We used mean spherical approximation (MSA) theory to evaluate ion binding thermodynamics within the proteins' EF-hand domains to account for the impact of ions' finite sizes and the surrounding electrolyte composition.  Instead, molecular mechanics generalized Born surface area (MM/GBSA) approximation energies, which we used to quantify the thermodynamic cost of structural rearrangement of the proteins upon binding ions, indicated that S55D/E59D αPV favored Ca2+ binding by -20 kcal/mol relative to WT versus 30 kcal/mol for D94S/G98E αPV. 
THERM