Striking stabilization of Arc repressor by an engineered disulfide bond

A solvent-exposed Cys11-Cys11' disulfide bond was designed to link the anti parallel strands of the beta sheet both in the Arc repressor dimer and in a single-chain variant in which the Arc subunits are connected by a 15-resid ue peptide tether. In both proteins, the presence of the disulfide bond inc reased the T-m by approximately 40 degrees C. In the single-chain backgroun d, the disulfide bond stabilized Are by 8.5 kcal/mol relative to the reduce d form, a significantly larger degree of stabilization than caused by other engineered disulfides and most natural disulfides. This exceptional stabil ization arises from a modest effective concentration of the Cys11-Cys11' di sulfide in the native state (71 M) and an anomalously low effective concent ration in the denatured state (40 mu M). Disulfide cross-linking of the two beta strands in the single-chain Arc background accelerated refolding by a factor of 170 into the sub-microsecond time scale. However, the major ener getic effect of the disulfide occurs after the transition state for Arc ref olding, slowing unfolding by 200 000-fold.