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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. ¹?N NMR relaxation analysis and CW-EPR experiments both confirm that Ca²?-bound recoverin forms a dimer at protein concentrations above 100 ?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.

SUBMITTER: Myers WK 

PROVIDER: S-EPMC3784611 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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

Myers William K WK   Xu Xianzhong X   Li Congmin C   Lagerstedt Jens O JO   Budamagunta Madhu S MS   Voss John C JC   Britt R David RD   Ames James B JB  

Biochemistry 20130816 34


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 recoveri  ...[more]

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