ORD OF RETINAL AND RETINYLIDENE PHOSPHORYL ETHANOLAMINE IN A MICELLE

Abstract— In the course of observing the formation of Schif bases of phosphatidyl ethanolamine and phosphoryl ethanolamine with all- trans retinal in a detergent micelle, we have studied their spectroscopic behavior and ORD properties. In so doing, we have demonstrated that all-rruns retinal itself, although having no intrinsic chirality at the aldehyde group, gives a marked Cotton effect, when dispersed in a CTAB micelle, at the absorption peak of the nπ* transition. This extrinsic chirality is attributed to the markedly asymmetric environment of the Stern layer of the micelle, and its significance is related to the ORD behavior of rod outer segments.     

Rhodopsin phosphorylation suggests biochemical heterogeneities of retinal rod disks.

Frogs (Rana pipiens) were injected subcutaneously with (3H)-leucine and allowed to incorporate the radioactive amino acid into newly assembled disks in the retinal rod outer segment. The labeled disks served as a temporal marker for following the turnover of rod outer segments. Animals were killed at different times after injection and outer segments were isolated and phosphorylated with ATP in the light. The visual pigment (as isorhodopsin) was regenerated with 9-cis retinal, extracted, and chromatographed on epichlorohydrin triethanolamine cellulose so that phosphorylated pigment could be separated from unphosphorylated pigment. The ratio of (3H)-radioactivity of phosphorylated pigment to that of unphosphorylated pigment was then plotted against the time after injection. The ratio was high when (3H)-labeled disks were largely associated with the basal region of the rod and decreased as the labeled disks moved toward the rod apical region. The results were interpreted as suggesting that newer disks are phosphorylated preferentially to older disks. Papain digestion of (3H)-labeled disks indicated that rhodopsin in newer disks is more susceptible to proteolysis than that in older disks.     

Patch-clamp recording of human retinal photoreceptors and bipolar cells

Photoreceptors and retinal bipolar cells are considered as nonspiking neurons; however, we recently showed that human rod photoreceptors can generate sodium action potentials in response to membrane depolarization from membrane potentials of -60 or -70 mV (Kawai et al., Neuron 30 [2001] 451). We performed patch-clamp recording of human cone photoreceptors and retinal bipolar cells to examine whether functional voltage-gated sodium channels are expressed in these cells as well as rod photoreceptors. Under current-clamp conditions, the injection of depolarizing current steps into a cone photoreceptor-induced marked action potentials. These action potentials were blocked by 1 microM tetrodotoxin, a voltage-gated sodium channel blocker. Under voltage-clamp conditions, depolarizing voltage steps-induced a fast transient inward current in several bipolar cells (n = 4/78). This current was activated from -70 to + 20 mV (maximal at -10 mV) and inactivated within 5 ms. The 10-90% rise time of this current was shorter than another inward current (less than one-hundredth). These results indicate that human cones and bipolar cells express voltage-gated sodium channels as rod photoreceptors. Sodium channels may serve to amplify the release of a neurotransmitter and to accelerate the light-dark change in photosignals.     

LIGHT-INDUCED PERTURBATION OF AROMATIC RESIDUES IN BOVINE RHODOPSIN AND BACTERIORHODOPSIN*

Light-induced changes in the UV absorption spectrum of bovine rod outer segment membranes were measured by conventional difference spectroscopy and by flash photolysis methods. Different thermal intermediates of rhodopsin (lumirhodopsin, metarhodopsin I, metarhodopsin II, and meta-rhodopsin III) have absorption spectra in the ultraviolet which differ from the rhodopsin spectrum and from each other. The spectra associated with metarhodopsin I, metarhodopsin II, and metarhodopsin III are characteristic of perturbation of a small number of tyr. and/or trp residues, most likely one trp residue. These aromatic residues are in the neighborhood of the retinal Schiff base and undergo coordinated changes of interaction with retinal during the bleaching sequence. At the metarhodopsin II stage, the magnitude of the UV spectral changes is consistent with the exposure of a previously shielded trp residue to an aqueous environment. The present results are consistent with previous spectral studies which limit the extent of light-induced conformational changes to regions of the protein in the neighborhood of the retinal Schiff base. An analogous study was made on light-adapted purple membranes of Halobacterium halobium. The UV absorption spectrum associated with the deprotonated Schiff base intermediate of the trans-bacteriorhodopsin cycle is indicative, in part, of aromatic residue perturbation. However, significant changes in the secondary and tertiary structures of the bacterio-rhodopsin protein characteristic of a delocalized conformational change are unlikely at this intermediate stage.     

