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Pavel P. Philippov - One of the best experts on this subject based on the ideXlab platform.
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Light-induced disulfide dimerization of Recoverin under ex vivo and in vivo conditions
Free radical biology & medicine, 2015Co-Authors: Evgeni Yu. Zernii, Pavel P. Philippov, Aliya A. Nazipova, Dmitry V. Zinchenko, Ivan I. Senin, N.k. Tikhomirova, O. S. Gancharova, Alexey S. Kazakov, Marina V. Serebryakova, Eugene A. PermyakovAbstract:Abstract 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.
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ca2 dependent regulatory activity of Recoverin in photoreceptor raft structures the role of caveolin 1
Biochemistry (moscow) Supplement Series A: Membrane and Cell Biology, 2014Co-Authors: Evgeni Yu. Zernii, Pavel P. Philippov, Dmitry V. Zinchenko, I. I. Grigoriev, Elena E. Skorikova, V M Lipkin, Vasiliy I Vladimirov, Viktoriia E Baksheeva, Ivan I. SeninAbstract: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.
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Synergetic Effect of Recoverin and Calmodulin on Regulation of Rhodopsin Kinase
Frontiers in molecular neuroscience, 2012Co-Authors: I. I. Grigoriev, Karl-wilhelm Koch, Sergei E. Permyakov, Ivan I. Senin, Konstantin E. Komolov, N.k. Tikhomirova, Evgeni Yurievich Zernii, Pavel P. PhilippovAbstract:Phosphorylation of photoactivated rhodopsin by rhodopsin kinase (RK or GRK1), a first step of the phototransduction cascade turnoff, is under the control of Ca2+/Recoverin. Here, we demonstrate that calmodulin, a ubiquitous Ca2+-sensor, can inhibit RK, though less effectively than Recoverin does. We have utilized the surface plasmon resonance (SPR) technology to map the calmodulin binding site in the RK molecule. Calmodulin does not interact with the Recoverin binding site within amino acid residues M1-S25 of the enzyme. Instead, the high affinity calmodulin binding site is localized within a stretch of amino acid residues V150-K175 in the N-terminal regulatory region of RK. Moreover, the inhibitory effect of calmodulin and Recoverin on RK activity is synergetic, which is in agreement with the existence of separate binding sites for each Ca2+-sensing protein. The synergetic inhibition of RK by both Ca2+-sensors occurs over a broader range of Ca2+-concentration than by Recoverin alone, indicating increased Ca2+-sensitivity of RK regulation in the presence of both Ca2+-sensors. Taken together, our data suggest that RK regulation by calmodulin in photoreceptor cells could complement the well-known inhibitory effect of Recoverin on RK.
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Aberrant demethylation of the Recoverin gene is involved in the aberrant expression of Recoverin in cancer cells.
Experimental dermatology, 2010Co-Authors: Alexandr V. Bazhin, Charles De Smet, Marina O. Golovastova, Jan Schmidt, Pavel P. PhilippovAbstract:The Ca2+-binding protein Recoverin is normally specific for the retina. Recoverin aberrantly expressed in lung and melanoma tumors can trigger the host immune response followed by the development of a paraneoplastic neurological syndrome represented by cancer- and melanoma-associated retinopathy, respectively. The mechanisms, underlying the aberrant expression of Recoverin in tumor cells, have remained unknown. The data obtained in this study suggest that (i) DNA methylation participates in the repression of synthesis of mRNA for Recoverin in normal tissues and (ii) aberrant hypomethylation of the Recoverin gene region, overlapping the promoter up-stream of the first exon and the first exon itself, is involved in the aberrant expression of Recoverin in tumor cells.
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Expression of Ca-binding protein Recoverin in normal, surviving, and regenerating retina of Pleurodeles waltl adult triton.
