Generation of a family of protein fragments for structure-folding studies. 2. Kinetics of association of the two chymotrypsin inhibitor-2 fragments.

The kinetics of association of the fragments of the barely chymotrypsin inhibitor-2, CI-2(20-59) and CI-2(60-83), to form a native-like structure follows two phases. There is a major second-order component with rate constant (3.7 +/- 0.3) x 10(3) M-1 s-1 and a slow first-order phase of rate constant 0.011 +/- 0.001 s-1. The major phase contains a cooperative folding process as judged by the secondary structure recovery in parallel with the fluorescence change. The time course for structure formation has uniform changes at all of the wavelengths of the circular dichroism spectra, suggesting that all elements of secondary structure are formed simultaneously. A series of kinetic experiments suggest that the association and folding occur in the second-order step and that the first-order step probably results from a cis-trans peptidylprolyl isomerization in the fragment CI-2(20-59). This was confirmed by experiments on fragments derived from two mutants whose parent proteins fold more slowly than wild-type CI-2. Those fragments display lower second-order rate constants, but the rate constants of the first-order phase are the same as for wild type. The experiments suggest that the mechanism of the association/folding of mutant fragments may be studied by a protein-engineering analysis.