Crystal structure of rhodopsin: a G-protein-coupled receptor.
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Animals↗
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Active states of rhodopsin.
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Amino Acid Sequence↗
Inherent chirality of the retinal chromophore in rhodopsin-A nonempirical theoretical analysis of chiroptical data.
CASSCF and GAUSSIAN CIS calculations were performed on ground and excited states of different conformations of 11-cis-retinal protonated Schiff bases, the chromophore of rhodopsin, in order to study their chiroptical properties and attempt a correlation between absolute conformation and CD-spectral data. Geometries were taken from molecular models, from published rhodopsin models, and from retinal conformations obtained from molecular dynamics with geometry restraints. In all the cases studied we find that a positive sense of twist about the C12-C13 bond correlates with a calculated positive CD of the long wavelength absorption band; the twist of the C6-C7 bond modulates this primary contribution of the C12-C13 bond. The correlation of the beta-band with structural features is not straightforward. Calculations on bathorhodopsin lend support to the idea that this intermediate is a highly twisted all-trans-conformation.
Circular Dichroism↗
On the bioactive conformation of the rhodopsin chromophore: absolute sense of twist around the 6-s-cis bond.
Incubation of opsin with synthetic 6-s-locked retinoids 2a and 2b only led to pigment formation from the alpha-locked 2a, the CD spectrum of which was similar to that of native rhodopsin (Rh). This establishes that the 6-s-bond of the chromophore in rhodopsin is cis, and that its helicity is negative. Earlier cross-linking studies showed that the 11-cis to all-trans photoisomerization occurring in the batho-Rh to lumi-Rh conversion induces a flip over of the side carrying the ring moiety. The GTP-binding assay of pigment Rh-(2a), incorporating retinal analogue 2a, has shown that its activity is 80% that of the native pigment. That is, the overall conformation around the 6-s bond is retained in the steps leading to G-protein activation.
Animals↗
Photoactivation of rhodopsin: interplay between protein and chromophore.
Data in the literature suggest a finely tuned interaction between ligand (11-cis-retinal) and protein (opsin) in order to allow very efficient photoactivation of the ligand and highly vectorial rhodopsin activation with a huge increase in receptor activity. We have further investigated this interaction using ligand homologues, 13C-ligand labelling or 15N-protein labelling, in combination with Fourier transform infrared (FT-IR) and solid-state magic angle spinning (ss-MAS)-NMR spectroscopy. Using 1D rotational resonance (RR) or double-quantum heteronuclear local field (2Q-HLF) ss-MAS-NMR we report the first structure refinement of the rhodopsin chromophore in situ. These measurements yield a specification of the torsional strain in the for isomerization essential C10-C13 segment of the chromophore. This strain is thought to contribute to the high rate and stereospecificity of the photoisomerization reaction. In agreement with previous data, the C10-C13 segment region reaches a relaxed all-trans configuration at the lumirhodopsin photointermediate. MAS-NMR analysis of [15N]lysine-labelled rhodopsin reveals the presence of a 'soft' counterion, requiring intermediate water molecules for stabilization. FT-IR studies on [2H]tyrosine-labelled rhodopsin demonstrate participation of several tyrosin(at)e residues in receptor activation. One of these, probably Tyr268, is already active at the bathorhodopsin stage. Finally, the effect of ligands with single additional methyl substituents in the C10-C12 region has been investigated. They do not affect the general activation pathway, but perturb the activation kinetics of rhodopsin, suggesting steric interference with protein residues. Possible implications of these results for a structural role of water residues will be discussed, as well.
Animals↗
The purple membrane of Halobacterium halobium: a new system for light energy conversion.
Patches of a distinctly different structure from the rest of the cell membrane occur in Halobacterium halobium. The isolated patches are called the purple membrane. It derives its colour from a retinal-protein complex, bacteriorhodopsin, which is the only protein species in this membrane serving a phototransducing function. Light energy is converted by a photochemical cycle going on continuously under illumination and accompanied by a cyclic release and uptake of protons. In the intact cell, this cycle operates as a vectorial process and therefore builds up an electrochemical gradient across the cell membrane conserving part of the absorbed light energy. The cell apparently uses this electrochemical gradient for the synthesis of ATP. Photophosphorylation is shown to be insensitive to cyanide but sensitive to dicyclohexylcarbodiimide (DCCD) and uncouplers. The concentration of ATP and the pH are tightly coupled but can be uncoupled by DCCD. Bacteriorhodopsin, as a light-driven proton pump, can then be studied in the cell as an isolated process. Quantitation of light energy conversion is possible by the indirect method of inhibition of respiration by light and the comparison of the number of absorbed quanta which prevent consumption of one molecule of oxygen.
