CONSERVED RESIDUES AND THE MECHANISM OF PROTEIN-FOLDING

EXPERIMENTAL(1-6) and simulation(7) studies show that small monomeric proteins fold in one kinetic step, which entails overcoming the free-e nergy barrier between the unfolded and the native protein through a tr ansition state(8,9). Two models of transition state formation have bee n proposed: a 'nonspecific' one in which it depends on the formation o f a sufficient number of native-like contacts regardless of what amino acids are involved(10-12) and a 'specific' one, in which it depends o n formation of a specific subset of the native structure (a folding nu cleus)(8,13,14). The latter requires that some amino acids form most o f their contacts in the transition state, whereas others only do so on reaching the native conformation. If so, mutations affecting the stab ility of the transition state nucleus should have a greater effect on the folding kinetics than mutations elsewhere, and the residues involv ed should be evolutionarily conserved. Lattice-model simulations and e xperiments(8,13-16) suggest that such mutations exist. Here we present a method for determining the folding nucleus of a protein with known structure with two-state folding kinetics. This method is based on the alignment of many sequences designed to fold into the native conforma tion of a protein to identify the positions where amino acids are most conserved in designed sequences. The method is applied to chymotrypsi n inhibitor 2 (CI2), a protein whose transition state has been previou sly studied by protein engineering(14-16). The involvement of residues in folding nucleus of CI2 is clearly correlated with their conservati on in design, and the residues forming the nucleus are highly conserve d in 23 natural sequences homologous to CI2.