STRAIN IN THE FOLDING NUCLEUS OF CHYMOTRYPSIN INHIBITOR-2

Background: Chymotrypsin inhibitor 2 (Cl2) is a member of the class of fast-folding small proteins, which is very suitable for testing theor ies of folding. Cl2 folds around a diffuse extended nucleus consisting of the single alpha helix and a set of hydrophobic residues. In parti cular, Ala16 has been predicted and independently found to interact wi th Leu49 and lle57, hydrophobic residues that are highly conserved amo ng homologues. We have characterised in detail the interactions betwee n these residues in the folding nucleus of the protein by using double -mutant cycles. Results: Surprisingly, we find that there is some dest abilising strain in the transition state for folding of the wild-type protein between Ala16 and lle57. Further, we find that the strain is l arger in the native state of the protein. This is shown directly in th e unfolding kinetics, which clearly show a release of strain. The net result of this is that the presence of both residues speeds up folding . Ala16 and Leu49 interact favourably in the transition state, but hav e no net interaction energy in the native state. Conclusions: Part of the folding nucleus of the protein fits together more snugly in the tr ansition state than it does in the native state. Interactions between some of the closely packed residues in the folding nucleus of Cl2 may perhaps be optimised for the rate of folding and not for stability.