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PubMed2003

Autoantibody-induced apoptosis as a possible mechanism of autoimmune retinopathy.

Circulating antibodies specific to retinal proteins have been associated with retinal dysfunction in patients with retinopathy. Anti-recoverin antibodies found in patients with cancer-associated retinopathy (CAR) represent a unique model to study the relationship between retinal degeneration and autoimmunity. A body of evidence from in vitro and in vivo studies indicates that anti-recoverin autoantibodies are cytotoxic to retinal cells and induce apoptotic death of retinal photoreceptor cells, which leads to the degeneration of the photoreceptor cell layer. Similar to anti-recoverin autoantibodies, antibodies with other retinal specificities induce their target retinal cell death by activating a caspase 3-dependent apoptotic pathway. Thus, autoantibody-induced apoptosis may be a common pathway that leads to retinal death and blindness.

Animals↗

PubMedDate not supplied

Recoverin immunoreactivity in mammalian cone bipolar cells.

Human, macaque monkey, and rat retinas were immunostained with a polyclonal antibody preparation against purified recoverin, a 23-kD calcium-binding protein isolated from bovine retina that localizes to rods and cones (Dizhoor et al., 1991). In addition to immunoreactive photoreceptors, we have identified subpopulations of recoverin-positive bipolar cells in all three species. Results from immunostaining with progressive dilutions of anti-recoverin and preadsorption of the antibody with a dilution series of purified recoverin showed that photoreceptors and bipolar cells had similar affinities for the antibody and suggested that the molecule recognized by the antibody in both cell types is recoverin. Immunoreactivity for recoverin and protein kinase C, a selective marker for all rod bipolar cells, was found in separate bipolar cell populations. Recoverin immunoreactivity is therefore a characteristic of certain cone bipolar cell types. In rat retina, anti-recoverin labeled two morphologically distinct subpopulations of cone bipolar cells whose axonal arbors stratified at different depths in the inner plexiform layer (IPL). The bipolar cells labeled with anti-recoverin did not correspond to those that were reactive for calbindin, another cone bipolar cell marker. Human and monkey retinas also had two populations of cone bipolar cells that were recoverin-positive. One population showed a distinct pattern of narrow bistratification at the outer border of the IPL and a regular mosaic arrangement of its axonal arbors, suggesting that the entire population of a single cone bipolar type was labeled. Cell density, dendritic morphology, and axonal-field size and stratification indicate that anti-recoverin selectively strains the flat midget (presumed OFF-center) cone bipolar cell type observed previously in Golgi preparations. By contrast the second bipolar cell population had axonal stratification in the inner half of the IPL and showed an unusual but consistent morphology and spatial distribution. Individual cells were intensely stained but were present at an extremely low density (approximately 2-5 cells/mm2). These cells had multibranched dendritic trees characteristic of the diffuse bipolar cell class, but very small axonal fields in the size range of the midget bipolar class. Neither of the two recoverin-positive bipolar cell types in monkey was labeled with anti-calbindin or anti-cholecystokinin. An antibody preparation against bovine pineal hydroxyindole-O-methyltransferase (HIOMT) labeled photoreceptors and bipolar cells that closely resembled the recoverin-positive bipolar cells in human and rat retinas.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMedDate not supplied

Developmental patterns of protein expression in photoreceptors implicate distinct environmental versus cell-intrinsic mechanisms.

The present study has examined the spatial and temporal expression patterns of various proteins associated with the structure and function of mature photoreceptor outer segments in the developing ferret's retina using immunocytochemistry and RT-PCR. One set of proteins, including rod opsin, arrestin, and recoverin, was detected progressively in photoreceptors as they became postmitotic, being expressed well before the differentiation of outer segments. A second set of proteins, including beta- and gamma-transducin, cGMP-phosphodiesterase, phosducin, rhodopsin kinase, rod cGMP-gated cation channel protein, and peripherin, displayed a contrasting temporal onset and pattern of spatial emergence. These latter proteins first became detectable either shortly before or coincident with outer segment formation, and were expressed simultaneously in both older and younger photoreceptor cells. A third set, the short wavelength-sensitive (SWS) and medium wavelength-sensitive (MWS) cone opsin proteins, was the last to be detected, but materialized in a spatio-temporal pattern reminiscent of the neurogenetic gradient of the cones. These different spatial and temporal patterns indicate that cellular maturation must play a primary role in regulating the onset of expression of some of these proteins, while extrinsic signals must act to coordinate the expression of other proteins across photoreceptors of different ages.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMedDate not supplied

Cholecystokinin-like immunoreactive amacrine cells in the rat retina.

