Effects of mutations in the calcium-binding sites of recoverin on its calcium affinity: evidence for successive filling of the calcium binding sites

A molecule of the photoreceptor Ca²⁺-binding protein recoverincontains four potential EF-hand Ca²⁺-binding sites, of whichonly two, the second and the third, are capable of binding calciumions. We have studied the effects of substitutions in the second,third and fourth EF-hand sites of recoverin on its Ca²⁺-bindingproperties and some other characteristics, using intrinsic fluorescence,circular dichroism spectroscopy and differential scanning microcalorimetry.The interaction of the two operating binding sites of wild-typerecoverin with calcium increases the protein's thermal stability,but makes the environment around the tryptophan residues moreflexible. The amino acid substitution in the EF-hand 3 (E121Q)totally abolishes the high calcium affinity of recoverin, whilethe mutation in the EF-hand 2 (E85Q) causes only a moderatedecrease in calcium binding. Based on this evidence, we suggestthat the binding of calcium ions to recoverin is a sequentialprocess with the EF-hand 3 being filled first. Estimation ofCa²⁺-binding constants according to the sequential binding schemegave the values 3.7 x 10⁶ and 3.1 x 10⁵ M^–1 for thirdand second EF-hands, respectively. The substitutions in theEF-hand 2 or 3 (or in both the sites simultaneously) do notdisturb significantly either tertiary or secondary structureof the apo-protein. Amino acid substitutions, which have beendesigned to restore the calcium affinity of the EF-hand 4 (G160D,K161E, K162N, D165G and K166Q), increase the calcium capacityand affinity of recoverin but also perturb the protein structureand decrease the thermostability of its apo-form.