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

Recoverin, a 23-kDa Ca²⁺-binding protein of the neuronal calcium sensing (NCS) family, inhibits rhodopsin kinase, a Ser/Thr kinase responsible for termination of photoactivated rhodopsin in rod photoreceptor cells. Recoverin has two functional EF hands and a myristoylated N terminus. The myristoyl chain imparts cooperativity to the Ca²⁺-binding sites through an allosteric mechanism involving a conformational equilibrium between R and T states of the protein. Ca²⁺ binds preferentially to the R state; the myristoyl chain binds preferentially to the T state. In the absence of myristoylation, the R state predominates, and consequently, binding of Ca²⁺ to the non-myristoylated protein is not cooperative. We show here that a mutation, C39A, of a highly conserved Cys residue among NCS proteins, increases the apparent cooperativity for binding of Ca²⁺ to non-myristoylated recoverin. The binding data can be explained by an effect on the T/R equilibrium to favor the T state without affecting the intrinsic binding constants for the two Ca²⁺ sites.     

Photoreceptor calcium sensor proteins in detergent-resistant membrane rafts are regulated via binding to caveolin-1

Rod cell membranes contain cholesterol-rich detergent-resistant membrane (DRM) rafts, which accumulate visual cascade proteins as well as proteins involved in regulation of phototransduction such as rhodopsin kinase and guanylate cyclases. Caveolin-1 is the major integral component of DRMs, possessing scaffolding and regulatory activities towards various signaling proteins. In this study, photoreceptor Ca²⁺-binding proteins recoverin, NCS1, GCAP1, and GCAP2, belonging to neuronal calcium sensor (NCS) family, were recognized as novel caveolin-1 interacting partners. All four NCS proteins co-fractionate with caveolin-1 in DRMs, isolated from illuminated bovine rod outer segments. According to pull-down assay, surface plasmon resonance spectroscopy and isothermal titration calorimetry data, they are capable of high-affinity binding to either N-terminal fragment of caveolin-1 (1–101), or its short scaffolding domain (81–101) via a novel structural site. In recoverin this site is localized in C-terminal domain in proximity to the third EF-hand motif and composed of aromatic amino acids conserved among NCS proteins. Remarkably, the binding of NCS proteins to caveolin-1 occurs only in the absence of calcium, which is in agreement with higher accessibility of the caveolin-1 binding site in their Ca²⁺-free forms. Consistently, the presence of caveolin-1 produces no effect on regulatory activity of Ca²⁺-saturated recoverin or NCS1 towards rhodopsin kinase, but upregulates GCAP2, which potentiates guanylate cyclase activity being in Ca²⁺-free conformation. In addition, the interaction with caveolin-1 decreases cooperativity and augments affinity of Ca2 + binding to recoverin apparently by facilitating exposure of its myristoyl group. We suggest that at low calcium NCS proteins are compartmentalized in photoreceptor rafts via binding to caveolin-1, which may enhance their activity or ensure their faster responses on Ca²⁺-signals thereby maintaining efficient phototransduction recovery and light adaptation.     

Recoverin as a redox-sensitive protein

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.     

Ca2+-dependent regulatory activity of recoverin in photoreceptor raft structures: The role of caveolin-1

Recoverin is a Ca²⁺-binding protein implicated in the Ca²⁺-dependent regulation of desensitization of visual receptor rhodopsin in vertebrate retinal rods. Here we report that Ca²⁺ sensitivity of recoverin regulating rhodopsin phosphorylation increases in the presence of the photoreceptor membranes enriched in raft structures. The observed effect is mediated by a key protein component of raft structures caveolin-1. The presence of recombinant fragment Phe⁸¹-Arg¹⁰¹ of the caveolin-1 cytoplasmic domain enhances Ca²⁺ affinity of recoverin, therefore affecting its Ca²⁺-dependent regulatory activity.     