High levels of endogenous cholecystokinin (CCK) are present in the rat retina (Eskay & Beinfeld, 1982), but the cellular localization and physiological actions of CCK in the rat retina are uncertain. The goals of this study were to characterize the cells containing CCK, identify cell types that interact with CCK cells, and investigate the effects of CCK on rod bipolar cells. Rat retinas were labeled with antibody to gastrin-CCK (gCCK) using standard immunofluorescence techniques. Patch-clamp methods were used to record from dissociated rod bipolar cells from rats and mice. Gastrin-CCK immunoreactive (-IR) axons were evenly distributed throughout the retina in stratum 5 of the inner plexiform layer of the rat retina. However, the gCCK-IR somata were only detected in the ganglion cell layer in the peripheral retina. The gCCK-IR cells contained glutamate decarboxylase, and some of them also contained immunoreactive substance P. Labeled axons contacted PKC-IR rod bipolar cells, and recoverin-IR ON-cone bipolar cells. CCK-octapeptide inhibits GABA(C) but not GABA(A) mediated currents in dissociated rod bipolar cells.

Amacrine Cells↗

PubMedDate not supplied

Localization of protein kinase C to UV-sensitive photoreceptors in the mouse retina.

The present study has identified a population of cone photoreceptors in the murine retina that are uniquely immunoreactive for protein kinase C (PKC). Wavelength-sensitive cone subtypes are segregated along the dorso-ventral axis in the mouse retina with ventral retina occupied exclusively by ultraviolet wavelength-sensitive (UVWS) cones, and dorsal retina dominated by middle wavelength-sensitive cones. PKC-positive cones are found primarily in the ventral retina, and double-label immunocytochemistry using a short wavelength-sensitive opsin antibody confirms that they specifically correspond to the UVWS cone subtype. The PKC antibody, as documented in other mammals, also identifies rod bipolar cells in the mouse retina. UVWS cones and bipolar cells have previously been shown to share transcriptional regulatory elements, as observed in transgenic mice encoding a portion of the human SWS-opsin promoter controlling the lacZ reporter gene. In such mice, the transgene product, beta-galactosidase, is expressed in populations of both cones and bipolar cells. The present study confirms that lacZ-expressing photoreceptors are indeed PKC-positive photoreceptors, but that the lacZ-expressing bipolar cells are not the PKC-positive rod bipolar cells. These cells must correspond to a type of cone bipolar cell.

Animals↗

PubMedDate not supplied

Morphological differentiation of bipolar cells in the ferret retina.

Bipolar cells are not only important for visual processing but input from these cells may underlie the reorganization of ganglion cell dendrites in the inner plexiform layer (IPL) during development. Because little is known about the development of bipolar cells, here we have used immunocytochemical markers and dye labeling to identify and follow their differentiation in the neonatal ferret retina. Putative cone bipolar cells were immunoreacted for calbindin and recoverin, and rod bipolar cells were immunostained for protein kinase C (PKC). Our results show that calbindin-immunoreactive cone bipolar cells appear at postnatal day 15 (P15), at which time their axonal terminals are already localized to the inner half of the IPL. By contrast, recoverin-immunoreactive cells with terminals in the IPL are present at birth, but many of these cells may be immature photoreceptors. By the second postnatal week, recoverin-positive cells resembling cone bipolar cells were clearly present, and with increasing age, two distinct strata of immunolabeled processes occupied the IPL. PKC-containing rod bipolar cells emerged by the fourth postnatal week and at this age have stratified arbors in the inner IPL. The early bias of bipolar axonal arbors in terminating in the inner or outer half of the IPL is confirmed by dye labeling of cells with somata in the inner nuclear layer. At P10, several days before ribbon synapses have been previously observed in the ferret IPL, the axon terminals of all dye-labeled bipolar cells were clearly stratified. The results suggest that bipolar cells could provide spatially localized interactions that are suitable for guiding dendritic lamination in the inner retina.

Animals↗

PubMed1995

Calcium-dependent solvation of the myristoyl group of recoverin.

