FOLDING OF CHYMOTRYPSIN INHIBITOR-2 .1. EVIDENCE FOR A 2-STATE TRANSITION

The reversible folding and unfolding of barley chymotrypsin inhibitor 2 (CI2) appears to be a rare example in which both equilibria and kinetics are described by a two-state model. Equilibrium denaturation by guanidinium chloride and heat is completely reversible, and the data can be fitted to a simple two-state model involving only native and denatured forms. The free energy of folding in the absence of denaturant, DELTA-G(H2O) at pH 6.3, is calculated to be 7.03 +/- 0.16 and 7.18 +/- 0.43 kcal mol-1 for guanidinium chloride and thermal denaturation, respectively. Scanning microcalorimetry shows that the ratio of the van't Hoff enthalpy of denaturation to the calorimetric enthalpy of denaturation does not deviate from unity, the value observed for a two-state transition, over the pH range 2.2-3.5. The heat capacity change for denaturation is found to be 0.789 kcal mol-1 K-1. The rate of unfolding of CI2 is first order and increases exponentially with increasing guanidinium chloride concentration. Refolding, however, is complex and involves at least three well-resolved phases. The three phases result from heterogeneity of the unfolded form due to proline isomerization. The fast phase, 77% of the amplitude, corresponds to the refolding of the fraction of the protein that has all its prolines in a native trans conformation. The rate of this major phase decreases exponentially with increasing guanidinium chloride concentration. The unfolding and refolding kinetics can also be fitted to a two-state model. Importantly, DELTA-G(H2O) and m, the constant of proportionality of the free energy of folding with respect to guanidinium chloride concentration, calculated from the kinetic experiments, 7.24 +/- 0.22 kcal mol-1 and 1.86 +/- 0.05 kcal mol-1 M-1, respectively, agree, within experimental error, with the values measured from the equilibrium experiments. This is perhaps the strongest evidence that the unfolding of CI2 follows a simple two-state transition.