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Amino Acids

, Volume 42, Issue 4, pp 1435–1442 | Cite as

Oxidation mimicking substitution of conservative cysteine in recoverin suppresses its membrane association

  • Sergei E. Permyakov
  • Evgeni Yu Zernii
  • Ekaterina L. Knyazeva
  • Alexander I. Denesyuk
  • Aliya A. Nazipova
  • Tatiana V. Kolpakova
  • Dmitry V. Zinchenko
  • Pavel P. Philippov
  • Eugene A. Permyakov
  • Ivan I. Senin
Original Article

Abstract

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.

Keywords

EF-hand NCS family Vision Recoverin Cysteine Redox regulation 

Abbreviations

NCS

Neuronal calcium sensor

ROS

Reactive oxygen species

WT

Recombinant wild-type recoverin

HEPES

N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)

Tris

Tris(hydroxymethyl) aminomethane

EDTA

Ethylenediaminetetraacetic acid

CD

Circular dichroism

GCAP

Guanylate cyclase activated protein

λmax

Position of fluorescence spectrum maximum

T1/2

Mid-transition temperature

FDPB

Finite difference Poisson–Boltzmann method

NMR

Nuclear magnetic resonance

Notes

Acknowledgments

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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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Sergei E. Permyakov
    • 1
    • 2
  • Evgeni Yu Zernii
    • 3
  • Ekaterina L. Knyazeva
    • 1
  • Alexander I. Denesyuk
    • 1
    • 4
  • Aliya A. Nazipova
    • 1
  • Tatiana V. Kolpakova
    • 3
  • Dmitry V. Zinchenko
    • 5
  • Pavel P. Philippov
    • 3
  • Eugene A. Permyakov
    • 1
    • 2
  • Ivan I. Senin
    • 3
  1. 1.Institute for Biological Instrumentation of the Russian Academy of SciencesPushchino, Moscow RegionRussia
  2. 2.Department of Biomedical EngineeringPushchino State UniversityPushchino, Moscow RegionRussia
  3. 3.Department of Cell SignallingA.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State UniversityMoscowRussia
  4. 4.Department of BiosciencesÅbo Akademi UniversityTurkuFinland
  5. 5.Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of SciencesPushchino, Moscow RegionRussia

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