Rhodopsin Chromophore Research Articles

Syntheses of 13C 2-labelled 11 Z-retinals

To enable solid-state NMR investigations of the rhodopsin chromophore and its photointermediates, a series of 11 Z-retinal isotopomers have been synthesised containing pairs of adjacent 13C labels at C9/C10, C10/C11 or C11/C12, respectively. The C9 labelled carbon atom was introduced through the Heck reaction of a 13C-labelled Weinreb acrylamide derivative, and the label at the C12 position derived from a 13C-containing ethoxy Bestmann–Ohira reagent. The 13C labels at C10 and C11 were introduced through the reaction of β-ionone with labelled triethyl phosphonoacetate.

Benchmarking the Approximate Second-Order Coupled-Cluster Method on Biochromophores

Extensive benchmarking calculations are presented to assess the accuracy of commonly used quantum chemical methods in studying excited state properties of biochromophores. The first few excited states of 12 common model chromophores of photoactive yellow protein, green fluorescent protein, and rhodopsin have been studied using approximate second-order coupled-cluster (CC2) and linear-response time-dependent density functional theory (TDDFT) calculations. The study comprises investigations of basis-set dependences on CC2 excitation energies as well as comparisons of the CC2 results with excitation energies obtained at other computational levels and with experimental data. The basis-set study shows that the accuracy of the two lowest excitation energies is generally sufficient when triple-ζ basis sets augmented with polarization functions are employed, whereas the third and higher excited states were found to require diffuse basis functions in the basis set. Augmenting the basis set with diffuse functions contributes less than 0.15 eV to the excitation energies of low-lying excited states, except for some of the studied anionic states and for Rydberg states. Calculations at the TDDFT level using the B3LYP functional show the necessity of stabilizing anions with point charges or counterions when aiming at reliable electronic excitation spectra. The two lowest excitation energies of the green fluorescent protein and rhodopsin chromophores calculated at the CC2 level agree within 0.15 eV with experimental excitation energies, whereas the B3LYP values are somewhat less accurate, with a maximum deviation of 0.27 eV. The computed excitation energies for the photoactive yellow protein chromophore models deviate from available experimental values by 0.3-0.4 eV and 0.1-0.5 eV, at the CC2 and B3LYP levels of theory, respectively.

Role of Bulk Water in Hydrolysis of the Rhodopsin Chromophore

Rhodopsin (Rho) is a prototypical G protein-coupled receptor that changes from an inactive conformational state to a G protein-activating state as a consequence of its retinal chromophore isomerization, 11-cis-retinal → all-trans-retinal. The photoisomerized chromophore covalently linked to Lys(296) by a Schiff base is subsequently hydrolyzed, but little is known about this reaction. Recent research indicates a significant role for tightly bound transmembrane water molecules in the Rho activation process. Atomic structures of Rho and hydroxyl radical footprinting reveal ordered waters within Rho transmembrane helices that are located close to highly conserved and functionally important receptor residues, forming a hydrogen bond network. Using (18)O-labeled H(2)O, we now report that water from bulk solvent, but not tightly bound water, is involved in the hydrolytic release of chromophore upon Rho activation by light. Moreover, small molecules (and presumably, water) enter the Rho structure from the cytoplasmic side of the membrane. Thus, this work indicates two distinct origins of water vital for Rho function.

On the role of the triplet state in the cis/trans isomerization of rhodopsin: A CASPT2//CASSCF study of a model chromophore

AbstractThe possibility of population of the lowest‐lying triplet state (T1) in the early events of the photochemical isomerization process of a model chromophore of Rhodopsin (Rh) has been analyzed using multireference perturbation theory (CASPT2//CASSCF) methods. It is shown that the characteristics of the isomerization process namely small S1−T1 gap, presence of hydrogen out of plane active vibrational modes, and existence of a dense manifold of vibrational states, render possible the fulfilment of the conditions needed for the population of T1. The possible consequences for the photochemistry and photophysics of Rh are also discussed. © 2011 Wiley Periodicals, Inc. Int J Quantum Chem 111:3431–3437, 2011

