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. Study holds ProTherm entries: 4648, 4649 Extra Details: additive : EDTA(0.2 mM), rate constants; two-state reaction; hydrophobic surface;,bimolecular rate-limiting step; Arrhenius plots
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:26 p.m.
|Number of data points||6|
|Proteins||Transcriptional repressor arc ; Transcriptional repressor arc|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG ; Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG|
|Libraries||Mutations for sequence MKGMSKMPQFNLRWPREVLDLVRKVAEENGRSVNSEIYQRVMESFKKEGRIGA|