Adenosine Triphosphate↗
A tiny excited-state barrier can induce a multiexponential decay of the retinal chromophore: a quantum dynamics investigation.
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Kinetics↗
Fourier transform IR spectroscopy study for new insights into molecular properties and activation mechanisms of visual pigment rhodopsin.
Fourier transform IR (FTIR) spectroscopy has been successfully applied in recent years to examine the functional and structural properties of the membrane protein rhodopsin, a prototype G protein coupled receptor. Unlike UV-visible spectroscopy, FTIR spectroscopy is structurally sensitive. It may give us both global information about the conformation of the protein and very detailed information about the retinal chromophore and all other functional groups, even when these are not directly related to the chromophore. Furthermore, it can be successfully applied to the photointermediates of rhodopsin, including the active receptor species, metarhodopsin II, and its decay products, which is not expected presently or even in the near future from crystallographic approaches. In this review we show how FTIR spectroscopy has significantly contributed to the understanding of very different aspects of rhodopsin, comprising both structural properties and the mechanisms leading to receptor activation and deactivation.
Mutagenesis, Site-Directed↗
Synthesis of N-heteroaryl retinals and their artificial bacteriorhodopsins.
N-Heteroaryl retinals derived from indole, 1-indolizine and 3-indolizine (10 a-c) have been synthesized after their UV/Vis red-shifted absorption properties had been predicted by time-dependent density functional theory (TD-DFT) computations. The three new analogues form artificial pigments upon recombination with bacterioopsin: indolyl retinal 10 a undergoes fast and efficient reconstitution to form a species with a UV/Vis absorbance maximum similar to that of wild-type bacteriorhodopsin, whilst the indolizinyl retinals 10 b and 10 c also reconstitute in significant proportion to give noticeably red-shifted, although unstable, pigments. Significant changes in the pK(a) values of these artificial bacteriorhodopsins are interpreted as arising from nonoptimal binding-site occupancy by the chromophore due to steric constraints.
Algorithms↗
The role of the 11-cis-retinal ring methyl substituents in visual pigment formation.
Artificial visual pigment formation from ring-demethylated retinals was studied in an effort to understand the effect that methyl groups on the chromophore cyclohexenyl ring have on the visual cycle. The stereoselective synthesis of the 11-cis-ring-demethylated analogues involves thallium-accelerated Suzuki cross-coupling reactions and highly stereocontrolled Wittig reactions to form key bonds. Only 11-cis-1,1,5-trisdemethylretinal (2) failed to form an artificial pigment, whilst variable pigment-formation yields were determined for the remaining analogues, increasing with the number (and location) of the chromophore hydrophobic ring methyl groups. Our results with the monodemethylated analogues 11-cis-5-demethylretinal (4) and 11-cis-1-demethylretinal (5) show that the C1-2-CH(3) groups are more important for pigment formation than the C5-CH(3) substituent. This is reflected in the absorption maxima of the artificial pigments, with values closer to that of native rhodopsin for 4. Docking studies based on a rhodopsin crystal structure, however, predict a lower pigment stability for 4 than for 5. Gas-phase DFT (B3LYP/6-31G*) computations of the free-ligand geometries, conformational searches about the C6--C7 bond, and docking studies revealed that, although the conformation of bound 5 is close to that of the native chromophore, the ligand needs to overcome the energy cost of shifting the unbound favored 6-s-trans conformation to the bound 6-s-cis form. In addition, the presence of an extra methyl group at C18 (11-cis-18-methylretinal, 7) is tolerated well and adds further stability to the complex, most probably due to increased hydrophobic interactions.
Binding Sites↗
(9Z)- and (11Z)-8-methylretinals for artificial visual pigment studies: stereoselective synthesis, structure, and binding models.
Artificial visual pigment formation was studied by using 8-methyl-substituted retinals in an effort to understand the effect that alkyl substitution of the chromophore side chain has on the visual cycle. The stereoselective synthesis of the 9-cis and 11-cis isomers of 8-methylretinal, as well as the 5-demethylated analogues is also described. The key bond formations consist of a thallium-accelerated Suzuki cross-coupling reaction between cyclohexenylboronic acids and dienyliodides (C6-C7), and a highly stereocontrolled Horner-Wadsworth-Emmons or Wittig condensation (C11-C12). The cyclohexenylboronic acid was prepared by trapping the precursor cyclohexenyllithium species with B(OiPr)(3) or B(OMe)(3). The cyclohexenyllithium species is itself obtained by nBuLi-induced elimination of a trisylhydrazone (Shapiro reaction), or depending upon the steric hindrance of the ring, by iodine-metal exchange. In binding experiments with the apoprotein opsin, only 9-cis-5-demethyl-8-methylretinal yielded an artificial pigment; 9-cis-8-methylretinal simply provided residual binding, while evidence of artificial pigment formation was not found for the 11-cis analogues. Molecular-mechanics-based docking simulations with the crystal structure of rhodopsin have allowed us to rationalize the lack of binding displayed by the 11-cis analogues. Our results indicate that these isomers are highly strained, especially when bound, due to steric clashes with the receptor, and that these interactions are undoubtedly alleviated when 9-cis-5-demethyl-8-methylretinal binds opsin.