Amino acid sequences of two immune-dominant epitopes of recoverin are involved in Ca<Superscript>2+</Superscript>/recoverin-dependent inhibition of phosphorylation of rhodopsin

Antibodies AB^60–72 and AB^80–92 against two immune-dominant epitopes of photoreceptor Ca²⁺-binding protein recoverin, 60-DPKAYAQHVFRSF-72 and 80-LDFKEYVIALHMT-92, which can be exposed in a Ca²⁺-dependent manner, were obtained. The presence of AB^60–72 or AB^80–92 results in a slight increase in Ca²⁺-affinity of recoverin and does not affect significantly a Ca²⁺-myristoyl switch mechanism of the protein. However in the presence of AB^60–72 or AB^80–92 recoverin loses its ability to interact with rhodopsin kinase and consequently to perform a function of Ca²⁺-sensitive inhibitor of rhodopsin phosphorylation in photoreceptor cells.     

The myristoylation of guanylate cyclase-activating protein-2 causes an increase in thermodynamic stability in the presence but not in the absence of Ca²⁺

Guanylate cyclase activating protein‐2 (GCAP‐2) is a Ca²⁺‐binding protein of the neuronal calcium sensor (NCS) family. Ca²⁺‐free GCAP‐2 activates the retinal rod outer segment guanylate cyclases ROS‐GC1 and 2. Native GCAP‐2 is N‐terminally myristoylated. Detailed structural information on the Ca²⁺‐dependent conformational switch of GCAP‐2 is missing so far, as no atomic resolution structures of the Ca²⁺‐free state have been determined. The role of the myristoyl moiety remains poorly understood. Available functional data is incompatible with a Ca²⁺‐myristoyl switch as observed in the prototype NCS protein, recoverin. For the homologous GCAP‐1, a Ca²⁺‐independent sequestration of the myristoyl moiety inside the proteins structure has been proposed. In this article, we compare the thermodynamic stabilities of myristoylated and non‐myristoylated GCAP‐2 in their Ca²⁺‐bound and Ca²⁺‐free forms, respectively, to gain information on the nature of the Ca²⁺‐dependent conformational switch of the protein and shed some light on the role of its myristoyl group. In the absence of Ca²⁺, the stability of the myristoylated and non‐myristoylated forms was indistinguishable. Ca²⁺ exerted a stabilizing effect on both forms of the protein, which was significantly stronger for myr GCAP‐2. The stability data were corroborated by dye binding experiments performed to probe the solvent‐accessible hydrophobic surface of the protein. Our results strongly suggest that the myristoyl moiety is permanently solvent‐exposed in Ca²⁺‐free GCAP‐2, whereas it interacts with a hydrophobic part of the protein's structure in the Ca²⁺‐bound state.     

Structural Insights for Activation of Retinal Guanylate Cyclase by GCAP1

Guanylyl cyclase activating protein 1 (GCAP1), a member of the neuronal calcium sensor (NCS) subclass of the calmodulin superfamily, confers Ca²⁺-sensitive activation of retinal guanylyl cyclase 1 (RetGC1) upon light activation of photoreceptor cells. Here we present NMR assignments and functional analysis to probe Ca²⁺-dependent structural changes in GCAP1 that control activation of RetGC. NMR assignments were obtained for both the Ca²⁺-saturated inhibitory state of GCAP1 versus a GCAP1 mutant (D144N/D148G, called EF4mut), which lacks Ca²⁺ binding in EF-hand 4 and models the Ca²⁺-free/Mg²⁺-bound activator state of GCAP1. NMR chemical shifts of backbone resonances for Ca²⁺-saturated wild type GCAP1 are overall similar to those of EF4mut, suggesting a similar main chain structure for assigned residues in both the Ca²⁺-free activator and Ca²⁺-bound inhibitor states. This contrasts with large Ca²⁺-induced chemical shift differences and hence dramatic structural changes seen for other NCS proteins including recoverin and NCS-1. The largest chemical shift differences between GCAP1 and EF4mut are seen for residues in EF4 (S141, K142, V145, N146, G147, G149, E150, L153, E154, M157, E158, Q161, L166), but mutagenesis of EF4 residues (F140A, K142D, L153R, L166R) had little effect on RetGC1 activation. A few GCAP1 residues in EF-hand 1 (K23, T27, G32) also show large chemical shift differences, and two of the mutations (K23D and G32N) each decrease the activation of RetGC, consistent with a functional conformational change in EF1. GCAP1 residues at the domain interface (V77, A78, L82) have NMR resonances that are exchange broadened, suggesting these residues may be conformationally dynamic, consistent with previous studies showing these residues are in a region essential for activating RetGC1.     

