FOLDING OF CIRCULAR AND PERMUTED CHYMOTRYPSIN INHIBITOR-2 - RETENTIONOF THE FOLDING NUCLEUS

The 64-residue chymotrypsin inhibitor 2 (CI2) folds by a two-state nuc leation-condensation mechanism, whereby secondary and tertiary structu re coalesce concomitantly in the transition state around Ala 16 in the helical N-cap. Permutation of the SH3-domain of alpha-spectrin appare ntly shifts its folding nucleus to another region of the protein, sugg esting that a protein's transition state may be altered by altering th e protein's connectivity. We have characterized the structure of the t ransition state of a circular and a permuted version of CI2 by a prote in engineering study encompassing 11 mutations. Circular CI2 was obtai ned by the introduction of cysteines at residues 3 and 63 and linking them by disulfide bond formation. Subsequent cyanogen-bromide cleavage of the scissile bond, Met 40-Glu 41, yielded permuted CI2. Circular a nd permuted CI2 also fold according to a two-state mechanism. Permutat ion does not affect the folding rate constant, but circularization inc reases it 7-fold. The transition states of circular and permuted CI2 a re essentially unchanged from that of wild-type CI2. Importantly, the folding nucleus around Ala16 is retained. These results complement a p revious observation that the transition state for association of two C I2 fragments (residues 1-40 and 41-64, generated by CNBr cleavage) is very similar to the folding transition state of intact CI2. The simila rity of rate constants for folding of wild-type and permuted CI2, and their value relative to that for the association of fragments, allows us to estimate the gain in entropy of activation on having the separat e fragments linked: 18.3 cal M-1 K-1; i.e. an effective molarity of 10 (4) M. The contrast between the retention of the folding nucleus on pe rmutation of CI2 and its change for the SH3-domain of alpha-spectrin p robably arises because the latter was cleaved in its folding nucleus w hereas cleavage at sites other than 40-41 in CI2 is very destabilizing . Whether or not a folding nucleus can be changed probably depends on the specific protein and its permissivity to permutation.