Identification of mutation sites by PROPKA
In order to screen possible mutations we used PROPKA to identify stabilizing motifs that were present in the thermophilic template but not in the mesophilic variant. PROPKA predicts protein pKa values, and can therefore be used to examine the electrostatic part of the protein folding free energy, ΔGuf → f. This is related to shifts in protein pKa values because the interactions that stabilize the folded protein compared to the unfolded protein are the same interactions that shifts the pKa values of the residues in the unfolded state to its folded state. The total electrostatic protein unfolding free energy can be made into a thermodynamic cycle where the unfolding free energy is calculated as