DESIGN AND STRUCTURES OF DISULFIDE CONTAINING SUBTILISIN VARIANTS

Abstract

The crystal structures of 4 variants of subtilisin, each one containing an engineered disulfide crosslink have been determined. The geometries of the engineered disulfide groups are atypical. For the Cys24-Cys87 and Cys22-Cys87 disulfides there is a relationship between their measured redox potentials and their calculated dihedral energies. Disulfide introduction produced cavities in the protein structures. The cavity produced by removal of Met119 in A29C/M119C (Ala29 to Cys, Met119 to Cys) was partially filled by a disordering of nearby Asn117. The cavities were often filled with ordered water molecules that replaced interactions of the removed groups. Molecular modelling provided insight into the location where a disulfide could be incorporated, and into its resulting geometry. The structures of A29C/M119C and of V26C/A232C showed that introduction of disulfides into buried hydrophobic regions resulted in long range concerted rearrangements.