Double electron-electron resonance probes Ca2+-induced conformational changes and dimerization of recoverin

William K. Myers, Xianzhong Xu, Congmin Li, Jens O. Lagerstedt, Madhu S. Budamagunta, John C Voss, R. David Britt, James B. Ames

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Abstract

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 Ca 2+-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 Ca 2+-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. 15N NMR relaxation analysis and CW-EPR experiments both confirm that Ca2+-bound recoverin forms a dimer at protein concentrations above 100 μM, whereas Ca2+-free recoverin is monomeric. We propose that Ca 2+-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.

Original languageEnglish (US)
Pages (from-to)5800-5808
Number of pages9
JournalBiochemistry
Volume52
Issue number34
DOIs
StatePublished - Aug 27 2013

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Myers, W. K., Xu, X., Li, C., Lagerstedt, J. O., Budamagunta, M. S., Voss, J. C., Britt, R. D., & Ames, J. B. (2013). Double electron-electron resonance probes Ca2+-induced conformational changes and dimerization of recoverin. Biochemistry, 52(34), 5800-5808. https://doi.org/10.1021/bi400538w