Stabilization of Bacillus stearothermophilus neutral protease by introduction of prolines

The thermostability of neutral proteases has been shown to depend on autolysis which presumably occurs in flexible regions of the protein. In an attempt to rigidify such a region in the neutral protease of Bacillus stearothermophilus, residues in the solvent-exposed 63-69 loop were replaced by proline. The mutations caused large positive (Ser-65-->Pro, Ala-69-->Pro) or negative (Thr-63-->Pro, Tyr-66-->Pro) changes in thermostability, which were explained on the basis of molecular modelling of the mutant proteins. The data show that the introduction of prolines at carefully selected positions in the protein can be a powerful method for stabilization.