Bulletin of experimental biology and medicine, 2009Co-Authors: E. N. Grigoryan, Alexandr V. Bazhin, M. S. Krasnov, Pavel P. PhilippovAbstract:Immunohistochemical study of the expression of Recoverin (photoreceptor protein) in the retina of Pleurodeles waltl adult triton was carried out in health, during regeneration after removal, and under conditions of long-lasting detachment. Studies with polyclonal (monospecific) antibodies to Recoverin showed that normally it is present in the internal segment, connective cilium, in distal portions of the external segments of cones and rods, and in Landolt clubs of displaced bipolar cells. Detachment of the retina is associated with translocation of Recoverin from the photoreceptor processes to perikaryons, and the content of Recoverin-positive displaced bipolar cells increases. During regeneration of the retina after its excision via conversion of the pigmented epithelial cells, Recoverin is synthesized in the prospective photoreceptor perikaryons and then accumulates in the forming inner segments. Hence, Recoverin can serve as a reliable marker in studies of photoreceptor differentiation and functioning during regeneration or survival of the retina.
Karl-wilhelm Koch - One of the best experts on this subject based on the ideXlab platform.
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Interaction of G protein-coupled receptor kinases and Recoverin isoforms is determined by localization in zebrafish photoreceptors.
Biochimica et biophysica acta. Molecular cell research, 2020Co-Authors: Nicole Ahrens, Dana Elbers, Helena Greb, Ulrike Janssen-bienhold, Karl-wilhelm KochAbstract:The zebrafish retina expresses four Recoverin genes (rcv1a, rcv1b, rcv2a and rcv2b) and four opsin kinase genes (grk1a, grk1b, grk7a and grk7b) coding for Recoverin and G protein-coupled receptor kinase (opsin kinase) paralogs, respectively. Both protein groups are suggested to form regulatory complexes in rod and cone outer segments, but at present, we lack information about co-localization of Recoverin and opsin kinases in zebrafish retinae and which protein-protein interacting pairs form. We analyzed the distribution and co-localization of Recoverin and opsin kinase expression in the zebrafish retina. For this purpose, we used custom-tailored monospecific antibodies revealing that the amount of Recoverin paralogs in a zebrafish retina can differ by more than one order of magnitude with the highest amount for Recoverin 1a and 2b. Further, immunohistochemical labelling showed presence of Recoverin 1a in all rod cell compartments, but it only co-localized with opsin kinase 1a in rod outer segments. In contrast, Recoverin 2b was only detected in double cones and co-localized with opsin kinases 1b, 7a and 7b. Further, we investigated the interaction between Recoverin and opsin kinase variants by surface plasmon resonance spectroscopy indicating interaction of Recoverin 1a and Recoverin 2b with all opsin kinases. However, binding kinetics for Recoverin 1a differed from those observed with Recoverin 2b that showed slower association and dissociation processes. Our results indicate diverse Recoverin and opsin kinase properties due to differential expression and interaction profiles.
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Zebrafish Recoverin isoforms display differences in calcium switch mechanisms
Frontiers in molecular neuroscience, 2018Co-Authors: Dana Elbers, Alexander Scholten, Karl-wilhelm KochAbstract:Primary steps in vertebrate vision occur in rod and cone cells of the retina and require precise molecular switches in excitation, recovery, and adaptation. In particular, recovery of the photoresponse and light adaptation processes are under control of neuronal Ca2+ sensor (NCS) proteins. Among them, the Ca2+ sensor Recoverin undergoes a pronounced Ca2+-dependent conformational change, a prototypical so-called Ca2+-myristoyl switch, which allows selective targeting of G protein-coupled receptor kinase. Zebrafish (Danio rerio) has gained attention as a model organism in vision research. It expresses four different Recoverin isoforms (zRec1a, zRec1b, zRec2a, and zRec2b) that are orthologs to the one known mammalian variant. The expression pattern of the four isoforms cover both rod and cone cells, but the differential distribution in cones points to versatile functions of Recoverin in these cell types. Initial functional studies on zebrafish larvae indicate different Ca2+-sensitive working modes for zebrafish Recoverins, but experimental evidence is lacking so far. The aims of the present study are (1) to measure specific Ca2+-sensing properties of the different Recoverin isoforms, (2) to ask whether switch mechanisms triggered by Ca2+ resemble that one observed with mammalian Recoverin, and (3) to investigate a possible impact of an attached myristoyl moiety. For addressing these questions, we employ fluorescence spectroscopy, surface plasmon resonance (SPR), dynamic light scattering, and equilibrium centrifugation. Exposure of hydrophobic amino acids, due to the myristoyl switch, differed among isoforms and depended also on the myristoylation state of the particular Recoverin. Ca2+-induced rearrangement of the protein-water shell was for all variants less pronounced than for the bovine ortholog indicating either a modified Ca2+-myristoyl switch or no switch. Our results have implications for a step-by-step response of Recoverin isoforms to changing intracellular Ca2+ during illumination.