Vitamin A as an Activator and Sensitizing Chromophore for Rhodopsin

Absorption of light by rhodopsin isomerizes its 11-cis retinal chromophore to the all-trans conformation. The rhodopsin then activates a biochemical cascade that produces an electrical response by the photoreceptor. Eventually, all-trans retinal dissociates from the opsin and is reduced to vitamin A. The truncated retinal analog, beta-ionone, can pharmacologically activate some types of visual pigment, mimicking the effects of light. Beta-ionone is not normally found in the retina, however, vitamin A is present within the photoreceptor and can reach millimolar concentrations after exposure to bright light. Can vitamin A activate rhodopsin? In suction electrode recordings from isolated green-sensitive rods of salamander, exogenous vitamin A decreased circulating current and flash sensitivity, and accelerated flash response kinetics, changes that can also be seen during exposure to background light. Microspectrophotometric measurements showed that vitamin A accumulated in the outer segments, and an in vitro binding assay confirmed the binding of vitamin A to rhodopsin. These results suggested that vitamin A activated rhodopsin. In addition, suction electrode recordings showed that vitamin A improved the relative sensitivity of rods to UV light and in an in vitro bleaching assay, vitamin A enhanced the bleaching of rhodopsin by UV light. Presumably, both effects involved Forster resonance energy transfer (FRET) from vitamin A to the 11-cis chromophore of rhodopsin. Next, we tested whether FRET could be induced by endogenous vitamin A. After bleaching a large fraction of the rhodopsin in green-sensitive rods to generate vitamin A, relative sensitivity to UV wavelengths did indeed increase. Therefore, vitamin A can bind rhodopsin, activate it and also serve as a sensitizing chromophore.

QM/MM calculation of protein magnetic shielding tensors with generalized hybrid-orbital method: A GIAO approach

A method to compute magnetic shielding tensors with generalized hybrid-orbital (GHO) QM/MM scheme is developed at the levels of Hartree-Fock and second-order Møller-Plesset perturbation theory using gauge-including atomic orbitals. A feature of the GHO method is utilized to ensure gauge-origin independency of GHO shielding tensors in a simple way. The benchmark calculations indicate that the GHO method reproduced full-QM shielding constants nearly quantitatively for atoms not directly coupled to the GHO linking atoms. As an application to a realistic protein, carbon chemical shifts are calculated for the retinal chromophore in visual rhodopsin.

Highly conserved tyrosine stabilizes the active state of rhodopsin

Light-induced isomerization of the 11-cis-retinal chromophore in the visual pigment rhodopsin triggers displacement of the second extracellular loop (EL2) and motion of transmembrane helices H5, H6, and H7 leading to the active intermediate metarhodopsin II (Meta II). We describe solid-state NMR measurements of rhodopsin and Meta II that target the molecular contacts in the region of the ionic lock involving these three helices. We show that a contact between Arg135(3.50) and Met257(6.40) forms in Meta II, consistent with the outward rotation of H6 and breaking of the dark-state Glu134(3.49)-Arg135(3.50)-Glu247(6.30) ionic lock. We also show that Tyr223(5.58) and Tyr306(7.53) form molecular contacts with Met257(6.40). Together these results reveal that the crystal structure of opsin in the region of the ionic lock reflects the active state of the receptor. We further demonstrate that Tyr223(5.58) and Ala132(3.47) in Meta II stabilize helix H5 in an active orientation. Mutation of Tyr223(5.58) to phenylalanine or mutation of Ala132(3.47) to leucine decreases the lifetime of the Meta II intermediate. Furthermore, the Y223F mutation is coupled to structural changes in EL2. In contrast, mutation of Tyr306(7.53) to phenylalanine shows only a moderate influence on the Meta II lifetime and is not coupled to EL2.

Seaming is believing

Do excited molecules relaxing to their ground state pass through a 'seam' connecting the potential energy profiles of the states? Experimental data suggest the answer to this long-standing question is 'yes'. See Letter p. 440 The primary photochemical event in vision, isomerization of the 11-cis chromophore in rhodopsin to the all-trans form, is one of the fastest natural photochemical processes known, taking less than a millionth of a millionth of a second. The molecular details of reactions of such rapidity are a stiff challenge to experimenters, but Polli et al. now report the characterization of the reaction using ultrafast optical spectroscopy with sub-20-femtosecond time resolution and spectral coverage from the visible to the near infrared. The data confirm that rhodopsin's extreme reactivity results from a molecular funnel mechanism that involves a 'conical intersection' between the potential energy surfaces of the starting and product molecules.

