P22 Arc repressor: transition state properties inferred from mutational effects on the rates of protein unfolding and...

The kinetics of unfolding and refolding have been measured for a set of Arc repressor mutants bearing single amino acid substitutions at 44 of the 53 residue positions. Roughly half of the mutations cause significant changes in the unfolding and/or refolding rate constants. These substitutions alter the hydrophobic core, tertiary hydrogen bonds and salt bridges, and glycines with restricted backbone conformations. Overall, the mutations cause larger changes in the unfolding rates than the refolding rates, indicating that significantly less side-chain information is used between the denatured state and transition state than between the transition state and native state. The set of mutants displays reasonable Brønsted behavior, suggesting that many native interactions are partially formed in the transition state. Taken together, these observations suggest that the overall structure of most of the protein must be somewhat native-like in the transition state but without close, complementary packing of the hydrophobic core or good hydrogen bond geometry. Such a transition state is inconsistent with a model in which monomers fold to their correct conformations and then dock to form the dimer but supports a model in which folding and dimerization are concurrent processes.