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 four potential EF-hand Ca2+-binding sites, of which only two, the second and the 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 intrinsic fluorescence, circular dichroism spectroscopy and differential scanning microcalorimetry. The interaction of the two operating binding sites of wild-type recoverin with calcium increases the protein\u27s 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 high 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 × 106 and 3.1 × 105 M–1 for third and second EF-hands, respectively. The substitutions in the EF-hand 2 or 3 (or in both the sites simultaneously) do not disturb significantly either tertiary or secondary structure of the apo-protein. Amino acid substitutions, which have been designed to restore the 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 apo-form

Point Amino Acid Substitutions in The Ca\u3csup\u3e2+\u3c/sup\u3e-binding Sites of Recoverin: III. A Mutant with The Fourth Reconstructed Ca\u3csup\u3e2+\u3c/sup\u3e-binding Site

Unlike wild type recoverin with only two (the second and the third) functioning Ca+2-binding sites out of four potential ones, the +EF4 mutant contains a third active Ca+2-binding site. This site was reconstructed from the fourth potential Ca+2-binding domain by the introduction of several amino acid substitutions in it by site-directed mutagenesis. The effect of these mutations in the fourth potential Ca+2-binding site of myristoylated recoverin on the structural features and conformational stability of the protein was studied by fluorimetry and circular dichroism. The apoform of the resulting mutant (free of Ca2+ ions) was shown to have a higher calcium capacity, significantly lower thermal stability, and noticeably different secondary and tertiary structures as compared with the apoform of wild-type recoverin