Stereochemical modeling of disulfide bridges. Criteria for introduction into proteins by site-directed mutagenesis

A computer modeling procedure for assessing the stereochemical suitability of pairs of residues in proteins as potential sites for introduction of cystine disulfide crosslinks has been developed. Residue pairs with Cα – Cα distances of ≤6.5 Å and Cbeta;–Cβ distances of ≤4.5 Å are chosen for geometrical fixation of S atoms using the program MODIP. The stereochemistry of the modeled disulfides is evaluated using limits for the structural parameters of the various torsion angles and S–S bond length in the disulfide bridge. The ability of the procedure to correctly model disulfides has been checked with examples of cystine peptides of known crystal structures and 103 disulfide bridges from 25 available protein crystal structures determined at ≤2 Å resolution. An analysis of results on three proteins with engineered disulfides, T4 lysozyme, dihydrofolate reductase and subtilisin, is presented. Two positions for the introduction of 'stereochemically optimal' disulfides are identified in subtilisin.