Electrostatic contributions to the binding of Ca2+ in calbindin mutants. A Monte Carlo study

Monte Carlo simulation is used to calculate the free energy of binding of calcium ions to the native and several mutant forms of bovine calbindin D(9K) in salt solution. The simulations are performed in the canonical ensemble wherein free energies are calculated with a modified Widom method. The protein is modelled as a set of fixed hard spheres of fractional or unit charge with the surrounding solution as a dielectric continuum containing counterions and added salt particles. The interior of the protein is assumed to have the same dielectric permittivity as the solvent, which turns out to be an excellent approximation. Indeed, this simple model is able to predict accurately experimentally measured shifts in the calcium binding constants of up to five orders of magnitude, due to mutations and added salt.