Sensory Rhodopsin I and Sensory Rhodopsin II Form Trimers of Dimers in Complex with their Cognate Transducers

Archaeal photoreceptors consist of sensory rhodopsins in complex with their cognate transducers. After light excitation, a two‐component signaling chain is activated, which is homologous to the chemotactic signaling cascades in enterobacteria. The latter system has been studied in detail. From structural and functional studies, a picture emerges which includes stable signaling complexes, which assemble to receptor arrays displaying hexagonal structural elements. At this higher order structural level, signal amplification and sensory adaptation occur. Here, we describe electron microscopy data, which show that also the archaeal phototaxis receptors sensory rhodopsin I and II in complex with their cognate transducers can form hexagonal lattices even in the presence of a detergent. This result could be confirmed by molecular dynamics calculations, which revealed similar structural elements. Calculations of the global modes of motion displayed one mode, which resembles the “U”‐”V” transition of the NpSRII:NpHtrII complex, which was previously argued to represent a functionally relevant global conformational change accompanying the activation process [Ishchenko et al. (2013) J. Photochem. Photobiol. B 123, 55‐58]. A model of cooperativity at the transmembrane level is discussed.     

Ca2+ -dependent regulation of phototransduction

Photon absorption by rhodopsin triggers the phototransduction signaling pathway that culminates in degradation of cGMP, closure of cGMP-gated ion channels and hyperpolarization of the photoreceptor membrane. This process is accompanied by a decrease in free Ca(2+) concentration in the photoreceptor cytosol sensed by Ca(2+)-binding proteins that modulate phototransduction and activate the recovery phase to reestablish the photoreceptor dark potential. Guanylate cyclase-activating proteins (GCAPs) belong to the neuronal calcium sensor (NCS) family and are responsible for activating retinal guanylate cyclases (retGCs) at low Ca(2+) concentrations triggering synthesis of cGMP and recovery of the dark potential. Here we review recent structural insight into the role of the N-terminal myristoylation in GCAPs and compare it to other NCS family members. We discuss previous studies identifying regions of GCAPs important for retGC1 regulation in the context of the new structural data available for myristoylated GCAP1. In addition, we present a hypothetical model for the Ca(2+)-triggered conformational change in GCAPs and retGC1 regulation. Finally, we briefly discuss the involvement of mutant GCAP1 proteins in the etiology of retinal degeneration as well as the importance of other Ca(2+) sensors in the modulation of phototransduction.     

Rhodopsin: its molecular substructure and phospholipid interactions.

Abstract—Rhodopsin in retinal rod outer segment disc membranes, was proteolyzed by treatment with papain. This treatment left three fragments of apparent mol wt of 26,000, 19,000 and 10,000 in the membrane. The circular dichroism (CD) of solubilized, proteolyzed rhodopsin, in both the UV and visible spectral regions, was essentially identical to that of native rhodopsin. This indicates that the retinal binding site configuration is essentially unchanged by proteolysis and that the proteolyzed form of rhodopsin retained the helical content of native rhodopsin. Far UV CD measurements on the fragments indicate that the secondary structural features of the proteolyzed complex were largely maintained when the complex was dissociated. This finding suggests that the proteolytic fragments represent independently stabilized domains within rhodopsin. Measurements of the dependence of the activation free energy of the unfolding of opsin (as determined by the rate of loss of regenerability of opsin) and the meta I to meta II transition on the level of phospholipid associated with opsin and rhodopsin. respectively, have allowed for a determination of the mode of stabilization of these proteins by phospholipid. This dependence has been shown to have a linear form for opsin and rhodopsin. Hence, it appears that the stabilization of the tertiary structure of both solubilized opsin and rhodopsin is attributable to the sum of their interactions with individual phospholipid molecules, interacting with the protein in a non-cooperative manner.     

Rhodopsin in a new model bilayer membrane

In search of a planar and uniform model membrane presenting a wide interaction-surface for the study of photobiological processes, we have succeeded in depositing lipid bilayer on a single face of a porous disc. Using a method that combines the Langmuir-Blodgett dipping and the touching procedures, a symmetrical or asymmetrical bilayer membrane has been obtained. The illumination of rhodopsin-containing membranes appears to increase the permeability to K⁺, Na⁺, and Ca²⁺ with a high selectivity for Ca²⁺. The bilayer adsorption on the porous disc is discussed and an attempt is made to explain the failure of the dipping method in our experimental conditions for preparing an artificial membrane. A comparative look at model membranes is also presented.     