Cleavage of the retinal pigment epithelium-specific protein RPE65 under oxidative stress

The regeneration of the 11- cis-retinyl imine chromophore of rhodopsin during the visual cycle and mechanisms that control this process are central questions in the field of vision research. The retinal pigment epithelium (RPE)-specific protein RPE65 is centrally involved in the isomerization and hydrolysis of all- trans-retinyl esters. In this study, we investigated RPE65 cleavage and potential regulatory mechanisms under oxidative stress conditions. The D407 RPE cell cultures were exposed to H 2O 2 (100–1000 μM). Changes in the levels of RPE65 and proteins related to apoptosis were investigated using gel electrophoresis and western blotting. Mass spectrometry was used to confirm the identity of RPE65. C57BL/6J (M450) and C3HeB/FeJ (L450) mice were used for in vivo experiments. We found that a novel 45 kDa truncated fragment of the RPE65 protein, designated RPE45, appears in RPE cells upon light exposure or oxidative stress. RPE45 is generated in vitro by recombinant caspases via an ubiquitination-dependent mechanism. Collectively, our results indicate that oxidative stress during the visual cycle results in cleavage of RPE65.

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A(2) treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment.

Fluoro Derivatives of Retinal Illuminate the Decisive Role of the C12-H Element in Photoisomerization and Rhodopsin Activation

Rhodopsin, the visual pigment of the vertebrate rod cell, is among the best investigated members of the G-protein-coupled receptor family. Within this family a unique characteristic of visual pigments is their covalently bound chromophore, 11-cis retinal, which acts as an inverse agonist. Upon illumination it can be transformed into the all-trans isomer that acts as a full agonist. This photoisomerization process is extremely efficient: 2 out of 3 photons are effective, full stereoselectivity is achieved, and stereoinversion occurs within 200 fs. The mechanism behind this process is still not really understood, although the available evidence points at the twisted C(9)-C(13) segment of the 11-cis ligand as the quintessence. To further dissect the role of this segment, we have generated the 10-fluoro, 12-fluoro, and 14-fluoro analogues of rhodopsin. A fluoro substituent brings in only little more volume than hydrogen, but considerably more mass and polarizability. The analogue pigments were compared to rhodopsin with respect to their photosensitivity (quantum yield), light-induced structural transitions (UV-vis and FT-IR spectroscopy), and signaling activity (G protein activation rate). We find that 14-F substitution is quite neutral, while 10-F and 12-F substitutions exert significant but distinct effects. The 10-F pigment exhibits a quantum yield similar to that of rhodopsin (0.65) but strongly perturbed thermodynamics of the structural transitions following photoactivation and only 20% of the native signaling activity. The 12-F pigment exhibits a significantly decreased quantum yield (0.47) and signaling activity (30%) but mixed effects on the structural transitions. These properties are compared to those of the corresponding methyl derivatives. We conclude that rotation of the C(12)-H bond of the rhodopsin chromophore is a major rate-limiting factor in the photoisomerization process, while the C(10)-H moiety plays a dominant role in ligand relaxation and further rearrangements following photoactivation.

To see in different seas: spatial variation in the rhodopsin gene of the sand goby (Pomatoschistus minutus)

Aquatic organisms living in a range of photic environments require specific mechanisms to tune their visual pigments. Maximum absorbance (lambda(max)) of retinal rods in populations of the marine demersal sand goby, (Pomatoschistus minutus; Gobiidae, Teleostei) correlates with the local optic environment. It has been shown that this is not regulated through a physiological response by exchanging the rhodopsin chromophore. To test for evolutionary adaptation, the sequence of the rhodopsin (RH1) gene was analysed in 165 Pomatoschistus minutus individuals from seven populations across its distribution range. Analysis showed a high level of intraspecific polymorphism at the RH1 gene, including nonsynonymous mutations on amino acids, known as spectral tuning sites. Population differentiation at these sites was in agreement with the observed differentiation in lambda(max) values. Analyses of d(N)/d(S) substitution rate ratios and likelihood ratio tests under site-specific models detected a significant signal of positive Darwinian selection on the RH1 gene. A strong discrepancy in differentiation was noticed between RH1 gene variation and the presumably neutral microsatellites and mitochondrial data. Samples did not cluster according to geographical or historical proximity with regards to RH1, but according to the general photic conditions of the habitat environment of the sand goby. This study highlights the usefulness of sensory genes, like rhodopsin, for studying the characteristics of local adaptation in marine nonmodel organisms.

