The crystallographically determined structures of atypical strained disulfides engineered into subtilisin.

The geometries of two disulfide bridges genetically engineered into subtilisin have been characterized by x-ray crystallography to determine the structural and energetic constraints involved in introducing disulfide bonds into proteins. Both disulfide bridges (Cys-24-Cys-87 and Cys-22-Cys-87) exhibit atypical sets of dihedral angles compared to those for other reported disulfide structures in proteins. The geometric trends for naturally occurring disulfides in protein crystal structures are examined. Comparison of the disulfide-containing mutant protein structures with the wild-type structure shows that, in both cases, disulfide incorporation is accommodated by relatively minor changes in local main-chain conformation. The Cys-22-Cys-87 disulfide has two high energy dihedral angles (X2 = 121 degrees, X2' = 143 degrees). Both disulfides produce short non-bonded contacts with the main-chain.