Conformational dynamics of recoverin's Ca2+-myristoyl switch probed by 15N NMR relaxation dispersion and chemical shift analysis

Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, serves as a calcium sensor in retinal rod cells. Ca²⁺‐induced conformational changes in recoverin promote extrusion of its covalently attached myristate, known as the Ca²⁺‐myristoyl switch. Here, we present nuclear magnetic resonance (NMR) relaxation dispersion and chemical shift analysis on ¹⁵N‐labeled recoverin to probe main chain conformational dynamics. ¹⁵N NMR relaxation data suggest that Ca²⁺‐free recoverin undergoes millisecond conformational dynamics at particular amide sites throughout the protein. The addition of trace Ca²⁺ levels (0.05 equivalents) increases the number of residues that show detectable relaxation dispersion. The Ca²⁺‐dependent chemical shifts and relaxation dispersion suggest that recoverin has an intermediate conformational state (I) between the sequestered apo state (T) and Ca²⁺ saturated extruded state (R): T ? I ? R. The first step is a fast conformational equilibrium ([T]/[I] < 100) on the millisecond time scale (τ_exδω < 1). The final step (I ? R) is much slower (τ_exδω > 1). The main chain structure of I is similar in part to the structure of half‐saturated E85Q recoverin with a sequestered myristoyl group. We propose that millisecond dynamics during T ? I may transiently increase the exposure of Ca²⁺‐binding sites to initiate Ca²⁺ binding that drives extrusion of the myristoyl group during I ? R. Proteins 2011; © 2011 Wiley‐Liss, Inc.     

Molecular tuning of calcium dependent processes by neuronal calcium sensor proteins in the retina

Vertebrate photoreceptor cells are exquisite light detectors operating under very dim and bright illumination mediated by phototransduction, which is under control of the two secondary messengers cGMP and Ca²⁺. Feedback mechanisms enable photoreceptor cells to regain their responsiveness after light stimulation and involve neuronal Ca²⁺-sensor proteins, named GCAPs (guanylate cyclase-activating proteins) and recoverins. This review compares the diversity in Ca²⁺-related signaling mediated by GCAP and recoverin variants that exhibit differences in Ca²⁺-sensing, protein conformational changes, myristoyl switch mechanisms, diversity in divalent cation binding and dimer formation. In summary, both subclasses of neuronal Ca²⁺-sensor proteins contribute to a complex signaling network in rod and cone cells, which is perfectly suited to match the requirements for sensitive cell responses and maintaining this responsiveness in the presence of different background light intensities.     

Point amino acid substitutions in the Ca2+-binding sites of recoverin: II. The unusual behavior of the protein upon the binding of calcium ions

The structural properties of myristoylated forms of recombinant recoverin of the wild type and of its mutants with damaged second and/or third Ca²⁺-binding sites were studied by fluorimetry and circular dichroism. The interaction of wild-type recoverin with calcium ions was shown to induce unusual structural rearrangements in its molecule. In particular, protein binding with Ca²⁺ ions results in an increase in the mobility of the environment of Trp residues, in hydrophobicity, and in thermal stability (its thermal transition shifts by 15°C to higher temperatures) but has almost no effect on its secondary structure. Similar structural changes induced by Ca²⁺ are also characteristic of the -EF2 mutant of recoverin whose second Ca²⁺-binding site is modified and cannot bind calcium ions. The structural properties of the -EF3 and -EF2,3 mutants (whose third or simultaneously second and third Ca²⁺-binding sites, respectively, are modified and damaged) are practically indifferent to the presence of calcium ions. For the communication I, see [1].     

