Herein we examine the origin of the high temperature behaviour of a thermophilic mutant enzyme (labelled 5-3H5) derived from subtilisin E by eight amino acid substitutions. Through the Use of Molecular dynamics (MD) we provided molecular insight level into how point mutations stabilise protein dynamics and structure. From our simulations we observed a reduced RMSD in several key regions, an increased overall flexibility, an increase in the number of hydrogen bonds and an increase in the number of stabilizing interactions in the thermophilic system. We also show that it is not a necessary requirement that thermophilic enzymes be less flexible than their mesophilic counterparts. However thermophilic enzymes must retain their three dimensional structure and flexibility at high temperature in order to retain activity. We suggest that a promising strategy towards the stabilisation of proteins is to identify fluxional regions within a protein through MD. Once localised focussed directed mutagenesis could be directed towards these regions, to stabilise them.

[1] Colombo, G. and Merz Jr., K. M., J. Am. Chem. Soc., 121, 6895-6903, 1999.