Steric Hindrance between Chromophore Substituents as the Driving Force of Rhodopsin Photoisomerization: 10-Methyl-13-Demethyl Retinal Containing Rhodopsin

Abstract— A visual chromophore analogue, 10-methyl-13-demethyl (dm) retinal, was synthesized and reconstituted with bleached bovine rhodopsin to form a visual pigment derivative with absorbance maximum at 505 nm. The investigations with this new compound were stimulated from recent results using 13-dm retinal as a chromophore that revealed a remarkable loss in quantum efficiency (φ of 13-dm retinal-containing rhodopsin: 0.30, Ternieden and Gartner, J. Photochem. Photobiol. B Biol. 33, 83–86, 1996). The quantum efficiency of the new pigment was determined as 0.59 by quantitative bleaching using reconstituted rhodopsin as a reference. The very similar quantum efficiencies of rhodopsin and the new pigment give experimental support for the recently presented hypothesis that a steric hindrance between the substituents at positions 10 and 13 in 11- cis -retinal is elevated during the photoisomerization and thus facilitates the rapid photoisomerization of the visual chromophore (Peteanu et al., Proc. Natl. Acad. Sci. USA 90, 11762–11766, 1993). Such steric hindrance is removed from the molecule by the elimination of the methyl group from position 13 and can be re-established via a rearrangement of the substitution pattern by introducing a methyl group at position 10 of 13-dm retinal.     

牛视紫红质蛋白质中视黄醛的活性位点

视紫红质蛋白是一个跨膜蛋白, 视黄醛(RET)在该蛋白中的活性结合位点涉及到视觉过程机理, 与一些眼科疾病病理有关. 基于牛视紫红质蛋白1U19的蛋白质晶体结构数据, 采用密度泛函理论的B3LYP方法计算RET-Lys296残基与视黄醛分子周围半径为0.6 nm的空间范围30个氨基酸残基相互作用和结合能. 数值显示1U19蛋白中的残基Glu113、Glu181和Glu122是质子化的RET-Lys296残基的活性结合位点, 结合能分别为-333.38、-205.67和-194.56 kJ·mol-1. 这些氨基酸残基带有一个负电荷, 与质子化的RET-Lys296残基发生强烈的离子静电相互作用. 另外几个残基Ala292、Cys187、Phe293、Pro291以及Trp265等与质子化RET-Lys296残基也有相互吸引作用. 当RET-Lys296残基非质子化, 上述相互作用消失, 促使视黄醛分子与视蛋白分离. 研究发现残基Glu113和Glu181周围各自有一个结晶水分子通过双氢键形式起着稳定作用.     

Identification of Two Palmitoyl Groups in Octopus Rhodopsin

Abstract— We determined the structure and site of fatty acid incorporated in octopus rhodopsin using a combination of fluorescence label and enzymatic cleavage methods in conjunction with fast-atom bombardment (FAB) mass spectrometry. A single peptide containing two adjacent cysteines, Cys337 and Cys338, was successfully isolated using the fluorescence from a dye conjugated to Cys345. The FAB mass spectrometric analysis of the peptide (323phe-³⁴⁰phe) revealed that two palmitoyl groups are linked to Cys337 and Cys338 via thioester bonds in octopus rhodopsin as in bovine rhodopsin.     

Rhodopsin phosphorylation in rats exposed to intense light

The damaging effects of intense light on the rat retina are known to vary depending on the time of day of exposure. The purpose of this study was to determine if rhodopsin phosphorylation patterns, a measure of the activity of the pigment, varied in a similar manner. After 10 min in strong light (1400 lux), all six threonine and serine sites in the rat rhodopsin C-terminus were phosphorylated, with mono- to tetraphosphorylation being substantially more prominent than penta- to hexaphosphorylation. The level and multiplicity of rhodopsin phosphorylations were reduced both with the duration of light exposure and the duration of subsequent darkness. Although showing vast differences in susceptibility to light damage, rats exposed at 5 P.M. or 1 A.M. showed similar rhodopsin phosphorylation levels and patterns. These data indicate that a process controlled by circadian rhythm other than rhodopsin phosphorylation is involved either in damaging or mediating the damage evoked by intense light exposure.     

Transmembrane Signaling Mediated by Water in Bovine Rhodopsin

Abstract— Unhydrated air-dried films of rhodopsin from bovine rod outer segment membranes do not produce its active state, metarhodopsin II. In order to reveal requirements for its formation, we studied changes in H-bonding of water, peptide carbonyl and carboxylic acid in the photochemical reactions by means of difference Fourier transform infrared spectroscopy, under both hydrated and unhydrated conditions. A water molecule near Glull3, which undergoes H-bonding change in bathorhodopsin, remained in the unhydrated film, but with a weaker H-bonding state than in the hydrated film. The other water molecules, which shift in lumirhodopsin and metarhodopsin I as well as in bathorhodopsin of the hydrated film, were not observed in the unhydrated film. Effects of the dehydration were detected in all the C=O stretching vibrations of the peptide backbone and of Asp83 in the formation of bathorhodopsin. The C=O stretching band of Asp83 of lumirhodopsin and metarhodopsin I is intensified in the unhydrated film. We propose that structural changes at the intradiscal site in the interaction between the Schiff base and Glull3 affect water molecules, the peptide backbone, Asp83 and Glul22 in helices B and C through consecutive photochemical processes to metarhodopsin II.     