Preparation and characteristics of Ca<Superscript>2+</Superscript>-dependent monoclonal antibodies to recoverin

Thirty-four primary hybridoma clones were prepared which expressed monoclonal antibodies to the Ca²⁺-binding protein recoverin. Among the resulting monoclonal antibodies, two Ca²⁺-dependent clones (mAb3 and mAb19) recognizing recoverin were detected by solid-phase immunoenzyme assay. In the presence of Ca²⁺, antibodies of the mAb3 and mAb19 clones bound to recoverin several times better than in the absence of Ca²⁺. The mAb3 and mAb19 antibodies recognized epitopes located inside the sequences Pro61-Met91 and Pro57-Tyr64 of the recoverin molecule, respectively. The possible mechanism of the Ca²⁺-dependent recognition of recoverin by the prepared monoclonal antibodies is discussed.Translated from Biokhimiya, Vol. 69, No. 12, 2004, pp. 1667–1674.Original Russian Text Copyright © 2004 by Tikhomirova, Goncharskaya, Senin.     

Structure and Calcium Binding Properties of a Neuronal Calcium-Myristoyl Switch Protein,Visinin-Like Protein 3

Visinin-like protein 3 (VILIP-3) belongs to a family of Ca²⁺-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca²⁺ binding, characterize Ca²⁺-induced conformational changes, and determine the NMR structure of myristoylated VILIP-3. Three Ca²⁺ bind cooperatively to VILIP-3 at EF2, EF3 and EF4 (K_D = 0.52 μM and Hill slope of 1.8). NMR assignments, mutagenesis and structural analysis indicate that the covalently attached myristoyl group is solvent exposed in Ca²⁺-bound VILIP-3, whereas Ca²⁺-free VILIP-3 contains a sequestered myristoyl group that interacts with protein residues (E26, Y64, V68), which are distinct from myristate contacts seen in other Ca²⁺-myristoyl switch proteins. The myristoyl group in VILIP-3 forms an unusual L-shaped structure that places the C₁₄ methyl group inside a shallow protein groove, in contrast to the much deeper myristoyl binding pockets observed for recoverin, NCS-1 and GCAP1. Thus, the myristoylated VILIP-3 protein structure determined in this study is quite different from those of other known myristoyl switch proteins (recoverin, NCS-1, and GCAP1). We propose that myristoylation serves to fine tune the three-dimensional structures of neuronal calcium sensor proteins as a means of generating functional diversity.     

Point amino acid substitutions in the Ca2+-binding sites of recoverin: III. A mutant with the fourth reconstructed Ca2+-binding site

Unlike wild type recoverin with only two (the second and the third) functioning Ca⁺²-binding sites out of four potential ones, the +EF4 mutant contains a third active Ca⁺²-binding site. This site was reconstructed from the fourth potential Ca⁺²-binding domain by the introduction of several amino acid substitutions in it by site-directed mutagenesis. The effect of these mutations in the fourth potential Ca⁺²-binding site of myristoylated recoverin on the structural features and conformational stability of the protein was studied by fluorimetry and circular dichroism. The apoform of the resulting mutant (free of Ca²⁺ ions) was shown to have a higher calcium capacity, significantly lower thermal stability, and noticeably different secondary and tertiary structures as compared with the apoform of wild-type recoverin. For communication II, see [1].     

The cone-specific calcium sensor guanylate cyclase activating protein 4 from the zebrafish retina

Guanylate cyclase activating proteins (GCAPs) serve as neuronal Ca²⁺-sensor proteins in vertebrate rod and cone photoreceptor cells. Zebrafish express in their retina a variety of six different GCAPs, of which four are specific for cone cells. One isoform, zGCAP4, is mainly expressed in double cones and long single cones. We cloned the zGCAP4 gene, purified non-myristoylated and myristoylated forms of the protein after heterologous expression in Escherichia coli and studied its properties: zGCAP4 was a strong activator of membrane-bound guanylate cyclases from bovine and zebrafish retina, showing half-maximal activation at 520–570 nM free Ca²⁺ concentration. Furthermore, the Ca²⁺-sensitive activation properties of non-myristoylated and myristoylated zGCAP4 were similar, indicating no influence of the myristoyl moiety on Ca²⁺-sensor function. Myristoylated zGCAP4 showed low affinity for membranes and did not exhibit a Ca²⁺–myristoyl switch, a feature typical of some but not all neuronal Ca²⁺-sensor proteins. However, tryptophan fluorescence studies and Ca²⁺-dependent differences in protease accessibility revealed Ca²⁺-induced conformational changes in myristoylated and non-myristoylated zGCAP4, indicating the operation as a Ca²⁺ sensor. Thus, expression and biochemical properties of zGCAP4 are in agreement with its function as an efficient Ca²⁺-sensitive regulator of guanylate cyclase activity in cone vision.     