EFFECT OF THE PHYSICAL STATE OF PHOSPHOLIPID ON RHODOPSIN REGENERATION FROM RETINYLIDENE PHOSPHOLIPID

Abstract— Rhodopsin regeneration in rod membranes involves reactions of all -trans retinal (released from bleached pigment) with phosphatidylethanolamine, photic isomerization of retinal, and binding of 11-cis retinal to opsin. This investigation demonstrated that formation of retinylidene phospholipid and retinal binding to opsin were both affected by the physical state of phospholipid. A fluid membraneous environment provided by the acyl chains of phospholipid was essential for these reactions to proceed efficiently. The retinal moiety of retinylidene phospholipid appeared to be directly transferred to opsin by transimination.     

X-ray crystallographic analysis of 9-cis-rhodopsin, a model analogue visual pigment

Recent progress in high-resolution structural study of rhodopsin has been enabled by a novel selective extraction procedure with rod photoreceptor cells. In this study, we applied the method for rapid and efficient preparation of a purified analogue pigment using bovine rod outer segment membranes with 9-cis-retinal. After complete bleaching of the membranes and subsequent regeneration with the exogenous retinal, 9-cis-rhodopsin is selectively extracted from the membranes using combination of zinc and heptylthioglucoside. The solubilized sample, even with a small amount of contaminating retinal oximes, is shown to be pure enough for three-dimensional crystallization. The X-ray diffraction from 9-cis-rhodopsin crystals was examined and the electron density map at 2.9 angstroms resolution in the chromophore region can be fitted well with the model of 9-cis-retinal Schiff base.     

Functional properties of interphotoreceptor retinoid-binding protein.

It has been hypothesized that interphotoreceptor retinoid-binding protein (IRBP) functions as a two-way carrier of retinoid between the retinal pigment epithelium (RPE) and rod photoreceptors in the vertebrate eye. This hypothesis has been tested in recent studies that have employed purified, initially ligand-free, bovine IRBP and the "RPE-eyecup" obtained from the toad (Bufo marinus) eye. The present experiments further characterize the IRBP/RPE-eyecup system with respect to (i) the solubilization and protection of retinol by IRBP, and (ii) the time course of IRBP-mediated release of 11-cis retinal by the RPE. The data, together with previous findings in the IRBP/RPE-eyecup preparation, support the view that 11-cis retinal is the principal retinoid released by the RPE into IRBP-supplemented aqueous medium, and that IRBP in vivo promotes the regeneration of rhodopsin by facilitating the exchange of retinoid between bleached rods and the RPE.     

INCORPORATION OF 11,12-DIHYDRORETINAL INTO THE RETINAE OF VITAMIN A DEPRIVED RATS

Abstract— The incorporation of 11,12-[15–³H]-dihydroretinal, a retinal in which the crucial 11-ene is saturated, into the retinae of vitamin A deficient rats as a result of intraperitoneal injection of the corresponding alcohol was shown by the presence of the tritium label in the rod outer segments and by identification of the extracted retinals using high pressure liquid chromatography. The amplitude of the electroretinogram (ERG) b-wave, diminished as the result of vitamin A deprivation, was not affected by administration of the analogue, although similar treatment of deprived litter mates with trans retinal restored the ERG b-wave amplitude to a normal level.
The evidence that the analogue is bound to opsin forming 11,12-dihydrorhodopsin is as follows: (1) when incubated with 11- cis retinal, extracts from vitamin A deficient rats regenerate 1.4 nmol rhodopsin while extracts from rats deficient in vitamin A and supplemented with 11,12-dihydroretinal regenerate 0.6 nmol rhodopsin indicating binding of the dihydroretinal blocks rhodopsin regeneration. (2) 11,12-dihydroretinal is shown to remain unchanged in hexane-washed retinae after extraction with methylene chloride and (3) injection of retinal into animals previously injected with 11,12-dihydroretinal also fails to restore visual sensitivity as measured by the ERG b-wave. Our results indicate that the dihydro-chromophore occupies the same binding site as the natural 11- cis retinal and that occupation of the chromophore binding site of opsin is not sufficient to restore the visual sensitivity in a vitamin-A-deprived animal.     

