Analysis of the mechanism of assembly of cleaved barnase from two peptide fragments and its relevance to the folding pathway of uncleaved barnase.

A peptide corresponding to residues 1-22 of barnase that contains its major alpha-helix (residues 6-18) binds rapidly to the complementary peptide (residues 23-110) to form a catalytically active complex with near-native structure. Peptide 1-22 is approximately 3% helical in aqueous solution at 25 degrees C. A set of mutations in the helical regions of (1-22) cause the helix to be destabilized. We have investigated the mechanism of assembly of the peptides by analyzing the kinetics and equilibria of association of those mutants of (1-22) with native (23-110). The association reaction follows second-order kinetics. Virtually all the change in stability of the complex on mutation is reflected in changes in the association rate constant with the dissociation rate constant being very little affected. Both Brønsted and theta-value analyses show that the helix is essentially fully formed in the transition state for the association at all the positions probed (residues 13-18). Peptide (23-110) contains all the residues necessary for catalysis. The complexes between all mutants peptides (1-22) with (23-110) are all only 10% active, however. The noncovalent complex is destabilized less by mutations in the helix than is the intact protein. Double-mutant cycle and other analyses show, however, that the intrahelical interactions are as strong in the noncovalent complex as in the intact protein and so the interactions between the helix and the rest of the protein must be weakened on cleavage of the 22-23 bond. This could well lead to effects on catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)