Ca2+ and Mg2+ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

Downstream Regulatory Element Antagonist Modulator (DREAM) belongs to the family of neuronal calcium sensors (NCS) that transduce the intracellular changes in Ca²⁺ concentration into a variety of responses including gene expression, regulation of Kv channel activity, and calcium homeostasis. Despite the significant sequence and structural similarities with other NCS members, DREAM shows several features unique among NCS such as formation of a tetramer in the apo-state, and interactions with various intracellular biomacromolecules including DNA, presenilin, Kv channels, and calmodulin. Here we use spectroscopic techniques in combination with molecular dynamics simulation to study conformational changes induced by Ca²⁺/Mg²⁺ association to DREAM. Our data indicate a minor impact of Ca²⁺ association on the overall structure of the N- and C-terminal domains, although Ca²⁺ binding decreases the conformational heterogeneity as evident from the decrease in the fluorescence lifetime distribution in the Ca²⁺ bound forms of the protein. Time-resolved fluorescence data indicate that Ca²⁺binding triggers a conformational transition that is characterized by more efficient quenching of Trp residue. The unfolding of DREAM occurs through an partially unfolded intermediate that is stabilized by Ca²⁺ association to EF-hand 3 and EF-hand 4. The native state is stabilized with respect to the partially unfolded state only in the presence of both Ca²⁺ and Mg²⁺ suggesting that, under physiological conditions, Ca²⁺ free DREAM exhibits a high conformational flexibility that may facilitate its physiological functions.     

CaMKII isoforms differ in their specific requirements for regulation by nitric oxide

The Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) mediates physiological and pathological functions by its Ca²⁺-independent autonomous activity. Two novel mechanisms for generating CaMKII autonomy include oxidation and S-nitrosylation, the latter requiring both Cys280 and Cys289 amino acid residues in the brain-specific isoform CaMKIIα. Even though the other CaMKII isoforms have a different amino acid in the position homologous to Cys280, we show here that nitric oxide (NO)-signaling generated autonomy also for the CaMKIIβ isoform. Furthermore, although oxidation of the Met280/281 residues is sufficient to generate autonomy for most CaMKII isoforms, oxidation-induced autonomy was also prevented by a Cys289-mutation in the CaMKIIα isoform. Thus, all CaMKII isoforms can be regulated by physiological NO-signaling, but CaMKIIα regulation by oxidation and S-nitrosylation is more stringent.     

Recoverin as a cancer-retina antigen

In photoreceptor cells the Ca²⁺-binding protein recoverin controls phosphorylation of the visual receptor rhodopsin by inhibiting rhodopsin kinase (GRK-1). It can also serve as a paraneoplastic antigen in the development of retinal degeneration in some patients with cancer. The aberrant expression of recoverin in cancer cells and the presence of autoantibodies against recoverin are essential for the occurrence of cancer-associated retinopathy, which finally results in the apoptosis of photoreceptor cells. Noteworthy in cancer patients, the aberrant recoverin expression and the appearance of autoantibodies against recoverin are more frequent than paraneoplastic syndromes. We suggest the term “cancer-retina antigens” for this kind of proteins like recoverin that are solely expressed in retina and tumor tissues and evoke antibodies and/or T cells in patients with cancer. The rare development of a paraneoplastic syndrome is possibly caused by this immune response and probably depends on further events allowing to overcome the blood–retina barrier and the immune privileged status of the retina. It is still unknown whether aberrantly expressed recoverin could have a specific function in cancer cells, though it is suggested that it can be functionally associated with G-protein-coupled receptor kinases. This paper reviews the present knowledge on paraneoplastic syndromes associated with the aberrant expression of recoverin. A possible application of recoverin as a potential target for immunotherapy of cancer is discussed.This article is a symposium paper from the conference “Progress in Vaccination against Cancer 2005 (PIVAC 5)”, held in Athens, Greece, on 20–21 September 2005.     