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Table_1_Zebrafish Recoverin Isoforms Display Differences in Calcium Switch Mechanisms.DOCX
2018Co-Authors: Dana Elbers, Alexander Scholten, Karl-wilhelm KochAbstract:Primary steps in vertebrate vision occur in rod and cone cells of the retina and require precise molecular switches in excitation, recovery, and adaptation. In particular, recovery of the photoresponse and light adaptation processes are under control of neuronal Ca2+ sensor (NCS) proteins. Among them, the Ca2+ sensor Recoverin undergoes a pronounced Ca2+-dependent conformational change, a prototypical so-called Ca2+-myristoyl switch, which allows selective targeting of G protein-coupled receptor kinase. Zebrafish (Danio rerio) has gained attention as a model organism in vision research. It expresses four different Recoverin isoforms (zRec1a, zRec1b, zRec2a, and zRec2b) that are orthologs to the one known mammalian variant. The expression pattern of the four isoforms cover both rod and cone cells, but the differential distribution in cones points to versatile functions of Recoverin in these cell types. Initial functional studies on zebrafish larvae indicate different Ca2+-sensitive working modes for zebrafish Recoverins, but experimental evidence is lacking so far. The aims of the present study are (1) to measure specific Ca2+-sensing properties of the different Recoverin isoforms, (2) to ask whether switch mechanisms triggered by Ca2+ resemble that one observed with mammalian Recoverin, and (3) to investigate a possible impact of an attached myristoyl moiety. For addressing these questions, we employ fluorescence spectroscopy, surface plasmon resonance (SPR), dynamic light scattering, and equilibrium centrifugation. Exposure of hydrophobic amino acids, due to the myristoyl switch, differed among isoforms and depended also on the myristoylation state of the particular Recoverin. Ca2+-induced rearrangement of the protein-water shell was for all variants less pronounced than for the bovine ortholog indicating either a modified Ca2+-myristoyl switch or no switch. Our results have implications for a step-by-step response of Recoverin isoforms to changing intracellular Ca2+ during illumination.
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Synergetic Effect of Recoverin and Calmodulin on Regulation of Rhodopsin Kinase
Frontiers in molecular neuroscience, 2012Co-Authors: I. I. Grigoriev, Karl-wilhelm Koch, Sergei E. Permyakov, Ivan I. Senin, Konstantin E. Komolov, N.k. Tikhomirova, Evgeni Yurievich Zernii, Pavel P. PhilippovAbstract:Phosphorylation of photoactivated rhodopsin by rhodopsin kinase (RK or GRK1), a first step of the phototransduction cascade turnoff, is under the control of Ca2+/Recoverin. Here, we demonstrate that calmodulin, a ubiquitous Ca2+-sensor, can inhibit RK, though less effectively than Recoverin does. We have utilized the surface plasmon resonance (SPR) technology to map the calmodulin binding site in the RK molecule. Calmodulin does not interact with the Recoverin binding site within amino acid residues M1-S25 of the enzyme. Instead, the high affinity calmodulin binding site is localized within a stretch of amino acid residues V150-K175 in the N-terminal regulatory region of RK. Moreover, the inhibitory effect of calmodulin and Recoverin on RK activity is synergetic, which is in agreement with the existence of separate binding sites for each Ca2+-sensing protein. The synergetic inhibition of RK by both Ca2+-sensors occurs over a broader range of Ca2+-concentration than by Recoverin alone, indicating increased Ca2+-sensitivity of RK regulation in the presence of both Ca2+-sensors. Taken together, our data suggest that RK regulation by calmodulin in photoreceptor cells could complement the well-known inhibitory effect of Recoverin on RK.
