P22 Arc repressor: folding kinetics of a single-domain, dimeric protein

Abstract

The rate constants for refolding and unfolding of the P22 Arc repressor dimer have been determined by stop-flow fluorescence experiments. Under most conditions, refolding is described well as a two-state reaction with a bimolecular rate-limiting step (kf approximately 10(7) M-1 s-1). A unimolecular step appears to become co-rate limiting at high protein concentrations. The urea dependence of the refolding reaction suggests that about 75% of the total burial of hydrophobic surface occurs between the unfolded state and the transition state for folding. Hydrophobic interactions are also evidenced by the temperature dependence of the refolding reaction; the rate increases with temperature and Arrhenius plots are curved, as expected for a reaction that proceeds with a significant heat capacity change. The refolding of Arc also proceeds more rapidly as the salt concentration is raised, presumably because repulsive interactions between monomers are screened. At a protein concentration of 10 microM, the apparent rate constant for refolding of the Arc dimer is approximately 100 s-1, as fast as the refolding of many monomeric proteins. The rate constant for unfolding is approximately 0.1 s-1, corresponding to a half-life of less than 10 s for the folded Arc dimer. This rate of unfolding is very fast in comparison to that of other characterized proteins and implies that a free Arc molecule must unfold and refold hundreds of times per generation in the cell.