Refined crystal structures of human Ca(2+)/Zn(2+)-binding S100A3 protein characterized by two disulfide bridges

S100A3, a member of the EF-hand-type Ca²⁺-binding S100 protein family, is unique in its exceptionally high cysteine content and Zn²⁺ affinity. We produced human S100A3 protein and its mutants in insect cells using a baculovirus expression system. The purified wild-type S100A3 and the pseudo-citrullinated form (R51A) were crystallized with ammonium sulfate in N,N-bis(2-hydroxyethyl)glycine buffer and, specifically for postrefolding treatment, with Ca²⁺/Zn²⁺ supplementation. We identified two previously undocumented disulfide bridges in the crystal structure of properly folded S100A3: one disulfide bridge is between Cys30 in the N-terminal pseudo-EF-hand and Cys68 in the C-terminal EF-hand (SS1), and another disulfide bridge attaches Cys99 in the C-terminal coil structure to Cys81 in helix IV (SS2). Mutational disruption of SS1 (C30A + C68A) abolished the Ca²⁺ binding property of S100A3 and retarded the citrullination of Arg51 by peptidylarginine deiminase type III (PAD3), while SS2 disruption inversely increased both Ca²⁺ affinity and PAD3 reactivity in vitro. Similar backbone structures of wild type, R51A, and C30A + C68A indicated that neither Arg51 conversion by PAD3 nor SS1 alters the overall dimer conformation. Comparative inspection of atomic coordinates refined to 2.15−1.40 Å resolution shows that SS1 renders the C-terminal classical Ca²⁺-binding loop flexible, which are essential for its Ca²⁺ binding properties, whereas SS2 structurally shelters Arg51 in the metal-free form. We propose a model of the tetrahedral coordination of a Zn²⁺ by (Cys)₃His residues that is compatible with SS2 formation in S100A3.     

Light-induced disulfide dimerization of recoverin under ex vivo and in vivo conditions

Despite vast knowledge of the molecular mechanisms underlying photochemical damage of photoreceptors, linked to progression of age-related macular degeneration, information on specific protein targets of the light-induced oxidative stress is scarce. Here, we demonstrate that prolonged intense illumination (halogen bulb, 1500 lx, 1–5 h) of mammalian eyes under ex vivo (cow) or in vivo (rabbit) conditions induces disulfide dimerization of recoverin, a Ca²⁺-dependent inhibitor of rhodopsin kinase. Western blotting and mass spectrometry analysis of retinal extracts reveals illumination time-dependent accumulation of disulfide homodimers of recoverin and its higher order disulfide cross-linked species, including a minor fraction of mixed disulfides with intracellular proteins (tubulins, etc.). Meanwhile, monomeric bovine recoverin remains mostly reduced. These effects are accompanied by accumulation of disulfide homodimers of visual arrestin. Histological studies demonstrate that the light-induced oxidation of recoverin and arrestin occurs in intact retina (illumination for 2 h), while illumination for 5 h is associated with damage of the photoreceptor layer. A comparison of ex vivo levels of disulfide homodimers of bovine recoverin with redox dependence of its in vitro thiol–disulfide equilibrium (glutathione redox pair) gives the lowest estimate of redox potential in rod outer segments under illumination from −160 to −155  mV. Chemical crosslinking and dynamic light scattering data demonstrate an increased propensity of disulfide dimer of bovine recoverin to multimerization/aggregation. Overall, the oxidative stress caused by the prolonged intense illumination of retina might affect rhodopsin desensitization via concerted disulfide dimerization of recoverin and arrestin. The developed herein models of eye illumination are useful for studies of the light-induced thiol oxidation of visual proteins.