Evidence for partial secondary structure formation in the transition statefor Arc repressor refolding and dimerization

Structure formation and dimerization are concerted processes in the refoldi ng of Arc repressor. The integrity of secondary structure in the transition state of Are refolding has been investigated here by determining the chang es in equilibrium stability and refolding/unfolding kinetics for a set of A la -> Gly mutations at residues that are solvent-exposed in the native Are dimer. At some sites, reduced stability was caused primarily by faster unfo lding, indicating that secondary structure at these positions is largely ab sent in the transition state. However, most of the Ala -> Gly substitutions in the alpha-helices of Are and a triple mutant in the beta-sheet also res ulted in decreased refolding rates, in some cases, accounting fur the major fraction of thermodynamic destabilization. Overall, these results suggest that some regions of native secondary structure are present but incompletel y formed in the transition state of Are refolding and dimerization. Consoli dation of this secondary structure, like close packing of the hydrophobic c ore, seems to occur later in the folding process. On average, Phi(F) values for the Ala -> Gly mutations were significantly larger than Phi(F) values previously determined for alanine-substitution mutants, suggesting that bac kbone interactions in the transition state may be stronger than side chain interactions. Mutations causing significant reductions in the Are refolding rate were found to cluster in the central turn of alpha-helix A and in the first two turns of alpha-helix B. In the Arc dimer, these elements pack to gether in a compact structure, which might serve as nucleus for further fol ding.