All‐Trans Retinal Mediates Light‐Induced Oxidation in Single Living Rod Photoreceptors†

All‐trans retinal is a potent photosensitizer that is released in photoreceptor outer segments by the photoactivated visual pigment following the detection of light. Photoreceptor outer segments also contain high concentrations of polyunsaturated fatty acids, and are thus particularly susceptible to oxidative damage such as that initiated by light via a photosensitizer. Upon its release, all‐trans retinal is reduced within the outer segment to all‐trans retinol, through a reaction requiring metabolic input in the form of NADPH. The phototoxic potential of physiologically generated all‐trans retinal was examined in single living rod photoreceptors obtained from frog (Rana pipiens) retinas. Light‐induced oxidation was measured with fluorescence imaging using an oxidation‐sensitive indicator dye from the shift in fluorescence between the intact and oxidized forms. Light‐induced oxidation was highest in metabolically compromised rod outer segments following photoactivation of the visual pigment rhodopsin, and after a time interval, sufficiently long to ensure the release of all‐trans retinal. Furthermore, light‐induced oxidation increased with the concentration of exogenously added all‐trans retinal. The results show that the all‐trans retinal generated during the detection of light can mediate light‐induced oxidation. Its removal through reduction to all‐trans retinol protects photoreceptor outer segments against light‐induced oxidative damage.     

A HIGHLY REACTIVE HETEROATOM ANALOG OF RETINAL AND ITS INTERACTION WITH BACTERIORHODOPSIN

Abstract— C₁₈ formate ester (5) [2-(6-methyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3E,5E,7E-octatrienyl formate], a highly reactive analog of retinal, was synthesized and its interaction with bacterioopsin studied. The formate ester, in the absence of purple or bleached membrane, undergoes very rapid reaction (t_l/2= 0.9 min) in neutral buffer but with membrane present it diffuses more rapidly into the membrane where it reacts slowly. Incorporation of 5 in the membrane results in a 38 nm (3900 cm^-1) red shift which remains after reconstitution with retinal. Similar experiments with the corresponding C₁₈ alcohol (4) results in a red shift, but this absorption blue shifts upon reconstitution with retinal. Washing the formate ester-treated membrane with bovine serum albumin or the corresponding lyophilized preparation with hexane, treatments that remove retinal oxime, fails to remove the UV-visible absorption, suggesting that a covalent bond between the C₁₈ moiety and a nucleophilic group of the protein has probably formed.     

A HIGHLY REACTIVE HETEROATOM ANALOG OF RETINAL AND ITS INTERACTION WITH BACTERIORHODOPSIN

Abstract
C₁₈ formate ester (5) [2-(6-methyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3E,5E,7E-octatrienyl formate], a highly reactive analog of retinal, was synthesized and its interaction with bacterioopsin studied. The formate ester, in the absence of purple or bleached membrane, undergoes very rapid reaction (t_l/2= 0.9 min) in neutral buffer but with membrane present it diffuses more rapidly into the membrane where it reacts slowly. Incorporation of 5 in the membrane results in a 38 nm (3900 cm^-1) red shift which remains after reconstitution with retinal. Similar experiments with the corresponding C₁₈ alcohol (4) results in a red shift, but this absorption blue shifts upon reconstitution with retinal. Washing the formate ester-treated membrane with bovine serum albumin or the corresponding lyophilized preparation with hexane, treatments that remove retinal oxime, fails to remove the UV-visible absorption, suggesting that a covalent bond between the C₁₈ moiety and a nucleophilic group of the protein has probably formed.     

