Recoverin Is a Zinc-binding Protein
Recoveri, Zinc-binding Sites, Site-directed Mutagenesis, Thermal Stability, Structure
Digital Object Identifier (DOI)
Recoverin is an N-myristoylated 23 kDa calcium-binding protein from retina, which modulates the Ca2+-sensitive deactivation of rhodopsin via Ca2+-dependent inhibition of rhodopsin kinase. It was shown by intrinsic and bis-ANS probe fluorescence, circular dichroism, and differential scanning calorimetry that myristoylated recombinant recoverin interacts specifically with zinc ions. Similar to the calcium binding, the binding of zinc to Ca2+-loaded recoverin additionally increases its α-helical content, hydrophobic surface area, and environmental mobility/polarity of its tryptophan residues. In contrast to the calcium binding, the binding of zinc decreases thermal stability of the Ca2+-loaded protein. Zn2+-titration of recoverin, traced by bis-ANS fluorescence, reveals binding of a single Zn2+ ion per protein molecule. It was shown that the double-mutant E85Q/E121Q with inactivated Ca2+-binding EF-hands 2 and 3 (Alekseev, A. M.; Shulga-Morskoy, S. V.; Zinchenko, D. V.; Shulga-Morskaya, S. A.; Suchkov, D. V.; Vaganova, S. A.; Senin, I. I.; Zargarov, A. A.; Lipkin, V. M.; Akhtar, M.; Philippov, P. P. FEBS Lett.1998, 440, 116−118), which can be considered as an analogue of the apo-protein, binds Zn2+ ion as well. Apparent zinc equilibrium binding constants evaluated from spectrofluorimetric Zn2+-titrations of the protein are 1.4 × 105 M-1 (dissociation constant 7.1 μM) for Ca2+-loaded wild-type recoverin and 3.3 × 104 M-1 (dissociation constant 30 μM) for the E85Q/E121Q mutant (analogue of apo-recoverin). Study of the binding of wild-type recoverin to ROS membranes showed a zinc-dependent increase of its affinity for the membranes, without regard to calcium content, suggesting further solvation of a protein myristoyl group upon Zn2+ binding. Possible implications of these findings to the functioning of recoverin are discussed.
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Citation / Publisher Attribution
Journal of Proteome Research, v. 2, issue 1, p. 51-57
Scholar Commons Citation
Permyakov, Sergei E.; Cherskaya, Alexandra M.; Wasserman, Lyubov A.; Khokhlova, Tatyana I.; Senin, Ivan I.; Zargarov, Aminullah A.; Zinchenko, Dmitry V.; Zernii, Eugene Yu.; Lipkin, Valery M.; Philippov, Pavel P.; Uversky, Vladimir N.; and Permyakov, Eugene A., "Recoverin Is a Zinc-binding Protein" (2003). Molecular Medicine Faculty Publications. 701.