title: Double electron-electron resonance probes Ca²⁺-induced conformational changes and dimerization of recoverin.
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 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. 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 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.
authors.affiliation: Department of Chemistry, University of California, Davis, California 95616, United States.
authors.forename: William K
authors.initials: WK
authors.lastname: Myers
authors.forename: Xianzhong
authors.initials: X
authors.lastname: Xu
authors.forename: Congmin
authors.initials: C
authors.lastname: Li
authors.forename: Jens O
authors.initials: JO
authors.lastname: Lagerstedt
authors.forename: Madhu S
authors.initials: MS
authors.lastname: Budamagunta
authors.forename: John C
authors.initials: JC
authors.lastname: Voss
authors.forename: R David
authors.initials: RD
authors.lastname: Britt
authors.forename: James B
authors.initials: JB
authors.lastname: Ames
mesh_terms: D002118:Calcium / Q000494:pharmacology*; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D009682:Magnetic Resonance Spectroscopy; D009227:Myristic Acids / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects*; D055503:Protein Multimerization / Q000187:drug effects*; D051603:Recoverin / Q000737:chemistry* / Q000378:metabolism; D013113:Spin Labels
chemical_list: D009227:Myristic Acids; D013113:Spin Labels; D051603:Recoverin; D002118:Calcium
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