title: Oxidation mimicking substitution of conservative cysteine in recoverin suppresses its membrane association.
abstract: Recoverin belongs to the family of intracellular Ca(2+)-binding proteins containing EF-hand domains, neuronal calcium sensors (NCS). In photoreceptor outer segments, recoverin is involved into the recovery of visual cycle via Ca(2+)-dependent interaction with disk membranes and inhibition of rhodopsin kinase. The function of a conservative within NCS family Cys residue in the inactive EF-loop 1 remains unclear, but previous study has shown its vulnerability to oxidation under mild oxidizing conditions. To elucidate the influence of oxidation of the conservative Cys39 in recoverin the properties of its C39D mutant, mimicking oxidative conversion of Cys39 into sulfenic, sulfinic or sulfonic acids have been studied using intrinsic fluorescence, circular dichroism, and equilibrium centrifugation methods. The C39D substitution results in essential changes in structural, physico-chemical and physiological properties of the protein: it reduces α-helical content, decreases thermal stability and suppresses protein affinity for photoreceptor membranes. The latter effect precludes proper functioning of the Ca(2+)-myristoyl switch in recoverin. The revealed significance of oxidation state of Cys39 for maintaining the protein functional status shows that it may serve as redox sensor in vision and suggests an explanation of the available data on localization and light-dependent translocation of recoverin in rod photoreceptors.
authors.affiliation: Institute for Biological Instrumentation of Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia.
authors.forename: Sergei E
authors.initials: SE
authors.lastname: Permyakov
authors.forename: Evgeni Yu
authors.initials: EY
authors.lastname: Zernii
authors.forename: Ekaterina L
authors.initials: EL
authors.lastname: Knyazeva
authors.forename: Alexander I
authors.initials: AI
authors.lastname: Denesyuk
authors.forename: Aliya A
authors.initials: AA
authors.lastname: Nazipova
authors.forename: Tatiana V
authors.initials: TV
authors.lastname: Kolpakova
authors.forename: Dmitry V
authors.initials: DV
authors.lastname: Zinchenko
authors.forename: Pavel P
authors.initials: PP
authors.lastname: Philippov
authors.forename: Eugene A
authors.initials: EA
authors.lastname: Permyakov
authors.forename: Ivan I
authors.initials: II
authors.lastname: Senin
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D015536:Down-Regulation; D006801:Humans; D007700:Kinetics; D010084:Oxidation-Reduction; D020419:Photoreceptor Cells, Vertebrate / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D055550:Protein Stability; D021381:Protein Transport; D051603:Recoverin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D051603:Recoverin; D003545:Cysteine; D002118:Calcium