Recoverin is an N-myristoylated calcium-binding protein present in the photoreceptor cells of the mammalian retina. It is believed to function as a calcium sensor in visual signal transduction by coupling the kinetics of the recovery phase of the photoresponse to changes in the levels of intracellular Ca2+. Upon binding Ca2+, recoverin undergoes a conformational change that allows it to associate with membranes in a manner that requires N-myristoyl modification. It has been proposed that, in the Ca(2+)-free conformation, the myristoyl group is sequestered in a hydrophobic part of the protein, and in the Ca(2+)-bound conformation, the myristoyl group is exposed to solution. The crystal structure of Ca(2+)-bound recoverin reveals an exposed cluster of hydrophobic residues, raising the possibility that residues in this region may function as part of an intramolecular myristoyl binding site. Fluorescence spectroscopy analysis of interactions between recoverin and 1-anilinonaphthalene-8-sulfonate (ANS) shows that an increase in solvent-accessible hydrophobic surface accompanies Ca2+ binding. 1H nuclear magnetic resonance (NMR) spectra of myristoyl protons show dispersed chemical shifts in the Ca(2+)-free conformation that become relatively uniform upon the addition of Ca2+. Two-dimensional nuclear Overhauser effect (NOE) spectra of Ca(2+)-free recoverin show NOE contacts between myristoyl protons and aromatic ring protons. Tryptophan fluorescence quenching by acrylamide indicates that the myristoyl group is in proximity to a tryptophan residue only in the Ca(2+)-free conformation. These results indicate that the myristoyl group is in contact with residues in the hydrophobic cluster in Ca(2+)-free recoverin and that it is exposed to solution in the Ca(2+)-bound conformation.

Anilino Naphthalenesulfonates↗

PubMed2000

How can Ca2+ selectively activate recoverin in the presence of Mg2+? Surface plasmon resonance and FT-IR spectroscopic studies.

We investigated the relationship between metal ion selective conformational changes of recoverin and its metal-bound coordination structures. Recoverin is a 23 kDa heterogeneously myristoylated Ca(2+)-binding protein that inhibits rhodopsin kinase. Upon accommodating two Ca(2+) ions, recoverin extrudes a myristoyl group and associates with the lipid bilayer membrane, which was monitored by the surface plasmon resonance (SPR) technique. Large changes in SPR signals were observed for Sr(2+), Ba(2+), Cd(2+), and Mn(2+) as well as Ca(2+), indicating that upon binding to these ions, recoverin underwent a large conformational change to extrude the myristoyl group, and thereby interacted with lipid membranes. In contrast, no SPR signal was induced by Mg(2+), confirming that even though it accommodates two Mg(2+) ions, recoverin does not induce the large conformational change. To investigate the coordination structures of metal-bound Ca(2+) binding sites, FT-IR studies were performed. The EF-hands, Ca(2+)-binding regions each comprising 12 residues, arrange to coordinate Ca(2+) with seven oxygen ligands, two of which are provided by a conserved bidentate Glu at the 12th relative position in the EF-hand. FT-IR analysis confirmed that Sr(2+), Ba(2+), Cd(2+), and Mn(2+) were coordinated to COO(-) of Glu by a bidentate state as well as Ca(2+), while coordination of COO(-) with Mg(2+) was a pseudobridging state with six-coordinate geometry. These SPR and FT-IR results taken together reveal that metal ions with seven-coordinate geometry in the EF-hands induce a large conformational change in recoverin so that it extrudes the myristoyl group, while metal ions with six-coordinate geometry in the EF-hands such as Mg(2+) remain the myristoyl group sequestered in recoverin.

Animals↗

PubMed1994

Secondary structure of myristoylated recoverin determined by three-dimensional heteronuclear NMR: implications for the calcium-myristoyl switch.

