Double electron-electron resonance probes Ca~(2+)-induced conformational changes and dimerization of recoverin

摘要

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. Ca~(2+)-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 Ca~(2+)-free recoverin and 3.7 ± 0.1 nm for Ca~(2+)-bound recoverin. An additional DEER distance (5-6 nm) observed for Ca~(2+)-bound recoverin may represent an intermolecular distance between C39 and N120. 15N NMR relaxation analysis and CW-EPR experiments both confirm that Ca~(2+)-bound recoverin forms a dimer at protein concentrations above 100 μM, whereas Ca~(2+)-free recoverin is monomeric. We propose that Ca ~(2+)-induced dimerization of recoverin at the disk membrane surface may play a role in regulating Ca~(2+)-dependent phosphorylation of dimeric rhodopsin. The DEER approach will be useful for elucidating dimeric structures of NCS proteins in general for which Ca~(2+)-induced dimerization is functionally important but not well understood.