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dynamic cellular translocation of caldendrin is facilitated by the ca2 myristoyl switch of Recoverin
Journal of Neurochemistry, 2010Co-Authors: Ramona Fries, Pasham Parameshwar Reddy, Marina Mikhaylova, Silke Haverkamp, Tao Wei, Michael Müller, Michael R. Kreutz, Karl-wilhelm KochAbstract:J. Neurochem. (2010) 113, 1150–1162. Abstract Caldendrin and Recoverin are Ca2+-sensor proteins operating in neuronal systems. In a search for novel binding partners of Recoverin, we employed an affinity column and identified caldendrin as a possible interaction partner. Caldendrin and Recoverin co-localized in the retina in a subset of bipolar cells and in the pineal gland as revealed by immunofluorescence studies. The binding process was controlled by Ca2+ as revealed by pull-down assays, and surface plasmon resonance studies. Importantly, caldendrin existed as a Ca2+-independent homodimer whereas a complex of Recoverin and caldendrin formed with low to moderate affinity in the presence of Ca2+. Co-transfection of COS-7 cells with plasmids harboring the gene for fluorescently labeled Recoverin and caldendrin was used to study the cellular distribution by time-lapse fluorescence microscopy. Apparently, the increase of intracellular Ca2+ facilitates the translocation of caldendrin to intracellular membranes, which is under control of complex formation with Recoverin.
Ivan I. Senin - One of the best experts on this subject based on the ideXlab platform.
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Light-induced disulfide dimerization of Recoverin under ex vivo and in vivo conditions
Free radical biology & medicine, 2015Co-Authors: Evgeni Yu. Zernii, Pavel P. Philippov, Aliya A. Nazipova, Dmitry V. Zinchenko, Ivan I. Senin, N.k. Tikhomirova, O. S. Gancharova, Alexey S. Kazakov, Marina V. Serebryakova, Eugene A. PermyakovAbstract:Abstract 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.
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ca2 dependent regulatory activity of Recoverin in photoreceptor raft structures the role of caveolin 1
Biochemistry (moscow) Supplement Series A: Membrane and Cell Biology, 2014Co-Authors: Evgeni Yu. Zernii, Pavel P. Philippov, Dmitry V. Zinchenko, I. I. Grigoriev, Elena E. Skorikova, V M Lipkin, Vasiliy I Vladimirov, Viktoriia E Baksheeva, Ivan I. SeninAbstract: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.
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Synergetic Effect of Recoverin and Calmodulin on Regulation of Rhodopsin Kinase
Frontiers in molecular neuroscience, 2012Co-Authors: I. I. Grigoriev, Karl-wilhelm Koch, Sergei E. Permyakov, Ivan I. Senin, Konstantin E. Komolov, N.k. Tikhomirova, Evgeni Yurievich Zernii, Pavel P. PhilippovAbstract:Phosphorylation of photoactivated rhodopsin by rhodopsin kinase (RK or GRK1), a first step of the phototransduction cascade turnoff, is under the control of Ca2+/Recoverin. Here, we demonstrate that calmodulin, a ubiquitous Ca2+-sensor, can inhibit RK, though less effectively than Recoverin does. We have utilized the surface plasmon resonance (SPR) technology to map the calmodulin binding site in the RK molecule. Calmodulin does not interact with the Recoverin binding site within amino acid residues M1-S25 of the enzyme. Instead, the high affinity calmodulin binding site is localized within a stretch of amino acid residues V150-K175 in the N-terminal regulatory region of RK. Moreover, the inhibitory effect of calmodulin and Recoverin on RK activity is synergetic, which is in agreement with the existence of separate binding sites for each Ca2+-sensing protein. The synergetic inhibition of RK by both Ca2+-sensors occurs over a broader range of Ca2+-concentration than by Recoverin alone, indicating increased Ca2+-sensitivity of RK regulation in the presence of both Ca2+-sensors. Taken together, our data suggest that RK regulation by calmodulin in photoreceptor cells could complement the well-known inhibitory effect of Recoverin on RK.