Recoverin, a new member of the EF-hand superfamily, serves as a Ca2+ sensor in vision. A myristoyl or related N-acyl group is covalently attached at its N-terminus and plays an essential role in Ca(2+)-dependent membrane targeting by a novel calcium-myristoyl switch mechanism. The structure of unmyristoylated recoverin containing a single bound Ca2+ has recently been solved by X-ray crystallography [Flaherty, K. M., Zozulya, S., Stryer, L., & McKay, D. B. (1993) Cell 75, 709-716]. We report here multidimensional heteronuclear NMR studies on Ca(2+)-free, myristoylated recoverin (201 residues, 23 kDa). Complete polypeptide backbone 1H, 15N, and 13C resonance assignments and secondary structure are presented. We find 11 helical segments and two pairs of antiparallel beta-sheets, in accord with the four EF-hands seen in the crystal structure. The present NMR study also reveals some distinct structural features of the Ca(2+)-free myristoylated protein. The N-terminal helix of EF-2 is flexible in the myristoylated Ca(2+)-free protein, whereas it has a well-defined structure in the unmyristoylated Ca(2+)-bound form. This difference suggests that the binding of Ca2+ to EF-3 induces EF-2 to adopt a conformation favorable for the binding of a second Ca2+ to recoverin. Furthermore, the N-terminal helix (K5-E16) of myristoylated Ca(2+)-free recoverin is significantly longer than that seen in the unmyristoylated Ca(2+)-bound protein. We propose that this helix is stabilized by the attached myristoyl group and may play a role in sequestering the myristoyl group within the protein in the Ca(2+)-free state.

Amino Acid Sequence↗

PubMed2002

Structure and calcium-binding studies of a recoverin mutant (E85Q) in an allosteric intermediate state.

Recoverin, a member of the EF-hand superfamily, serves as a calcium sensor in retinal rod cells. A myristoyl or related fatty acyl group covalently attached to the N-terminus of recoverin facilitates the binding of recoverin to retinal disk membranes by a mechanism known as the Ca2+-myristoyl switch. Previous structural studies revealed that the myristoyl group of recoverin is sequestered inside the protein core in the absence of calcium. The cooperative binding of two calcium ions to the second and third EF-hands (EF-2 and EF-3) of recoverin leads to the extrusion of the fatty acid. Here we present nuclear magnetic resonance (NMR), fluorescence, and calcium-binding studies of a myristoylated recoverin mutant (myr-E85Q) designed to abolish high-affinity calcium binding to EF-2 and thereby trap the myristoylated protein with calcium bound solely to EF-3. Equilibrium calcium-binding studies confirm that only one Ca2+ binds to myr-E85Q under the conditions of this study with a dissociation constant of 100 microM. Fluorescence and NMR spectra of the Ca2+-free myr-E85Q are identical to those of Ca2+-free wild type, indicating that the E85Q mutation does not alter the stability and structure of the Ca2+-free protein. In contrast, the fluorescence and NMR spectra of half-saturated myr-E85Q (one bound Ca2+) look different from those of Ca2+-saturated wild type (two bound Ca2+), suggesting that half-saturated myr-E85Q may represent a structural intermediate. We report here the three-dimensional structure of Ca2+-bound myr-E85Q as determined by NMR spectroscopy. The N-terminal myristoyl group of Ca2+-bound myr-E85Q is sequestered within a hydrophobic cavity lined by many aromatic residues (F23, W31, Y53, F56, F83, and Y86) resembling that of Ca2+-free recoverin. The structure of Ca2+-bound myr-E85Q in the N-terminal region (residues 2-90) is similar to that of Ca2+-free recoverin, whereas the C-terminal region (residues 100-202) is more similar to that of Ca2+-bound wild type. Hence, the structure of Ca2+-bound myr-E85Q represents a hybrid between the structures of recoverin with zero and two Ca2+ bound. The binding of Ca2+ to EF-3 leads to local structural changes within the EF-hand that alter the domain interface and cause a 45 degrees swiveling of the N- and C-terminal domains, resulting in a partial unclamping of the myristoyl group. We propose that Ca2+-bound myr-E85Q may represent a stable intermediate state in the kinetic mechanism of the calcium-myristoyl switch.

Allosteric Regulation↗

PubMed2003

Structure, topology, and dynamics of myristoylated recoverin bound to phospholipid bilayers.

