Increasing thermal stability of subtilisin from mutations suggested by strongly interacting side-chain clusters.

In this paper we present for seven subtilisin structures a systematic comparison of densely packed side-group clusters (defined as an ensemble of side chains with extensive internal atomic contacts as compared with those made with the surrounding protein environment and measured relative to the maximum possible for each residue type). Spatially consistent clusters are observed at structurally equivalent positions in the proteins, as revealed by careful multiple superpositioning of the respective backbone atoms. The clusters are positioned at strategic loop-connecting sites near the protein surfaces. The residues within consistent clusters displaying extensive association show varying conservation at structurally equivalent alignment sites. Suggestions for residue substitutions, as observed over the seven tertiary structures, were taken from the cluster positions and were shown to be consistent with a number of point mutations in one of the seven structures (savinase) that result in increased thermal stability.