Novel disulfide engineering in human carbonic anhydrase II using the PAIRWISE side-chain geometry database

An analysis of the pairwise side-chain packing geometries of cysteine resid ues observed in high-resolution protein crystal structures indicates that c ysteine pairs have pronounced orientational preferences due to the geometri c constraints of disulfide bond formation. A potential function was generat ed from these observations and used to evaluate models for novel disulfide bonds in human carbonic anhydrase CI (HCAII). Three double-cysteine variant s of HCAII were purified and the effective concentrations of their thiol gr oups were determined by titrations with glutathione and dithiothreitol. The effects of the cysteine mutations on the native state structure and stabil ity were characterized by circular dichroism, enzymatic activity, sulfonami de binding, and guanidine hydrochloride titration. These analyses indicate that the PAIRWISE potential is a good predictor of the strength of the disu lfide bond itself, but the overall structural and thermodynamic effects on the protein are complicated by additional factors. In particular, Che effec ts of cysteine substitutions an the native state and the stabilization of c ompact nonnative slates by the disulfide can override any stabilizing effec t of the cross-link.