Recoverin, a member of the EF-hand protein superfamily, serves as a calcium sensor in retinal rod cells. A myristoyl group covalently attached to the N-terminus of recoverin facilitates its binding to retinal disk membranes by a mechanism known as the Ca(2+)-myristoyl switch. Samples of (15)N-labeled Ca(2+)-bound myristoylated recoverin bind anisotropically to phospholipid membranes as judged by analysis of (15)N and (31)P chemical shifts observed in solid-state NMR spectra. On the basis of a (2)H NMR order parameter analysis performed on recoverin containing a fully deuterated myristoyl group, the N-terminal myristoyl group appears to be located within the lipid bilayer. Two-dimensional solid-state NMR ((1)H-(15)N PISEMA) spectra of uniformly and selectively (15)N-labeled recoverin show that the Ca(2+)-bound protein is positioned on the membrane surface such that its long molecular axis is oriented approximately 45 degrees with respect to the membrane normal. The N-terminal region of recoverin points toward the membrane surface, with close contacts formed by basic residues K5, K11, K22, K37, R43, and K84. This orientation of the membrane-bound protein allows an exposed hydrophobic crevice, near the membrane surface, to serve as a potential binding site for the target protein, rhodopsin kinase. Close agreement between experimental and calculated solid-state NMR spectra of recoverin suggests that membrane-bound recoverin retains the same overall three-dimensional structure that it has in solution. These results demonstrate that membrane binding by recoverin is achieved primarily by insertion of the myristoyl group inside the bilayer with apparently little rearrangement of the protein structure.

Calcium↗

PubMed1997

Calcium-dependent binding of recoverin to membranes monitored by surface plasmon resonance spectroscopy in real time.

Recoverin is an N-myristoylated Ca2+-binding protein that serves as a Ca2+-sensor in visual transduction. We studied the dynamics of its Ca2+-dependent membrane association which depends on the myristoyl modification (Ca2+-myristoyl switch) by surface plasmon resonance spectroscopy. Either recoverin or phospholipid vesicles were immobilized on a sensor chip surface, and the respective binding partner was supplied in the mobile phase. Binding of recoverin to artificial liposomes or rod outer segment membranes was strictly dependent on Ca2+ and the myristoyl group. The Ca2+-myristoyl switch was half-maximal between 4.0 and 7.7 microM Ca2+, depending on whether recoverin or liposomes were in the mobile phase. At saturating [Ca2+], the dissociation constant (KD) of recoverin for phospholipid liposomes was approximately 150 microM. The association and dissociation of recoverin to membranes was fast and biphasic (fast and slow components) with time constants on the order of 0.1 s-1 and 0.01 s-1, respectively. Dissociation of the recoverin-membrane complex was 3-fold faster at low than at high free [Ca2+]. We discuss the analogy between the liposome-sensor chip and the disk surface and conclude that a minor fraction of the total recoverin in a rod outer segment is associated with membranes at resting dark levels of free [Ca2+].

Animals↗

PubMed1998

Autophosphorylation and ADP regulate the Ca2+-dependent interaction of recoverin with rhodopsin kinase.

Recoverin is a 23 kDa myristoylated Ca2+-binding protein that inhibits rhodopsin kinase. We have used surface plasmon resonance to investigate the influences of Ca2+, myristoylation, and adenine nucleotides on the recoverin-rhodopsin kinase interaction. Our analyses confirmed that Ca2+ is required for recoverin to bind RK. Myristoylation had little effect on the affinity of recoverin for the kinase, but it raised the K0.5 for Ca2+ from 150 nM for nonacylated recoverin to 400 nM for myristoylated recoverin. Finally, our studies also revealed two separate and previously unreported effects of adenine nucleotides on the recoverin-rhodopsin kinase binding. The interaction is weakened by autophosphorylation of the kinase, and it is strengthened by the presence of ADP.

Adenosine Diphosphate↗

PubMed1998

Core mutations that promote the calcium-induced allosteric transition of bovine recoverin.