Studies on the conformational state of the chromophore group (11-cis-retinal) in rhodopsin by computer molecular simulation methods

Based on computer simulation methods, the molecular dynamics of the rhodopsin chromophore group (11-cis-retinal) has been analyzed. The molecular dynamics has been traced within a 3-ns time interval; thereby 3 x 10(6) discrete conformational states of opsin and rhodopsin were compared and analyzed. It was shown that, within a short time of about 0.3-0.4 ns from the start of simulation, the retinal beta-ionone ring becomes twisted around the C6-C7 bond by approximately 60 degrees compared with that of the initial configuration. The influence of retinal conformation on the positions of the maximum of the absorption band of rhodopsin at the conformational states of t=0 and t=3 ns were estimated using the ab initio methods. The results indicated that the absorption maximum for the final (3-ns) state is shifted by 10 nm toward the long wavelength region compared with the initial state. This suggests that the rhodopsin molecule with its twisted chromophore will possess a considerably lower activation energy than the rhodopsin molecule where the beta-ionone ring is in a planar orientation to the retinal polyene chain.

Electrostatic Control of the Photoisomerization Efficiency and Optical Properties in Visual Pigments: On the Role of Counterion Quenching

Hybrid QM(CASPT2//CASSCF/6-31G*)/MM(Amber) computations have been used to map the photoisomerization path of the retinal chromophore in Rhodopsin and explore the reasons behind the photoactivity efficiency and spectral control in the visual pigments. It is shown that while the electrostatic environment plays a central role in properly tuning the optical properties of the chromophore, it is also critical in biasing the ultrafast photochemical event: it controls the slope of the photoisomerization channel as well as the accessibility of the S(1)/S(0) crossing space triggering the ultrafast decay. The roles of the E113 counterion, the E181 residue, and the other amino acids of the protein pocket are explicitly analyzed: it appears that counterion quenching by the protein environment plays a key role in setting up the chromophore's optical properties and its photochemical efficiency. A unified scenario is presented that discloses the relationship between spectroscopic and mechanistic properties in rhodopsins and allows us to draw a solid mechanism for spectral tuning in color vision pigments: a tunable counterion shielding appears as the elective mechanism for L<-->M spectral modulation, while a retinal conformational control must dictate S absorption. Finally, it is suggested that this model may contribute to shed new light into mutations-related vision deficiencies that opens innovative perspectives for experimental biomolecular investigations in this field.

Towards an interpretation of 13C chemical shifts in bathorhodopsin, a functional intermediate of a G-protein coupled receptor

Photoisomerization of the membrane-bound light receptor protein rhodopsin leads to an energy-rich photostate called bathorhodopsin, which may be trapped at temperatures of 120 K or lower. We recently studied bathorhodopsin by low-temperature solid-state NMR, using in situ illumination of the sample in a purpose-built NMR probe. In this way we acquired 13C chemical shifts along the retinylidene chain of the chromophore. Here we compare these results with the chemical shifts of the dark state chromophore in rhodopsin, as well as with the chemical shifts of retinylidene model compounds in solution. An earlier solid-state NMR study of bathorhodopsin found only small changes in the 13C chemical shifts upon isomerization, suggesting only minor perturbations of the electronic structure in the isomerized retinylidene chain. This is at variance with our recent measurements which show much larger perturbations of the 13C chemical shifts. Here we present a tentative interpretation of our NMR results involving an increased charge delocalization inside the polyene chain of the bathorhodopsin chromophore. Our results suggest that the bathochromic shift of bathorhodopsin is due to modified electrostatic interactions between the chromophore and the binding pocket, whereas both electrostatic interactions and torsional strain are involved in the energy storage mechanism of bathorhodopsin.

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.