Identification of a Single Phosphorylation Site Within Octopus Rhodopsin

Abstract— Light-dependent phosphorylation of rhodopsin (Rho) is a first step in the desensitization of the signaling state of the receptor during vertebrate and invertebrate visual transduction. We found that only ³⁵⁸Ser of the photoac-tivated octopus Rho (oRho*) was phosphorylated by octopus rhodopsin kinase (oRK). Tryptic truncation of the C-terminal PPQGY repeats of oRho that follow the phosphorylation region did not influence spectral or G-protein activation properties of oRho but abolished phos phorylation. Despite significant structural differences between oRK and mammalian RK, these results provide i further evidence of the importance of singly phosphorylated species of Rho* in the generation of arrestin binding sites.     

Influence of Arrestin on the Photodecay of Bovine Rhodopsin

Continued activation of the photocycle of the dim‐light receptor rhodopsin leads to the accumulation of all‐trans‐retinal in the rod outer segments (ROS). This accumulation can damage the photoreceptor cell. For retinal homeostasis, deactivation processes are initiated in which the release of retinal is delayed. One of these processes involves the binding of arrestin to rhodopsin. Here, the interaction of pre‐activated truncated bovine visual arrestin (Arr^Tr) with rhodopsin in 1,2‐diheptanoyl‐sn‐glycero‐3‐phosphocholine (DHPC) micelles is investigated by solution NMR techniques and flash photolysis spectroscopy. Our results show that formation of the rhodopsin–arrestin complex markedly influences partitioning in the decay kinetics of rhodopsin, which involves the simultaneous formation of a meta II and a meta III state from the meta I state. Binding of Arr^Tr leads to an increase in the population of the meta III state and consequently to an approximately twofold slower release of all‐trans‐retinal from rhodopsin.     

Rhodopsin deactivation is affected by mutations of Tyrl91

The 9-methyl group of 11-cis retinal is important in the efficient formation of the active conformation of rhodopsin, Meta II. Here, Tyrl91 rhodopsin mutants were generated because of its proximity to that methyl group in the dark structure. If photoactivation results in movement of the 9-methyl group toward Tyrl91, the steric interactions involved with activation and/or deactivation might not be as tightly coupled in mutant proteins with smaller amino acids at that position. Tyrl91 mutations have no effect on the dark pigment. However, after photobleaching, the lifetime of Meta II is shorter; Meta II decays quickly into two inactive species: (1) a Meta III or Meta III-like species and (2) opsin and free retinal. The Meta III-like fraction maintains the covalent Schiff base linkage of the chromophore much longer than the wild type. On the other hand, the fast chromophore release is similar to cone pigments. Taken together, the data suggest that the role of the 9-methyl group after photo-isomerization is not only to form Meta II efficiently, but also to maintain its active conformation and allow for the timely hydrolysis of the Schiff base. Perturbation of this interaction effects changes in the hydrolysis of the Schiff base and for the case of the Y191A mutation the folded structure of the protein after photobleaching.     

Water‐mediated Spectral Shifts in Rhodopsin and Bathorhodopsin†

The role of water molecules in spectral tuning of proteins has been left largely unexplored. This topic is important because changing hydrogen bond patterns during the activation process may lead to spectral shifts which can be of diagnostic value for the underlying structures. Arguments put forward in this article are based on spectral shift calculations of the rhodopsin and bathorhodopsin chromophore due to wat2a and 2b in the presence and absence of the counterion and of the amino acids lining the rhodopsin binding pocket. They show, among others, that a single water molecule can shift the absorbance by up to 0.1 eV or 34 nm depending on the environment of the chromophore.     

Rhodopsin exhibits a preference for solvation by polyunsaturated docosohexaenoic acid

An all-atom molecular dynamics simulation of rhodopsin in a membrane environment has been carried out with lipid composition similar to that of the retinal membrane. The initial conformation of the protein was taken from the X-ray crystallographic structure (1F88), while those of the lipids came from a previous molecular dynamics simulation. During the course of the 12.5 ns simulation, the initially randomly placed lipids adopt an anisotropic solvation structure around the protein. The lipids, having one saturated stearic acid chain and one polyunsaturated docosohexaenoic acid chain with a zwitterionic phosphatidylcholine headgroup, arrange themselves to maximize contact between the polyunsaturated chain and the protein surface. This organization is driven by energetically favorable interactions between the transmembrance helices and the docosohexaenoyl chains that are largely of the van der Waals type. These observations are consistent with various experimental studies on rhodopsin and other G-protein coupled receptors and with the picture of extreme flexibility in polyunsaturated fatty acid chains that has arisen from recent NMR and computational work.     

