CONFORMATIONAL PATHWAY OF THE POLYPEPTIDE-CHAIN OF CHYMOTRYPSIN INHIBITOR-2 GROWING FROM ITS N-TERMINUS IN-VITRO - PARALLELS WITH THE PROTEIN-FOLDING PATHWAY

We have obtained a series of fragments growing from the N terminus of the protein chymotrypsin inhibitor-2 (CI2) in order to study the devel opment of structure on elongation of the polypeptide in solution. We p resent an extensive biophysical characterization of ten fragments usin g different-conformational probes. Small fragments up to residue 40 of the 64-residue protein are disordered. Fragment (1-40) has non-native local hydrophobic clusters, but nevertheless does not bind 8-anilinon aphthalene-1-sulphonate (ANS). Hydrophobic regions in longer fragments become gradually more capable of binding ANS as the chain grows to co mpletion, with a tendency to form native structures. Major changes in secondary structure and accessibility to hydrophobic sites occur in pa rallel, between (1-40) and (1-53), together with changes in hydrodynam ic volume and flexibility NMR studies of (1-53), the first fragment di splaying tertiary interactions, show that a subcore is fully formed an d the alpha-helix (residues 12 to 24) is of fluctuating structure. Fra gments (1-53) and (1-60) share many properties with molten globule-lik e structures, with varying degrees or order. Fluorescence properties o f the native fold are gradually recovered from fragments (1-60) to ful l-length CI2, together with a decrease in hydrophobic exposure. A smal l degree of co-operativity of formation of structure appears when resi due 60 is added, gradually increasing as residue 62 is added, but a fu ll two-state co-operative transition appears only on addition of Arg62 and Val63. We believe this is the result of correct side-chain packin g of the hydrophobic core, capping the major elements of secondary str ucture in CI2 at this late stage, which is probed by the complete reco very of the fluorescence of the unique Trp5. The structures that devel op as the poly-peptide chain increases in length parallel the structur al features present in the nucleus for the folding of intact protein, which develops in the transition state. The folding nucleus consists o f much of the helix and the interactions made by Ala16 in the helix wi th residues in the core, especially with Leu49 and Ile57, with the res t of the structure being formed only very weakly in the transition sta te. (C) 1995 Academic Press Limited