Many recent approaches involving site-directed mutants have succeeded
in increasing the thermostability of proteins. It is well known that r
eplacements with proline residues reduce the conformational degrees of
freedom in the main polypeptide chain and thus can increase protein t
hermostabilization. We have studied protein thermostabilization by int
roducing proline substitutions in the homologous oligo-1,6-glucosidase
s from various Bacillus strains which grow within different temperatur
e ranges. As a consequence, the 'proline rule' was proposed for protei
n thermostabilization. The principle of this rule is that an increase
in the frequency of proline occurrence at beta-turns and/or an increas
e in the total number of hydrophobic residues can enhance protein ther
mostability. We have generated several lines of evidence supporting th
e theory from the comparative analysis of oligo-1,6-glucosidases in th
eir primary and secondary structures and molecular properties, the X-r
ay crystal structure analysis of the Bacillus cereus oligo-1,6-glucosi
dase, and the enhancement in thermostability of the oligo-l,6-glucosid
ase by cumulative replacements with prolines. As a new finding from th
e studies, two specific sites (second positions at beta-turns and N1 p
ositions of alpha-helices) were found to be the most critical to prote
in thermostabilization dependent on several structural prerequisites f
or proline substitution. (C) 1998 Elsevier Science B.V. All rights res
erved.