Striking stabilization of Arc repressor by an engineered disulfide bond.

A solvent-exposed Cys11-Cys11' disulfide bond was designed to link the antiparallel 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-residue peptide tether. In both proteins, the presence of the disulfide bond increased the T(m) by approximately 40 degrees C. In the single-chain background, the disulfide bond stabilized Arc by 8.5 kcal/mol relative to the reduced form, a significantly larger degree of stabilization than caused by other engineered disulfides and most natural disulfides. This exceptional stabilization arises from a modest effective concentration of the Cys11-Cys11' disulfide in the native state (71 M) and an anomalously low effective concentration in the denatured state (40 microM). 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 energetic effect of the disulfide occurs after the transition state for Arc refolding, slowing unfolding by 200 000-fold.