The role of protein surface charges in ion binding

Protein engineering is a means of probing the role of electrostatic interactions in protein functions; this elegant technique has been applied to the elucidation of electrostatic effects in enzyme catalysis. Here we show how the use of mutant proteins allows the determination of the contributions of individual charges to the free energy of ion binding to proteins. We have investigated the importance of three negatively charged side chains in the binding of Ca2+ to bovine calbindin D9K (ref.2): these are clustered around the calcium sites but are not directly involved as ligands. Each of these charges is found to contribute approximately 7 kJ mol-1 to the free energy of binding of two Ca2+ ions and to affect the cooperativity of Ca2+ binding. The influence of surface charges on ion binding to proteins may be more common than generally supposed and could have important consequences for protein function.