH jump and the sequential pH jump, from a native condition (pH 7.0) to pHs beyond unfolding transition zones (pH 3.0 and pH 12), and vice versa, supports the mechanism, D3 half arrow right over half arrow left D2 half arrow right over half arrow left D1 half arrow right over half arrow left N0, in which N0 is the native state and D's are the three substates of the denatured form [Chen, H. M., You, J. L., Markin, V. S., & Tsong, T. Y. (I 990) J. Mol. Biol. 220, 771-778; Chen, H. M., Markin, V.S., & Tsong, T.Y. (1992) Biochemistry 31, 1483-1491]. Here we show that both the single- and the double-pH jump kinetics of folding and unfolding to the intermediate pHs (3.4-5.0, i.e., in the transition zone), in which both the native and the denatured states coexist, are not compatible with this simple sequential model. At 25-degrees-C, log tau1(-1) (for the D1 half arrow right over half arrow left N0 step) and log tau2(-1) (for the D2 half arrow right over half arrow left D1 step) vs pH show a square-root-shaped dependence on the final pH, with minimal values (tau1(-1) of 0.56 s-1 and tau2(-1) of around pH 3.9. The third relaxation tau3 (for the D3 half arrow right over half arrow left D2 step, 35 s) was independent of pH in the range 3.4-8.5. The square-root-shaped dependence on pH of log tau1(-1) and log tau2(-1) cannot be reproduced by the above ut can be accounted for if each of N0, D1, and D2 is composed of many microscopic states in rapid equilibrium. These microscopic states are designated as two subpopulations, alpha(i) (i = 0, 1, and 2), from which unfolding can take place, and beta(i) (i = 1, 2, and 3), from which folding can take place. Analysis of kinetic data indicates that alpha1 = 1 and alpha2 = 1 for the whole pH range. However, beta1 = 1 only for pH > 5.5 and decreases to 0 for pH < 3 (pK(a) around 4.5), and beta2 = 1 only for pH < 2 and decreases to small values for pH > 3 (pK(a) around 1.2). Analysis also shows that equilibrium unfolding of the protein is triggered by the absorption of 2.6 +/- 0.3 protons, of which 0.8 +/- 0.3 occurs in the N0 to D1 step and 1.7 +/- 0.3 occurs in the D1 to D2 step. We conclude that acidic unfolding can take place from the whole population of microscopic states while folding at pH 7 can take place only through those subpopulations which are folding-permitting. pH-dependent equilibrium transitions of all kinetically distinctive intermediates in the folding pathway are also determined and shown to be consistent with the above interpretation.