Recoverin is a small calcium binding protein involved in regulation of the phototransduction cascade in retinal rod cells. It functions as a calcium sensor by undergoing a cooperative, ligand-dependent conformational change, resulting in the extrusion of the N-terminal myristoyl group from a hydrophobic pocket. To test the role of certain core residues in tuning this allosteric switch, we have made and characterized two mutants: W31K, which replaces Trp31 with Lys; and a double mutant, I52A/Y53A, in which Ile52 and Tyr53 are both replaced by Ala. These mutations decrease the hydrophobicity of the myristoyl binding pocket. They are thus expected to make sequestering of the myristoyl group less favorable and destabilize the Ca2+-free state. As predicted, the myristoylated forms of the mutants exhibit increased affinity for Ca2+, whether monitored by equilibrium binding of 45Ca2+ (Kd = 17.2, 7.9, and 8.1 microM for wild type, W31K, and I52A/Y53A, respectively) or by the change in tryptophan fluorescence associated with the conformational change (Kd = 17.9, 3.6, and 4.4 microM for wild type, W31K, and I52A/Y53A, respectively). The mutants also exhibit decreased cooperativity of binding (Hill coefficient = 1.2 and 1.0 for W31K and I52A/Y53A vs 1. 4 for wild type). Binding of the mutant proteins to rod outer segment membranes occurs at lower Ca2+ concentrations compared to wild-type protein (K1/2 = 5.6, 2.2, and 1.0 microM for wild type, W31K, and I52A/Y53A, respectively). The unmyristoylated forms of the mutants exhibit biphasic Ca2+ binding curves, nearly identical to that observed for wild type. The binding data for the two mutants can be explained by a concerted allosteric model in which the mutations affect only the equilibrium constant L between the two allosteric forms, T (the Ca2+-free form) and R (the Ca2+-bound form), without affecting the intrinsic binding constants for the two Ca2+ sites. Two-dimensional NMR spectra of the Ca2+-free forms of the mutants have been compared to the wild-type spectrum, whose peaks have been assigned to specific residues (1). Many resonances assigned to residues in the C-terminal domain (residues 100-202) in the wild-type spectrum are identical in the mutant spectra, suggesting that the backbone structure of the C-terminal domain is probably unchanged in both mutants. The N-terminal domain, in which both mutations are located, reveals in each case numerous changes of undetermined spatial extent.

Allosteric Site↗

PubMed1998

Localization of the sites for Ca2+-binding proteins on G protein-coupled receptor kinases.

Inhibition of G protein-coupled receptor kinases (GRKs) by Ca2+-binding proteins has recently emerged as a general mechanism of GRK regulation. While GRK1 (rhodopsin kinase) is inhibited by the photoreceptor-specific Ca2+-binding protein recoverin, other GRKs can be inhibited by Ca2+-calmodulin. To dissect the mechanism of this inhibition at the molecular level, we localized the GRK domains involved in Ca2+-binding protein interaction using a series of GST-GRK fusion proteins. GRK1, GRK2, and GRK5, which represent the three known GRK subclasses, were each found to possess two distinct calmodulin-binding sites. These sites were localized to the N- and C-terminal regulatory regions within domains rich in positively charged and hydrophobic residues. In contrast, the unique N-terminally localized GRK1 site for recoverin had no clearly defined structural characteristics. Interestingly, while the recoverin and calmodulin-binding sites in GRK1 do not overlap, recoverin-GRK1 interaction is inhibited by calmodulin, most likely via an allosteric mechanism. Further analysis of the individual calmodulin sites in GRK5 suggests that the C-terminal site plays the major role in GRK5-calmodulin interaction. While specific mutation within the N-terminal site had no effect on calmodulin-mediated inhibition of GRK5 activity, deletion of the C-terminal site attenuated the effect of calmodulin on GRK5, and the simultaneous mutation of both sites rendered the enzyme calmodulin-insensitive. These studies provide new insight into the mechanism of Ca2+-dependent regulation of GRKs.

Amino Acid Sequence↗

PubMed1999

Role of carboxyl-terminal charges on S-modulin membrane affinity and inhibition of rhodopsin phosphorylation.

S-Modulin shows a higher affinity for urea-stripped frog rod outer segment membranes than s26 (a cone homologue of S-modulin). NaCl at a concentration of several hundred millimolar reduced the membrane affinity of S-modulin to the s26 level. Chimeric S-modulin and s26 whose respective 23 and 29 amino acids at the carboxyl terminus were swapped showed membrane affinites similar to those of s26 and S-modulin, respectively. The membrane affinity of an S-modulin mutant lacking C-terminal positive charges was reduced to the s26 level, while another S-modulin mutant lacking C-terminal negative charges has a higher membrane affinity than wild-type S-modulin. When the molar ratio of recombinant S-modulins to rhodopsin is 0.5, there was no large difference in the inhibition efficiency. However, S-modulin and mutants with high membrane affinities inhibit rhodopsin phosphorylation more efficiently than s26 and mutants with low membrane affinities at the molar ratio of 0.1. These results indicate that the C-terminal positive charges of these Ca2+-binding proteins enhance the membrane affinity and the inhibitory effect on rhodopsin phosphorylation by increasing the concentration of S-modulin on the membrane.

