Double Electron-Electron Resonance Probes Ca2+-Induced Conformational Changes and Dimerization of Recoverin

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Recoverin a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, is expressed in retinal photoreceptor cells and serves as a calcium sensor in vision. Ca2+-induced conformational changes in recoverin cause extrusion of its covalently attached myristate (termed Ca2+-myristoyl switch) that promotes translocation of recoverin to disk membranes during phototransduction in retinal rod cells. Here we report double electron electron resonance (DEER) experiments on recoverin that probe Ca2+-induced changes in distance as measured by the dipolar coupling between spin-labels strategically positioned at engineered cysteine residues on the protein surface. The DEER distance between nitroxide spin-labels attached at C39 and N120C is 2.5 +/- 0.1 nm for Ca2+-free recoverin and 3.7 +/- 0.1 nm for Ca2+-bound recoverin. An additional DEER distance (5-6 nm) observed for Ca2+-bound recoverin may represent an intermolecular distance between C39 and N120. N-15 NMR relaxation analysis and CW-EPR experiments both confirm that Ca2+-bound recoverin forms a dimer at protein concentrations above 100 mu M, whereas Ca2+-free recoverin is monomeric We propose that Ca2+-induced dimerization of recoverin at the disk membrane surface may play a role in regulating Ca2+-dependent phosphorylation of dimeric rhodopsin. The DEER approach will be useful for elucidating dimeric structures of NCS proteins in general for which Ca2+-induced dimerization is functionally important but not well understood.


  • William K. Myers
  • Xianzhong Xu
  • Congmin Li
  • Jens Lagerstedt
  • Madhu S. Budamagunta
  • John C. Voss
  • R. David Britt
  • James B. Ames
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Biochemistry and Molecular Biology
Original languageEnglish
Pages (from-to)5800-5808
Issue number34
Publication statusPublished - 2013
Publication categoryResearch