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 barsta r by guanidine hydrochloride (GdnHCl) in the folding transition zone, at pH 7, 25 degrees C, have been quantitatively analyzed using a 3-sta te mechanism: U-S reversible arrow U-F reversible arrow 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 tra ns and cis conformations, respectively, of the Tyr 47-Pro 48 bond. The 3-state model could be used because the kinetics of folding and unfol ding of barstar show 2 phases, a fast phase and a slow phase, and beca use the relative amplitudes of the 2 phases depend only on the final r efolding 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 betwee n 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 G dnHCl. K-21 has a value of 2.1 +/- 0.1, and k(12) and k(21) have value s of 5.3 x 10(-3) s(-1) and 11.2 x 10(-3) s(-1), respectively. Double- jump experiments that monitor reactions that are silent to fluorescenc e 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 fo lding 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 inter mediate. 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 rea ction, which involves the same trans-cia isomerization process as the U-S --> U-F reaction, occurs with a rate constant of 16 x 10(-3) s(-1) and is independent of GdnHCl concentration. Thus, trans-cis isomeriza tion occurs 3 times faster in the folding intermediate than in the unf olded state.