The Influence Of Rhodopsin Chromophore Binding On Protein Biosynthesis Examined In Vivo

Over 100 mutations in the rhodopsin gene are associated with retinitis pigmentosa (RP) and other retinal disorders. A subset of mutations found in the N-terminus of rhodopsin cause sector RP in which the lower retina is preferentially affected, suggesting that in these cases retinal degeneration (RD) is influenced by light exposure. One such example, P23H, is the most prevalent RP-causing rhodopsin mutation in North America. Recently we have developed X. laevis (frog) models of RP based on human and bovine P23H rhodopsin which demonstrate light sensitivity. In these models, dark rearing either partially or completely rescues RD. We have shown that the rescuing effect of dark rearing is associated with chromophore binding, since blocking binding also prevents rescue. Light exposure is associated with decreased expression of P23H rhodopsin and decreased transport of P23H rhodopsin to the rod outer segment, suggesting a defect in export of the mutant protein from the ER. In order to define the role of chromophore binding in the rescue of P23H-induced RD, we have performed an extensive characterization of light sensitivity in these models. We raised transgenic F1 tadpoles in varying intensities, durations and wavelengths of light and determined the influence of these factors on RD. Our results suggest that the rescuing effects of dark rearing are not mediated by increased chromophore availablility, but rather by increased stability of rhodopsin in the secretory pathway. Our results have significant implications for the design of molecular chaperone therapies for RP.

The Green-absorbing Drosophila Rh6 Visual Pigment Contains a Blue-shifting Amino Acid Substitution That Is Conserved in Vertebrates

The molecular mechanisms that regulate invertebrate visual pigment absorption are poorly understood. Through sequence analysis and functional investigation of vertebrate visual pigments, numerous amino acid substitutions important for this adaptive process have been identified. Here we describe a serine/alanine (S/A) substitution in long wavelength-absorbing Drosophila visual pigments that occurs at a site corresponding to Ala-292 in bovine rhodopsin. This S/A substitution accounts for a 10-17-nm absorption shift in visual pigments of this class. Additionally, we demonstrate that substitution of a cysteine at the same site, as occurs in the blue-absorbing Rh5 pigment, accounts for a 4-nm shift. Substitutions at this site are the first spectrally significant amino acid changes to be identified for invertebrate pigments sensitive to visible light and are the first evidence of a conserved tuning mechanism in vertebrate and invertebrate pigments of this class.

Functional evolution of photochemical energy transformations in oxygen-producing organisms.

Chlorophyll a is the photochemical agent accounting for most oxygenic photosynthesis, that is, over 99.9% of photosynthetic primary activity on Earth. The spectral and energetic properties of chlorophyll a can, at least in part, be rationalised in terms of the solar spectral output and the energetics of oxygen production and carbon dioxide reduction with two photochemical reactions. The long wavelength limit on in vivo chlorophyll a absorption is probably close to the energetic limit: longer wavelengths could not support a high rate and efficiency of oxygenic photosynthesis. Retinal, a β-carotene derivative that is the chromophore of rhodopsin, acts not only as a sensory pigment, but also as an ion-pumping photochemical transducer. Both sensory and energy-transforming rhodopsins occur in oxygenic phototrophs, although the extent of expression and the function of the latter are not well understood.

Photochemical Reaction Dynamics of the Primary Event of Vision Studied by Means of a Hybrid Molecular Simulation

The photoisomerization reaction dynamics of a retinal chromophore in the visual receptor rhodopsin was investigated by means of hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations. The photoisomerization reaction of retinal constitutes the primary step of vision and is known as one of the fastest reactions in nature. To elucidate the molecular mechanism of the high efficiency of the reaction, we carried out hybrid ab initio QM/MM MD simulations of the complete reaction process from the vertically excited state to the photoproduct via electronic transition in the entire chromophore-protein complex. An ensemble of reaction trajectories reveal that the excited-state dynamics is dynamically homogeneous and synchronous even in the presence of thermal fluctuation of the protein, giving rise to the very fast formation of the photoproduct. The synchronous nature of the reaction dynamics in rhodopsin is found to originate from weak perturbation of the protein surroundings and from dynamic regulation of volume-conserving motions of the chromophore. The simulations also provide a detailed view of time-dependent modulations of hydrogen-out-of-plane vibrations during the reaction process, and identify molecular motions underlying the experimentally observed dynamic spectral modulations.