P22 ARE REPRESSOR - FOLDING KINETICS OF A SINGLE-DOMAIN, DIMERIC PROTEIN

The rate constants for refolding and unfolding of the P22 Arc represso r dimer have been determined by stop-flow fluorescence experiments. Un der most conditions, refolding is described well as a two-state reacti on with a bimolecular rate-limiting step (k(f) approximate to 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 reac tion suggests that about 75% of the total burial of hydrophobic surfac e occurs between the unfolded state and the transition state for foldi ng. Hydrophobic interactions are also evidenced by the temperature dep endence of the refolding reaction; the rate increases with temperature and Arrhenius plots are curved, as expected for a reaction that proce eds 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 pr otein concentration of 10 mu M, the apparent rate constant for refoldi ng of the Are dimer is approximate to 100 s(-1), as fast as the refold ing of many monomeric proteins. The rate constant for unfolding is app roximate to 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 compa rison to that of other characterized proteins and implies that a free Arc molecule must unfold and refold hundreds of times per generation i n the cell.