title: Recoverin is a zinc-binding protein.
abstract: 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 alpha-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 x 10(5) M(-1) (dissociation constant 7.1 microM) for Ca2+-loaded wild-type recoverin and 3.3 x 10(4) M(-1) (dissociation constant 30 microM) 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.
authors.affiliation: Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia.
authors.forename: Sergei E
authors.initials: SE
authors.lastname: Permyakov
authors.forename: Alexandra M
authors.initials: AM
authors.lastname: Cherskaya
authors.forename: Lyubov A
authors.initials: LA
authors.lastname: Wasserman
authors.forename: Tatyana I
authors.initials: TI
authors.lastname: Khokhlova
authors.forename: Ivan I
authors.initials: II
authors.lastname: Senin
authors.forename: Aminullah A
authors.initials: AA
authors.lastname: Zargarov
authors.forename: Dmitry V
authors.initials: DV
authors.lastname: Zinchenko
authors.forename: Eugene Yu
authors.initials: EY
authors.lastname: Zernii
authors.forename: Valery M
authors.initials: VM
authors.lastname: Lipkin
authors.forename: Pavel P
authors.initials: PP
authors.lastname: Philippov
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Uversky
authors.forename: Eugene A
authors.initials: EA
authors.lastname: Permyakov
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000502:physiology*; D002151:Calorimetry; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005136:Eye Proteins; D051597:Hippocalcin; D007477:Ions; D008074:Lipoproteins; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009419:Nerve Tissue Proteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051603:Recoverin; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013696:Temperature; D013816:Thermodynamics; D015032:Zinc / Q000378:metabolism*
chemical_list: D000817:Anilino Naphthalenesulfonates; D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D007477:Ions; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D051603:Recoverin; D051597:Hippocalcin; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D015032:Zinc; D002118:Calcium