GENERATION OF A FAMILY OF PROTEIN-FRAGMENTS FOR STRUCTURE-FOLDING STUDIES .2. KINETICS OF ASSOCIATION OF THE 2 CHYMOTRYPSIN INHIBITOR-2 FRAGMENTS

The kinetics of association of the fragments of the barley chymotrypsi n inhibitor-2, CI-2(20-59) and CI-2(60-83), to form a native-like stru cture 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-orde r phase of rate constant 0.011 +/- 0.001 s(-1). The major phase contai ns 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 second ary structure are formed simultaneously. A series of kinetic experimen ts suggest that the association and foldingoccur in the second-order s tep and that the first-order step probably results from a cis-trans pe ptidylprolyl isomerization in the fragment CI-2(20-59). This was confi rmed by experiments on fragments derived from two mutants whose parent proteins fold more slowly than wild-type CI-2. Those fragments displa y lower second-order rate constants, but the rate constants of the fir st-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-engin eering analysis.