DISTRIBUTION OF ENZYMES INVOLVED IN NUCLEOTIDE METABOLISM IN THE DISK AND OTHER ROD MEMBRANES

Abstract— Intact disks and inverted disks were prepared from bovine retinal rods and the distribution in the disk membrane of such enzymes as guanyl cyclase, cyclic nucleotide phosphodiesterase, GTP binding protein (GTPase), 5'-nucleotidase and rhodopsin kinase was investigated. Guanyl cyclase was not detected in the disk; the enzyme activity was high in a membranous fraction containing the cilium or axoneme and the rod outer segment plasma membrane. Cyclic nucleotide phosphodiesterase, GTP binding protein (GTPase) and rhodopsin kinase were associated on the external surface of disk in the presence of 2 m M Mg²⁺. The enzymes dissociated from the membrane when Mg²⁺ was depleted. Thus, magnesium ion seems to regulate the state of these enzymes in the outer segment. 5'-Nucleotidase activity was low in intact disks but was significantly enhanced after inversion of the disk. The catalytic site of the enzyme, therefore, must be located on the internal (intradiscal) surface. Since the disks are known to be formed by invagination of the plasma membrane, 5'-nucleotidase, by inference, would have its catalytic site exposed on the external surface of the plasma membrane. Preliminary experiments showed that the capability of light-activated rhodopsin to activate cyclic nucleotide phosphodiesterase was inhibited by phosphorylation of the pigment. This supports the idea that rhodopsin kinase, cyclic nucleotide phosphodiesterase and GTPase exist as a functional complex on rod membranes.     

Activity switches of rhodopsin

Rhodopsin, the visual pigment of the rod photoreceptor cell contains as its light-sensitive cofactor 11-cis retinal, which is bound by a protonated Schiff base between its aldehyde group and the Lys296 side chain of the apoprotein. Light activation is achieved by 11-cis to all-trans isomerization and subsequent thermal relaxation into the active, G protein-binding metarhodopsin II state. Metarhodopsin II decays via two parallel pathways, which both involve hydrolysis of the Schiff base eventually to opsin and released all-trans retinal. Subsequently, rhodopsin's dark state is regenerated by a complicated retinal metabolism, termed the retinoid cycle. Unlike other retinal proteins, such as bacteriorhodopsin, this regeneration cycle cannot be short cut by light, because blue illumination of active metarhodopsin II does not lead back to the ground state but to the formation of largely inactive metarhodopsin III. In this review, mechanistic details of activating and deactivating pathways of rhodopsin, particularly concerning the roles of the retinal, are compared. Based on static and time-resolved UV/Vis and FTIR spectroscopic data, we discuss a model of the light-induced deactivation. We describe properties and photoreactions of metarhodopsin III and suggest potential roles of this intermediate for vision.     

Mechanism of G-protein activation by rhodopsin

Rhodopsin is a member of the family of G-protein-coupled receptors (GPCRs), and is an excellent molecular switch for converting light signals into electrical response of the rod photoreceptor cells. Light initiates cis-trans isomerization of the retinal chromophore of rhodopsin and leads to the formation of several thermolabile intermediates during the bleaching process. Recent investigations have identified spectrally distinguishable two intermediate states that can interact with the retinal G-protein, transducin, and have elucidated the functional sharing of these intermediates. The initial contact with GDP-bound G-protein occurs in the meta-Ib intermediate state, which has a protonated Schiff base as its chromophore. The meta-Ib intermediate in the complex with the G-protein converts to the meta-II intermediate with releasing GDP from the alpha-subunit of the G protein. Meta-II has a de-protonated Schiff base chromophore and induces binding of GTP to the alpha-subunit of the G-protein. Thus, the GDP-GTP exchange reaction, namely G-protein activation, by rhodopsin proceeds through at least two steps, with conformational changes in both rhodopsin and the G-protein.     

Studies on the Stability of the Human Cone Visual Pigments†

The retina of vertebrates contains two kinds of photoreceptor cells, rods and cones, which contain their specific visual pigments that are responsible for scotopic and photopic vision, respectively. In cone photoreceptor cells, there are three types of color pigments: blue, green and red, each with a distinctive absorption maximum. The goal of this investigation was to identify optimal conditions under which these pigments could be obtained and isolated in a stable form, thereby facilitating structural studies using high‐resolution approaches. For this purpose, all three human cone opsins were initially expressed in mammalian cells, reconstituted with 11‐cis retinal, detergent solubilized, purified and their stability compared with rod rhodopsin. As all three pigments showed dramatically reduced stability relative to rhodopsin, site‐directed mutagenesis was used in an attempt to engineer stability into the green cone pigment. The mutations introduced some structural motifs and sites of posttranslational modification present in rhodopsin, as well as amino acid substitutions that have been found to stabilize the rod opsin apo‐protein. We also modified the hydrophobic environment of the green cone pigment by varying the detergent and detergent/lipid composition used during solubilization and purification, and compared them with the retinal reconstituted pigment in membranes. Our results show that these changes do not significantly improve the inherent instability of the human cone pigments, and in some cases, lead to a decrease in stability and protein aggregation. We conclude that further efforts are required to stabilize the human cone pigments in a form suitable for high‐resolution structural studies.     

