Spectroscopic Studies of Site 3 Mutants of Calmodulin

Calmodulin is the quintessential member of the EF-hand family of calcium binding proteins. Two mutants of the C-domain of Calmodulin were created as a series of proteins that can be used as model 1-site calcium binding systems, terminal glutamate in Site 3 was mutated to either glutamine or aspartate, mutations were expected to abrogate binding in the mutated site such that only Site 4 is functional. There was minimal overall perturbation to the folding and stability of the apo-state. Calcium binding properties of these mutant proteins were investigated by 1) direct titrations with calcium were performed under equilibrium conditions monitored by fluorescence and circular dichroism signals, 2) calcium binding constants were deduced from calcium-dependent urea denaturation experiments, 3) the proteins were titrated under stoichiometric conditions monitored by the fluorescence of Y138. The critical These These experiments showed that the glutamine mutant was higher affinity than the aspartate mutant. The aspartate in Site 3 apparently disrupted the structure of Site 4 leading to lower affinity for calcium, significantly lower affinity than calcium-binding constants derived from the calciumdependent urea denaturation studies. This discrepancy is not due to the structural phenomena that are monitored by different spectroscopic signals. It is likely that the higher affinity calcium-binding constants derived from the urea denaturation experiments are due to urea destabilization of the native state leading to a greater value for the free energy of binding