THE STRUCTURE OF THE TRANSITION-STATE FOR FOLDING OF CHYMOTRYPSIN INHIBITOR-2 ANALYZED BY PROTEIN ENGINEERING METHODS - EVIDENCE FOR A NUCLEATION-CONDENSATION MECHANISM FOR PROTEIN-FOLDING

The 64-residue protein chymotrypsin inhibitor 2 (CI2) is a single modu le of structure. It folds and unfolds as a single co-operative unit by simple two-state kinetics via a single rate determining transition st ate. This transition state has been characterized at the level of indi vidual residues by analysis of the rates and equilibria of folding of some 100 mutants strategically distributed at 15 sites throughout the protein. Only one residue, a helical residue (Ala16) buried in the hyd rophobic core, has its full native interaction energy in the transitio n state. The only region of structure which is well developed in the t ransition state is the alpha-helix (residues 12 to 24). But, the inter actions within it are weakened, especially at the C-terminal region. T he rest of the protein has varying degrees of weakly formed structure. Thus, secondary and tertiary interactions appear to form concurrently These data, reinforced by studies on the structures of peptide fragme nts, fit a ''nucleation-condensation'' model in which the overall stru cture condenses around an element of structure, the nucleus, that itse lf consolidates during the condensation. The high energy transition st ate is composed of the whole of the molecule making a variety of weak interactions, the nucleus being those residues that make the strongest Interactions. The nucleus here is part of the cc-helix and some dista nt residues in the sequence with which it makes contacts. The remainde r of the protein has to be sufficiently ordered that it provides the n ecessary interactions to stabilize the nucleus. The nucleus is only we akly formed in the denatured state but develops in the transition stat e. The onrush of stability as the nucleus consolidates its local and l ong range interactions is so rapid that it is not yet fully formed in the transition state. The formation of the nucleus is thus coupled wit h the condensation. These results are consistent with a recent simulat ion of the folding of a computer model protein on a lattice which is f ound to proceed by a nucleation-growth mechanism. We suggest that the mechanism of folding of CI2 may be a common theme in protein folding w hereby fundamental folding units of larger proteins, which are modelle d by the folding of CI2, form by nucleation-condensation events and co alesce, perhaps in a hierarchical manner. (C) 1995 Academic Press Limi ted