Rhodopsin Activation Switches in a Native Membrane Environment†

The elucidation of structure–function relationships of membrane proteins still poses a considerable challenge due to the sometimes profound influence of the lipid bilayer on the functional properties of the protein. The visual pigment rhodopsin is a prototype of the family of G protein‐coupled transmembrane receptors and a considerable part of our knowledge on its activation mechanisms has been derived from studies on detergent‐solubilized proteins. This includes in particular the events associated with the conformational transitions of the receptor from the still inactive Meta I to the Meta II photoproduct states, which are involved in signaling. These events involve disruption of an internal salt bridge of the retinal protonated Schiff base, movement of helices and proton uptake from the solvent by the conserved cytoplasmic E(D)RY network around Glu134. As the equilibria associated with these events are considerably altered by the detergent environment, we set out to investigate these equilibria in the native membrane environment and to develop a coherent thermodynamic model of these activating steps using UV–visible and Fourier‐transform infrared spectroscopy as complementary techniques. Particular emphasis is put on the role of protonation of Glu134 from the solvent, which is a thermodynamic prerequisite for full receptor activation in membranes, but not in detergent. In view of the conservation of this carboxylate group in family A G protein‐coupled receptors, it may also play a similar role in the activation of other family members.     

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.     

The Effects of Octanol on the Late Photointermediates of Rhodopsin

Membrane suspensions of unperturbed rhodopsin and rhodopsin perturbed with 2.5 niM octanol were photo-lyzed with 477 nm laser pulses at 20^OC and 35°C. Changes in absorbance were monitored at times ranging from 1 u-s to 80 ms after excitation. The data were analyzed using singular value decomposition, global exponential fitting and kinetic modeling. A recently proposed model involving the photointermediate Meta-I₃₈₀ (T. E. Thorgeirsson, J. W. Lewis, S. E. Wallace-Williams, and D. S. Kliger, Biochemistry 32,13861–13872, 1993) fits data for samples with and without octanol. Comparison of the microscopic rates shows this alcohol accelerates the formation of Meta-II via Meta-I₃₈₀. Activation and equilibrium thermodynamic parameters obtained from Ar-rhenius plots suggest that octanol reduces the entropy increase in forming both Meta-I_3g0 and Meta-II. It also lowers the enthalpy of Meta-1, _SI, relative to Lumi and of Meta-II relative to Meta-I₄₈₀. To help determine whether octanol affects the protein directly or indirectly through the lipid bilayer, similar experiments were conducted using rhodopsin solubilized in 0.13% dodecyl maltoside with and without octanol. Spectral shifts in the presence of octanol suggest that a direct protein interaction exists in addition to previously reported effects dependent on membrane free volume.     

Characterization of Lamprey Rhodopsin by Isolation from Lamprey Retina and Expression in Mammalian Cells

Abstract— A visual pigment was extracted from lamprey retina and was expressed in cultured mammalian cells (293S) using a cDNA fragment isolated from lamprey retina. The extracted pigment, a putative lamprey rhodopsin, had an absorption maximum at 503 nm. The recombinant lamprey rhodopsin, reconstituted with 11- cis -retinal, showed an absorption maximum at about 500 nm. Both pigments reacted with an anti-bovine rhodopsin antibody (Rh29), which recognizes the short photoreceptor cells in lamprey retina. Unlike rhodopsins of higher vertebrates, the lamprey rhodopsin bleached gradually in the presence of 100 m M hydroxylamine even in the dark. Our results suggest that, despite its high similarities with other vertebrate rhodopsins, lamprey rhodopsin has a character different from those of higher vertebrates.     

Light Dynamics of the Retinal-Disease-Relevant G90D Bovine Rhodopsin Mutant

The RHO gene encodes the G-protein-coupled receptor (GPCR) rhodopsin. Numerous mutations associated with impaired visual cycle have been reported; the G90D mutation leads to a constitutively active mutant form of rhodopsin that causes CSNB disease. We report on the structural investigation of the retinal configuration and conformation in the binding pocket in the dark and light-activated state by solution and MAS-NMR spectroscopy. We found two long-lived dark states for the G90D mutant with the 11-cis retinal bound as Schiff base in both populations. The second minor population in the dark state is attributed to a slight shift in conformation of the covalently bound 11-cis retinal caused by the mutation-induced distortion on the salt bridge formation in the binding pocket. Time-resolved UV/Vis spectroscopy was used to monitor the functional dynamics of the G90D mutant rhodopsin for all relevant time scales of the photocycle. The G90D mutant retains its conformational heterogeneity during the photocycle.