Amino Acid Sequence↗

PubMedDate not supplied

Recoverin is a zinc-binding protein.

Recoverin is an N-myristoylated 23 kDa calcium-binding protein from retina, which modulates the Ca2+-sensitive deactivation of rhodopsin via Ca2+-dependent inhibition of rhodopsin kinase. It was shown by intrinsic and bis-ANS probe fluorescence, circular dichroism, and differential scanning calorimetry that myristoylated recombinant recoverin interacts specifically with zinc ions. Similar to the calcium binding, the binding of zinc to Ca2+-loaded recoverin additionally increases its alpha-helical content, hydrophobic surface area, and environmental mobility/polarity of its tryptophan residues. In contrast to the calcium binding, the binding of zinc decreases thermal stability of the Ca2+-loaded protein. Zn2+-titration of recoverin, traced by bis-ANS fluorescence, reveals binding of a single Zn2+ ion per protein molecule. It was shown that the double-mutant E85Q/E121Q with inactivated Ca2+-binding EF-hands 2 and 3 (Alekseev, A. M.; Shulga-Morskoy, S. V.; Zinchenko, D. V.; Shulga-Morskaya, S. A.; Suchkov, D. V.; Vaganova, S. A.; Senin, I. I.; Zargarov, A. A.; Lipkin, V. M.; Akhtar, M.; Philippov, P. P. FEBS Lett. 1998, 440, 116-118), which can be considered as an analogue of the apo-protein, binds Zn2+ ion as well. Apparent zinc equilibrium binding constants evaluated from spectrofluorimetric Zn2+-titrations of the protein are 1.4 x 10(5) M(-1) (dissociation constant 7.1 microM) for Ca2+-loaded wild-type recoverin and 3.3 x 10(4) M(-1) (dissociation constant 30 microM) for the E85Q/E121Q mutant (analogue of apo-recoverin). Study of the binding of wild-type recoverin to ROS membranes showed a zinc-dependent increase of its affinity for the membranes, without regard to calcium content, suggesting further solvation of a protein myristoyl group upon Zn2+ binding. Possible implications of these findings to the functioning of recoverin are discussed.

Anilino Naphthalenesulfonates↗

PubMed1998

Differential isotype labeling strategy for determining the structure of myristoylated recoverin by NMR spectroscopy.

The three-dimensional solution structure of recombinant bovine myristoylated recoverin in the Ca(2+)-free state has been refined using an array of isotope-assisted multidimensional heteronuclear NMR techniques. In some experiments, the myristoyl group covalently attached to the protein N-terminus was labeled with C and the protein was unlabeled or vice versa; in others, both were C-labeled. This differential labeling strategy was essential for structural refinement and can be applied to other acylated proteins. Stereospecific assignments of 41 pairs of beta-methylene protons and 48 methyl groups of valine and leucine were included in the structure refinement. The refined structure was constructed using a total of 3679 experimental NMR restraints, comprising 3242 approximate interproton distance restraints (including 153 between the myristoyl group and the polypeptide), 140 distance restraints for 70 backbone hydrogen bonds, and 297 torsion angle restraints. The atomic rms deviations about the average minimized coordinate positions for the secondary structure region of the N-terminal and C-terminal domains are 0.44 +/- 0.07 and 0.55 +/- 0.18 A for backbone atoms, and the 1.09 +/- 0.07 and 1.10 +/- 0.15 A for all heavy atoms, respectively. The refined structure allows for a detailed analysis of the myristoyl binding pocket. The myristoyl group is in a slightly bent conformation: the average distance between C1 and C14 atoms of the myristoyl group is 14.6 A. Hydrophobic residues Leu28, Trp31, and Tyr32 from a cluster that interacts with the front end of the myristoyl (C1-C8), whereas residues Phe49, Phe56, Tyr86, Val87, and Leu90 interact with the tail end (C9-C14). The relatively deep hydrophobic pocket that binds the myristoyl group (C14:0) could also accommodate other naturally occurring acyl groups such as C12:0, C14:1, C14:2 chains.

Animals↗