Recoverin as a Redox-Sensitive Protein

Abstract

Recoverin is a member of the neuronal calcium sensor (NCS) family of EF-hand calcium binding proteins. In a visual cycle of photoreceptor cells, recoverin regulates activity of rhodopsin kinase in a Ca2+-dependent manner. Like all members of the NSC family, recoverin contains a conserved cysteine (Cys38) in nonfunctional EF-hand 1. This residue was shown to be critical for activation of target proteins in some members of the NCS family but not for interaction of recoverin with rhodopsin kinase. Spectrophotometric titration of Ca2+-loaded recoverin gave 7.6 for the pKa value of Cys38 thiol, suggesting partial deprotonation of the thiol in vivo conditions. An ability of recoverin to form a disulfide dimer and thiol-oxidized monomer under mild oxidizing conditions was found using SDS-PAGE in reducing and nonreducing conditions and Ellman's test. Both processes are reversible and modulated by Ca2+. Although formation of the disulfide dimer takes place only for Ca2+-loaded recoverin, accumulation of the oxidized monomer proceeds more effectively for apo-recoverin. The Ca2+ modulated susceptibility of the recoverin thiol to reversible oxidation may be of potential importance for functioning of recoverin in photoreceptor cells.

A Highly Conserved Cysteine of Neuronal Calcium-sensing Proteins Controls Cooperative Binding of Ca2+ to Recoverin

“…These data are of interest because Cys-39 is the only cysteine residue in recoverin and is strictly conserved among all members of the NCS family. Permyakov et al showed that recoverin lost the ability to react with the thiol-specific Ellman's reagent (5,5Ј-dithiobis-(2-nitrobenzoic acid)) upon incubation under non-reducing conditions, suggesting that the sulfur of Cys-39 underwent oxidation (17). For this reason, noting also that the retina is one of the most vascularized tissues in the body, they further proposed that Cys-39 may function as a redox sensor in rod photoreceptor cells.…”

“…Those fractions containing recoverin were combined and concentrated with a YM-10 Centricon to 1-2 ml and then dialyzed against Buffer C at 4°C to remove EGTA. Purified protein was quantified spectrally by ⑀ 280 (WT, 24,075 M Ϫ1 cm Ϫ1 ; C39A, 24,000 M Ϫ1 cm Ϫ1 ) (17,21) and frozen at Ϫ80°C until use. Preparation of mRv-mRv was prepared from T7 Express E. coli cells coexpressing yeast N-myristoyltransferase1 in pBB131 (20) with recoverin.…”

“…Cys-39 is the only cysteine residue in recoverin and has been suggested to function as a redox sensor in rod photoreceptor cells through formation of derivatives with higher oxidation states of the sulfur (i.e. sulfenic, sulfinic, or sulfonic acids) (17,18).…”

Biological chemistry and functionality of protein sulfenic acids and related thiol modifications

“…Mild oxidative environments regulate the activity of recoverin by acting on a conserved Cys residue (Cys39 in the human homolog) [195–197]. Experiments using site-directed mutagenesis of Cys39 to Asp (-SO 2 H mimetic) showed that hyperoxidation of myristoylated protein did not change the affinity for Ca 2+ but drastically decreased the binding of recoverin to photoreceptor cell membranes [196].…”

Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein

“…Importantly, the free cysteine in position 39 (Cys39) is critically important for recoverin’s function and contributes to its ability to bind Ca 2+ . In fact, Cys39 is one of the most highly conserved residues and part of the CPXG motif in the Neuronal Calcium Sensor (NCS) family proteins10 and plays functional roles in redox sensing, dimerization, and ligand interactions1112. For example, mutation of Cys39 to aspartic acid results in a significant reduction of photoreceptor membrane affinity13.…”