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Involvement of the Recoverin C-terminal segment in recognition of the target enzyme rhodopsin kinase.
The Biochemical journal, 2011Co-Authors: Evgeni Yu. Zernii, Sergei E. Permyakov, Eugene A. Permyakov, Konstantin E. Komolov, Tatiana V. Kolpakova, Daniele Dell'orco, Annika Poetzsch, Ekaterina L. Knyazeva, I. I. Grigoriev, Ivan I. SeninAbstract:-dependent manner. In the present study, we investigated a series of Recoverin forms that were mutated at the C-terminus. Using pull-down assays, surface plasmon resonance spectroscopy and rhodopsin phosphorylation assays, we demonstrated that truncation of Recoverin at the C-terminus significantly reduced the affinity of Recoverin for rhodopsin kinase. Site-directed mutagenesis of single amino acids in combination with structural analysis and computational modelling of the Recoverin-kinase complex provided insight into the protein-protein interface between the kinase and the C-terminus of Recoverin. Based on these results we suggest that Phe 3 from the N-terminal helix of rhodopsin kinase and Lys 192 from the C-terminal segment of Recoverin form a cation-π interaction pair which is essential for target recognitionbyRecoverin.Takentogether,theresultsofthepresent study reveal a novel rhodopsin-kinase-binding site within the C- terminal region of Recoverin, and highlights its significance for target recognition and regulation.
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Mechanism of rhodopsin kinase regulation by Recoverin.
Journal of neurochemistry, 2009Co-Authors: Konstantin E. Komolov, Pavel P. Philippov, Ivan I. Senin, Valeriya A. Churumova, I. I. Grigoriev, Nadezda A. Kovaleva, Mathias P. Christoph, Muhammad Akhtar, Karl-wilhelm KochAbstract:Recoverin is suggested to inhibit rhodopsin kinase (GRK1) at high [Ca(2+)] in the dark state of the photoreceptor cell. Decreasing [Ca(2+)] terminates inhibition and facilitates phosphorylation of illuminated rhodopsin (Rh*). When Recoverin formed a complex with GRK1, it did not interfere with the phosphorylation of a C-terminal peptide of rhodopsin (S338-A348) by GRK1. Furthermore, while GRK1 competed with transducin on interaction with rhodopsin and thereby suppressed GTPase activity of transducin, Recoverin in the complex with GRK1 did not influence this competition. Constructs of GRK1 that encompass its N-terminal, catalytic or C-terminal domains were used in pull-down assays and surface plasmon resonance analysis to monitor interaction. Ca(2+)-Recoverin bound to the N-terminus of GRK1, but did not bind to the other constructs. GRK1 interacted with rhodopsin also by its N-terminus in a light-dependent manner. No interaction was observed with the C-terminus. We conclude that inhibition of GRK1 by Recoverin is not the result of their direct competition for the same docking site on Rh*, although the interaction sites of GRK1/Rh* and GRK1/Recoverin partially overlap. The N-terminus of GRK1 is recognized by Rh* leading to a conformational change which moves the C-terminus of Rh* into the catalytic kinase groove. Ca(2+)-Recoverin interacting with the N-terminus of GRK1 prevents this conformational change and thus blocks Rh* phosphorylation by GRK1.
