STRUCTURE OF THE HYDROPHOBIC CORE IN THE TRANSITION-STATE FOR FOLDINGOF CHYMOTRYPSIN INHIBITOR-2 - A CRITICAL TEST OF THE PROTEIN ENGINEERING METHOD OF ANALYSIS

Chymotrypsin inhibitor 2 (CI2) unfolds and refolds according to a simp le two-state kinetic mechanism. The single rate-determining transition state may thus be studied by kinetics of both unfolding and refolding . This has allowed the direct testing of some facets of the protein en gineering procedure (phi-value analysis). The structure of the hydroph obic core of CI2 in the transition state was analyzed from kinetic and thermodynamic measurements of guanidinium chloride-induced unfolding of 11 mutants and of their rates of refolding. In all cases, the stren gths of the interactions measured from refolding kinetics in water are in excellent agreement with those measured from unfolding kinetics in guanidinium chloride solutions and extrapolated to zero molar denatur ant. Changes in the free energies of unfolding on mutation, as well as other equilibrium properties calculated from the rate constants, are also in excellent agreement with those measured directly from equilibr ium studies. These data provide further evidence for application of th e principle of microscopic reversibility to aspects of protein folding in the presence of denaturant and the validity of extrapolation to th e absence of denaturant. The edges of the hydrophobic core of CI2 are significantly weakened in the transition state, and, in many cases, th e interactions are totally lost. The center of the core remains partia lly intact; the interaction energy is lowered by about 50%. In one cas e, Val --> Ala38, a residue which lies on the edge of the core and who se side chain is 44% solvent-exposed, the interaction energy in the tr ansition state (0.86 kcal mol-1) is found to be larger than in the nat ive state (0.46 kcal mol-1), suggesting that this region is more burie d in the transition state than in the native state. The results are co nsistent with the proposal that one of the final events in protein fol ding is the consolidation of the hydrophobic core and that the rate-de termining step involves the close packing of the side chains within th e hydrophobic core.