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 recoverin contains fou r potential EF-hand Ca2+-binding sites, of which only two, the second and t he third, are capable of binding calcium ions. We have studied the effects of substitutions in the second, third and fourth EF-hand sites of recoverin on its Ca2+-binding properties and some other characteristics, using intri nsic fluorescence, circular dichroism spectroscopy and differential scannin g microcalorimetry. The interaction of the two operating binding sites of w ild-type recoverin with calcium increases the protein's thermal stability, but makes the environment around the tryptophan residues more flexible, The amino acid substitution in the EF-hand 3 (E121Q) totally abolishes the hig h calcium affinity of recoverin, while the mutation in the EF-hand 2 (E85Q) causes only a moderate decrease in calcium binding, Based on this evidence , we suggest that the binding of calcium ions to recoverin is a sequential process with the EF-hand 3 being filled first. Estimation of Ca2+ -binding constants according to the sequential binding scheme gave the values 3.7 x 10(6) and 3.1 x 10(5) M-l for third and second EF-hands, respectively. The substitutions in the EP-hand 2 or 3 (or in both the sites simultaneously) d o not disturb significantly either tertiary or secondary structure of the a pe-protein. Amino acid substitutions, which have been designed to restore t he calcium affinity of the EF-hand 4 (G160D, K161E, K162N, D165G and K166Q) , increase the calcium capacity and affinity of recoverin but also perturb the protein structure and decrease the thermostability of its ape-form.