Lubert Stryer - One of the best experts on this subject based on the ideXlab platform.
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Molecular structure of membrane-targeting calcium sensors in vision: Recoverin and guanylate cyclase-activating protein 2.
Methods in enzymology, 2000Co-Authors: James B. Ames, Mitsuhiko Ikura, Lubert StryerAbstract:Publisher Summary An important class of calcium-binding proteins present in retinal photoreceptor cells includes Recoverin from mammalian rods, guanylate cyclase-activating proteins (GCAP-1, GCAP-2, and GCAP-3) from mammalian rods and cones, and guanylate cyclase inhibitory protein (GCIP) from frog rods. The Recoverin branch of the EF-hand superfamily includes neuronal Ca 2+ sensors such as neurocalcin, frequenin, and hippocalcin. Mass spectrometric analysis of retinal Recoverin and the GCAP proteins revealed that they are myristoylated at the amino terminus. GCAP-2 exhibits different membrane-targeting properties from those of Recoverin. The myristoylated and unmyristoylated forms of GCAP-2 bind to membranes at low Ca 2+ concentrations ( 50 mM NaCl).This chapter presents a detailed structural analysis of Recoverin and GCAP-2. The Ca 2+ -induced structural changes in these proteins are important for elucidating their membrane-targeting mechanisms and for understanding the molecular mechanism of Ca 2+ -sensitive regulation of phototransduction.
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The effect of recombinant Recoverin on the photoresponse of truncated rod photoreceptors.
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Martha A. Erickson, Thomas A. Neubert, Lubert Stryer, Leon Lagnado, Sergey Zozulya, Denis A. BaylorAbstract:Recoverin is a heterogeneously acylated calcium-binding protein thought to regulate visual transduction. Its effect on the photoresponse was investigated by dialyzing the recombinant protein into truncated salamander rod outer segments. At high Ca2+ (Ca), myristoylated Recoverin (Ca-Recoverin) prolonged the recovery phase of the bright flash response but had less effect on the dim flash response. The prolongation of recovery had an apparent Kd for Ca of 13 μM and a Hill coefficient of 2. The prolongation was shown to be mediated by inhibition of rhodopsin deactivation. After a sudden imposed drop in Ca concentration, the effect of Recoverin switched off with little lag. The myristoyl (C14:0) modification of Recoverin increased its activity 12-fold, and the C12:0 or C14:2 acyl group gave similar effects. These experiments support the notion that Recoverin mediates Ca-dependent inhibition of rhodopsin phosphorylation and thereby controls light-triggered phosphodiesterase activity, particularly at high light levels.
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Nuclear Magnetic Resonance Evidence for Ca2+-induced Extrusion of the Myristoyl Group of Recoverin
The Journal of biological chemistry, 1995Co-Authors: James B. Ames, Toshiyuki Tanaka, Mitsuhiko Ikura, Lubert StryerAbstract:Abstract Recoverin, a recently discovered member of the EF-hand protein superfamily, serves as a Ca sensor in vision. A myristoyl or related N-acyl group covalently attached to the amino terminus of Recoverin enables it to translocate to retinal disc membranes when the Ca level is elevated. Two-dimensional 1H-C shift correlation NMR spectra of Recoverin containing a C-labeled myristoyl group were obtained to selectively probe the effect of Ca on the environment of the attached myristoyl group. In the Ca-free state, each pair of methylene protons bonded to carbon atoms 2, 3, 11, and 12 of the myristoyl group gives rise to two peaks. The splittings, caused by nonequivalent methylene proton chemical shifts, indicate that the myristoyl group interacts intimately with the protein in the Ca-free state. By contrast, only one peak is seen for each pair of methylene protons in the Ca-bound state, indicating that the myristoyl group is located in an isotropic environment in this form. Furthermore, the 1H-C shift correlation NMR spectrum of Ca-bound Recoverin is very similar to that of myristic acid in solution. 1H-C shift correlation NMR experiments were also performed with C-labeled Recoverin to selectively probe the resonances of methyl groups in the hydrophobic core of the protein. The spectrum of Ca-bound myristoylated Recoverin is different from that of Ca-free myristoylated Recoverin but similar to that of Ca-bound unmyristoylated Recoverin. Hence, the myristoyl group interacts little with the hydrophobic core of myristoylated Recoverin in the Ca-bound state. Three-dimensional (C/F1)-edited (C/F3)-filtered heteronuclear multiple quantum correlation-nuclear Overhauser effect spectroscopy spectra of Recoverin containing a C-labeled myristoyl group were obtained to selectively probe protein residues located within 5 A of the myristoyl group. The myristoyl group makes close contact with a number of aromatic residues in Ca-free Recoverin, whereas the myristoyl group makes no observable contacts with the protein in the Ca-bound state. These NMR data demonstrate that the binding of Ca to Recoverin induces the extrusion of its myristoyl group into the solvent, which would enable it to interact with a lipid bilayer or a hydrophobic site of a target protein.
