Crystallographic investigations of subtilisin BPN' mutants engineered for studying thermal stability

Abstract

Subtilisin BPN' (BPN) is an industrially important serine protease that has been extensively investigated in many laboratories. In an effort to improve the thermal stability of the enzyme, researchers at Procter & Gamble have used site-directed mutagenesis techniques to produce several variants of BPN in which residues at the surface of the enzyme have been substituted. We initiated crystallographic studies to determine the structural consequences of these amino acid substitutions. In the course of this work we obtained excellent crystals that correspond to the C2 crystal form of native BPN that has been previously reported. Since the structure reported in that work was of only medium resolution, high-resolution X-ray data for this crystal form of native BPN have been collected and the refinement of the structure has been extended using these new data. Isomorphous crystals of two variants, Q19E and Q271E, have also been grown, high-resolution X-ray data have been collected for these crystals, and the experimental results are described. The structures of the native enzyme and the Q271E variant have been refined and are described.