Oxidation mimicking substitution of conservative cysteine in recoverin suppresses its membrane association
Recoverin belongs to the family of intracellular Ca2+-binding proteins containing EF-hand domains, neuronal calcium sensors (NCS). In photoreceptor outer segments, recoverin is involved into the recovery of visual cycle via Ca2+-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 Ca2+-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.
KeywordsEF-hand NCS family Vision Recoverin Cysteine Redox regulation
Neuronal calcium sensor
Reactive oxygen species
Recombinant wild-type recoverin
Guanylate cyclase activated protein
Position of fluorescence spectrum maximum
Finite difference Poisson–Boltzmann method
Nuclear magnetic resonance
This work was supported by Grant to P.E.A. from the Program of the Russian Academy of Sciences “Molecular and Cellular Biology”, Grant to S.E.P. from the President of Russia (No. MK-4581.2007.4), Grants to A.I.D. from Stiftelsens för Åbo Akademi Forskningsinstitut and the Sigrid Jusélius Foundation, Grants from Russian Foundation for Basic Research to I.I.S. (09-04-01778-a) and E.Yu.Z (09-04-00666-a) and Grant to I.I.S. from the President of Russia (No. MD-4423.2010.4).
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