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Sequestration of the membrane-targeting myristoyl group of Recoverin in the calcium-free state
Nature, 1995Co-Authors: Toshiyuki Tanaka, James B. Ames, Lubert Stryer, Timothy S. Harvey, Mitsuhiko IkuraAbstract:Recoverin, a retinal calcium-binding protein of relative molecular mass (M(r)) 23K, participates in the recovery phase of visual excitation and in adaptation to background light. The Ca(2+)-bound form of Recoverin prolongs the photoresponse, probably by blocking phosphorylation of photoexcited rhodopsin. Retinal Recoverin contains a covalently attached myristoyl group or related acyl group at its amino terminus and two Ca(2+)-binding sites. Ca2+ binding to myristoylated, but not unmyristoylated, Recoverin induces its translocation to bilayer membranes, indicating that the myristoyl group is essential to the read-out of calcium signals (calcium-myristoyl switch). Here we present the solution structure of Ca(2+)-free, myristoylated recombinant Recoverin obtained by heteronuclear multidimensional NMR spectroscopy. The myristoyl group is sequestered in a deep hydrophobic pocket formed by many aromatic and other hydrophobic residues from five flanking helices.
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Amino-terminal Myristoylation Induces Cooperative Calcium Binding to Recoverin
The Journal of biological chemistry, 1995Co-Authors: James B. Ames, Toshiyuki Tanaka, Mitsuhiko Ikura, Tudor Porumb, Lubert StryerAbstract:Recoverin, a new member of the EF-hand protein superfamily, serves as a Ca2+ sensor in vision. A myristoyl or related N-acyl group covalently attached to the amino terminus of Recoverin enables it to bind to disc membranes when the Ca2+ level is elevated. Ca2+-bound Recoverin prolongs the lifetime of photoexcited rhodopsin, most likely by blocking its phosphorylation. We report here Ca2+ binding studies of myristoylated and unmyristoylated recombinant Recoverin using flow dialysis, fluorescence, and NMR spectroscopy. Unmyristoylated Recoverin exhibits heterogeneous and uncooperative binding of two Ca2+ with dissociation constants of 0.11 and 6.9 μM. In contrast, two Ca2+ bind cooperatively to myristoylated Recoverin with a Hill coefficient of 1.75 and an apparent dissociation constant of 17 μM. Thus, the attached myristoyl group lowers the calcium affinity of the protein and induces cooperativity in Ca2+ binding. One-dimensional 1H and two-dimensional 15N-1H shift correlation NMR spectra of myristoylated Recoverin measured as a function of Ca2+ concentration show that a concerted conformational change occurs when two Ca2+ are bound. The Ca2+ binding and NMR data can be fit to a concerted allosteric model in which the two Ca2+ binding sites have different affinities in both the T and R states. The T and R conformational states are defined in terms of the Ca2+-myristoyl switch; in the T state, the myristoyl group is sequestered inside the protein, whereas in the R state, the myristoyl group is extruded. Ca2+ binds to the R state at least 10,000-fold more tightly than to T. In this model, the dissociation constants of the two sites in the R state of the myristoylated protein are 0.11 and 6.9 μM, as in unmyristoylated Recoverin. The ratio of the unliganded form of T to that of R is estimated to be 400 for myristoylated and
James B. Ames - One of the best experts on this subject based on the ideXlab platform.
