Search for nucleation sites in smaller fragments of chymotrypsin inhibitor 2.

There is a region of well-ordered structure in the transition state of folding of chymotrypsin inhibitor 2 (CI2) that consists of N-terminal residues in the unique alpha-helix (residues 12 to 24) plus some long range interactions, in particular those of Ala16 with Ile57 and Leu49 in the hydrophobic core. This is proposed to be a nucleation site. A crucial question for understanding the initiation of protein folding is: when is the nucleation site formed? Is the alpha-helix pre-formed in the nominally unfolded state, or does it require long-range interactions to be stabilized? To answer this question, we have characterized a series of N-terminal fragments of CI2, each containing an increasing number of subsets of the regular secondary structure. Four small fragments have been examined by circular dichroism and two-dimensional 1H and 15N NMR spectroscopy. The smallest, [1-5], comprises the sequence corresponding to the first beta-strand of the intact protein; the second, [1-13], contains also a type III reverse turn, the second beta-strand, and a type II reverse turn; the third [1-25], consists additionally of the sequence corresponding to the alpha-helix (residues 12 to 24); the fourth, [1-28], contains, in addition, the turn following the alpha-helix. All the fragments have disordered, non-compact structure in aqueous solution. In the structure-promoting co-solvent, trifluoroethanol, alpha-helical structure is stabilized in [1-25] and [1-28] in the region corresponding to the alpha-helix in the intact protein; however, the helix is frayed at both ends and is only fractionally populated, being in dynamic equilibrium with extended conformations. These observations indicate that there is little drive for independent formation of local secondary structure in CI2, and this is reflected in the highly concerted nature of the folding reaction of this protein. The nucleation site of folding of CI2 does not accumulate in the starting state for the folding reaction, but remains embryonic until there are sufficient long range interactions to stabilize it.