Protein thermostabilization by proline substitutions

Many recent approaches involving site-directed mutants have succeeded in increasing the thermostability of proteins. It is well known that replacements with proline residues reduce the conformational degrees of freedom in the main polypeptide chain and thus can increase protein thermostabilization. We have studied protein thermostabilization by introducing proline substitutions in the homologous oligo-1,6-glucosidases from various Bacillus strains which grow within different temperature ranges. As a consequence, the 'proline rule' was proposed for protein thermostabilization. The principle of this rule is that an increase in the frequency of proline occurrence at beta-turns and/or an increase in the total number of hydrophobic residues can enhance protein thermostability. We have generated several lines of evidence supporting the theory from the comparative analysis of oligo-1,6-glucosidases in their primary and secondary structures and molecular properties, the X-ray crystal structure analysis of the Bacillus cereus oligo-1,6-glucosidase, and the enhancement in thermostability of the oligo-l,6-glucosidase by cumulative replacements with prolines. As a new finding from the studies, two specific sites (second positions at beta-turns and N1 positions of alpha-helices) were found to be the most critical to protein thermostabilization dependent on several structural prerequisites for proline substitution. (C) 1998 Elsevier Science B.V. All rights reserved.