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Double Electron–Electron Resonance Probes Ca2+-Induced Conformational Changes and Dimerization of Recoverin
2016Co-Authors: William K. Myers, Jens O. Lagerstedt, Madhu S. Budamagunta, John C. Voss, David R. Britt, James B. AmesAbstract: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
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Double electron-electron resonance probes Ca2+-induced conformational changes and dimerization of Recoverin
Biochemistry, 2013Co-Authors: William K. Myers, Jens O. Lagerstedt, Madhu S. Budamagunta, John C. Voss, R. David Britt, James B. AmesAbstract: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.
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conformational dynamics of Recoverin s ca2 myristoyl switch probed by 15n nmr relaxation dispersion and chemical shift analysis
Proteins, 2011Co-Authors: Rieko Ishima, James B. AmesAbstract: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] 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.
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structural basis for calcium induced inhibition of rhodopsin kinase by Recoverin
Journal of Biological Chemistry, 2006Co-Authors: James B. Ames, Konstantin Levay, Jennifer N. Wingard, Jacqueline D. Lusin, Vladlen Z. SlepakAbstract:Recoverin, a member of the neuronal calcium sensor branch of the EF-hand superfamily, serves as a calcium sensor that regulates rhodopsin kinase (RK) activity in retinal rod cells. We report here the NMR structure of Ca(2+)-bound Recoverin bound to a functional N-terminal fragment of rhodopsin kinase (residues 1-25, called RK25). The overall main-chain structure of Recoverin in the complex is similar to structures of Ca(2+)-bound Recoverin in the absence of target (<1.8A root-mean-square deviation). The first eight residues of Recoverin at the N terminus are solvent-exposed, enabling the N-terminal myristoyl group to interact with target membranes, and Ca(2+) is bound at the second and third EF-hands of the protein. RK25 in the complex forms an amphipathic helix (residues 4-16). The hydrophobic face of the RK25 helix (Val-9, Val-10, Ala-11, Ala-14, and Phe-15) interacts with an exposed hydrophobic groove on the surface of Recoverin lined by side-chain atoms of Trp-31, Phe-35, Phe-49, Ile-52, Tyr-53, Phe-56, Phe-57, Tyr-86, and Leu-90. Residues of Recoverin that contact RK25 are highly conserved, suggesting a similar target binding site structure in all neuronal calcium sensor proteins. Site-specific mutagenesis and deletion analysis confirm that the hydrophobic residues at the interface are necessary and sufficient for binding. The Recoverin-RK25 complex exhibits Ca(2+)-induced binding to rhodopsin immobilized on concanavalin-A resin. We propose that Ca(2+)-bound Recoverin is bound between rhodopsin and RK in a ternary complex on rod outer segment disk membranes, thereby blocking RK interaction with rhodopsin at high Ca(2+).
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Structural basis for calcium-induced inhibition of rhodopsin kinase by Recoverin.
The Journal of biological chemistry, 2006Co-Authors: James B. Ames, Konstantin Levay, Jennifer N. Wingard, Jacqueline D. Lusin, Vladlen Z. SlepakAbstract:Recoverin, a member of the neuronal calcium sensor branch of the EF-hand superfamily, serves as a calcium sensor that regulates rhodopsin kinase (RK) activity in retinal rod cells. We report here the NMR structure of Ca(2+)-bound Recoverin bound to a functional N-terminal fragment of rhodopsin kinase (residues 1-25, called RK25). The overall main-chain structure of Recoverin in the complex is similar to structures of Ca(2+)-bound Recoverin in the absence of target (