AZIDOTETRAFLUOROPHENYL RETINAL ANALOGUE: SYNTHESIS AND BACTERIORHODOPSIN PIGMENT FORMATION

Abstract —The retinal derivative, all-truns-9–(4-azido-2,3,5,6-tetrafluorophenyl)-3,7-dimethyl-2,4,6,8-nonatetraenal, was synthesized by two routes as a potential photoactivatable cross-linking agent for studies in bacteriorhodopsin (BR) of the chromophore interaction with its apoprotein. The retinal analogue formed a stable, moderately functional BR pigment confirming that the ring cavity of the retinal binding site has a significant tolerance for derivatization on that portion of the molecule. Attempts to cross-link the azido chromophore to the protein by photoactivation were unsuccessful. The electron delocalization effect of the conjugated polyene side chain of the retinal appears to interfere with the formation or reactivity of the nitrene intermediate to the extent that photoactivated cross-linking is not achieved. These results demonstrate a limitation to the use of fluorinated aryl azides as photoaffinity reagents.     

POWER- AND TIME-RESOLVED RESONANCE RAMAN STUDIES AND CONFORMATIONAL CHANGES IN BACTERIORHODOPSIN

Abstract— By comparing the resonance Raman spectra of the retinal of the intermediates of bacteriorho-dopsin (obtained by using fixed flow with residence of time of 10 ps. variable laser power and frequency as well as computer subtraction techniques) with those of model compounds and with each other, the following possible conclusions can be obtained: (1) There exists stronger interaction between the retinal and the opsin in bacteriorhodopsin than that present in rhodopsin. (2) Conformational changes seem to take place during the dark light adaptation process as well as during the photosynthetic cycle. (3) The appearance of the spectrum of the retinal in the fingerprint region for the bL₅₅₀ and bM₄₁₂ intermediates is similar despite large shifts in their optical absorption maxima. This might argue against the theory that proposes ground state retinal conformational changes to explain the observed red shift in the optical spectra of retinal upon combining with the opsin. (4) Contrary to bM ₄₁₂ , the bL₅₅₀ species seems to be protonated. The fact that loss of proton does not seem to change the retinal conformation greatly might suggest that the protein and its ionic environment might carry the larger share of the load in the deprotonation process.     

A LINEAR DICHROISM STUDY OF THE ORIENTATION OF AROMATIC PROTEIN RESIDUES IN MAGNETICALLY ORIENTED BOVINE ROD OUTER SEGMENTS

Abstract— Linear dichroism measurements using magnetic field oriented bovine visual rod outer segments have been made in the UV and visible spectral regions. The results indicate that the planes of the aromatic amino acid residues of rhodopsin tend to be oriented normal to the membrane plane both before and after bleaching. In contrast, the retinal chromophore which tends to be oriented with its absorption oscillator parallel to the membrane plane before bleaching is randomly oriented about 10min after bleaching whereas the membranes remain oriented. Estimates of the anisotropy in the diamagnetic susceptibility of rhodopsin aromatic residues indicate that the anisotropic magnetic properties of these protein residues are sufficient to account for the observed orientation of visual rod outer segments in a homogenous magnetic field.     

Synthesis and self-organization of rod–dendron and dendron–rod–dendron molecules

We synthesized molecules containing one or two dendritic segments and a rigid-rod-like segment with their structures in the solid state. The molecules with rod–dendron or dendron–rod–dendron architecture had biphenyl ester rigid segments and 3,4,5 tris(n-dodecyloxy)benzoate of first or second generation as their dendritic segments. The variables investigated included the rod segment length as well as dendron generation, and all materials obtained were characterized by optical microscopy, differential scanning calorimetry, and X-ray scattering. Depending on the size of the rod segment and generation number of the dendritic segment, molecules organized into smectic, columnar, or cubic phases, and the symmetries observed were dominated by the anisotropic rod–rod interactions. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 3501–3518, 2003