FOLLOWING COOPERATIVE FORMATION OF SECONDARY AND TERTIARY STRUCTURE IN A SINGLE PROTEIN MODULE

We have prepared a family of peptide fragments of the 64 amino acid pr otein chymotrypsin inhibitor (CI2), corresponding to progressive elong ation from the N terminus, in order to elucidate the basis of conforma tional preferences in single-domain proteins and to obtain insights in to their conformational pathway. Structural analysis of the fragment c omprising the first 50 residues, CI2(1-50), indicates that it is mainl y disordered, with patches of hydrophobic residues exposed to the solv ent. Structural characterisation of the fragment CI2(1-63) which lacks only the C-terminal glycine, Gly64, shows native-like structure in al l regions of the fragment. The study provides insights into the contri bution of specific residues to the stability and co-operativity of the intact protein. We define a Phi(NMR) value, derived from chemical shi ft analysis, which describes the build-up of structure at the level of individual residues (protons). All the macroscopic probes used to stu dy the growth of structure in CI2 on elongation of the chain (circular dichroism, fluorescence and gel filtration) are in agreement with the residue-by-residue description by NMR. It is seen that secondary and tertiary structure build up in parallel in the fragments and show simi lar structures to those developed in the transition state for folding of the intact protein. (C) 1997 Academic Press Limited.