PROTEIN-FRAGMENTS AS MODELS FOR EVENTS IN PROTEIN-FOLDING PATHWAYS - PROTEIN ENGINEERING ANALYSIS OF THE ASSOCIATION OF 2 COMPLEMENTARY FRAGMENTS OF THE BARLEY CHYMOTRYPSIN INHIBITOR-2 (CI-2)

Two fragments of chymotrypsin inhibitor-2, CI-2(20-59) and CI-2(60-83) , derived from cyanogen bromide cleavage at Met-59, associate to give a native-like structure. We analyze the kinetics and equilibria of ass ociation of mutant fragments derived from cleaving mutant proteins at the same methionine residue. The changes in free energy of association have been measured both from isothermal studies of the binding of fra gments and from thermal denaturation of the complexes. In general, the re is a good correlation between the changes on mutation of the free e nergy of association of fragments and the changes in free energy of fo lding of the uncleaved parent protein. The notable exceptions are for residues in regions of the fragments that form nonnative hydrophobic c lusters in the isolated fragments, mutation of the hydrophobic residue s involved in these clusters decreases the equilibrium constant for fo rmation of the noncovalent complex less than it does the equilibrium c onstant for folding, of intact protein. The dissociated fragments must be destabilized by mutation of those hydrophobic residues, but to a l esser extent than is the complex itself. These clusters are thus less important energetically in the denatured state of the intact protein. The second-order rate constants for the major phase of association cha nge with mutation, similar results being obtained from fluorescence me asurements of the regain of tertiary structure and from circular dichr oism measurements of the regain of secondary structure. The rate const ants for association correlate well, in general, with the rate constan ts of refolding of the respective uncleaved proteins. Fragments that h ave mutations in the regions of nonnative hydrophobic clusters associa te faster than expected from the correlation. Thus, breaking up the cl usters facilitates the rate of folding. It is remarkable that the two fragments associate via a transition state that is very similar to tha t for the folding of the intact protein.