Binding Sites↗
Synthesis of 11-cis-locked bicyclo[5.1.0]octanyl retinal and an enantioselective binding to bovine opsin.
Both enantiomers of 13-(E) and 13-(Z) isomers of 11-cis-locked bicyclo[5.1.0]octanyl retinal were prepared by an improved synthesis and incubated with bovine opsin. The synthesis also establishes the absolute configuration of the enantiomers. Only one of the enantiomers binds to opsin, thus showing the steric restrictions regarding the middle polyene moiety of the retinoid molecule; this is in sharp contrast to the known leniency of the ring moiety binding site of retinoids. However, although one enantiomer is incorporated into the pigment, the circular dichroic spectrum of the pigment incorporating the bound enantiomer yields only a very weak Cotton effect, showing that, once incorporated, the bicyclo[5.1.0]octanyl chromophore is flattened by the opsin binding site. The titled retinoid was synthesized for study of the absolute conformation of the retinal pigment in rhodopsin.
Animals↗
Absolute configurational determination of an all-trans-retinal dimer isolated from photoreceptor outer segments.
An all-trans-retinal (ATR) dimer (1) isolated from photoreceptor outer segments was found to have a stereogenic center at C13' flanked by tetraene (295 nm) and hexaenal (438 nm) chromophores. Analytical chiral HPLC (Chiralcel OD) revealed that the isolated retinoid had formed in 13% enantiomeric excess. Using a combination of (1)H-(1)H NOESY constraints, molecular modeling, and CD exciton coupling analysis, it was determined that the favored enantiomer was 13'(R). Three low-energy conformers of the 13'(S) model were found with MMFF/DFT and were used to calculate the CD spectrum of the ATR dimer (DeVoe method). The Boltzmann weighted spectrum was found to exhibit a positive exciton couplet, in excellent agreement with the experimental spectrum for the first eluted enantiomer. This further suggested that despite the large energy difference between the two interacting chromophores, the dominant source of optical activity in the CD spectrum is the nondegenerate exciton mechanism.
Computational Biology↗
Circular dichroism of heterochromophoric and partially regenerated purple membrane: search for exciton coupling.
In order to determine the origin of the bisignate CD spectra of native purple membrane, heterochromophoric analogues containing bacteriorhodopsin regenerated with native all-trans-retinal and retinal analogues were investigated. The data collected for the purple membrane samples containing two different chromophores suggest the additive character of the CD spectra. This conclusion was supported by a series of spectra using 5,6-dihydroretinal and 3-dehydroretinal and by using 33% regenerated PM in buffer and in presence of osmolytes. Our results support the idea of conformational heterogeneity of the chromophores in the bR in the trimer, suggesting that the three bR subunits in the trimer are not conformationally equal, and therefore, the bisignate CD spectrum of bR in the purple membrane occurs rather due to a superposition of the CD spectra from variously distorted bR subunits in the trimer than interchromophoric exciton-coupling interactions.
Bacteriorhodopsins↗
Gain setting in Chlamydomonas reinhardtii: mechanism of phototaxis and the role of the photophobic response.
The unicellular green alga (Chlamydomonas reinhardtii maintains sensitivity of its phototaxis response (alignment of swimming direction along the axis of a light beam) over several orders of magnitude of light intensities. It is widely accepted that the rotation of the swimming cell provides temporal comparisons of light intensities via periodic contrast generated by its asymmetrically positioned refractile eyespot organelle. The cells also exhibit a second behavioral response to light called the photophobic (or stop) response, which is a brief cessation of swimming caused by a temporal change in light intensity. The cells are desensitized to photophobic stimuli by light exposure. Through comparative measurements of both responses, we explain the behavioral basis of the large dynamic range of phototaxis in terms of precise desensitization of the photophobic response. The basis of the explanation is that the flagellar beat changes which cause phototactic orientation are the residual of the photophobic response after desensitization (i.e., "mini-photophobic" reactions which cause brief reorienting motions without a full stop). This interpretation predicts quantitatively the dependence of the extent of desensitization on light intensity and the dependence of onset and maintenance of phototaxis on extent of desensitization. These predictions are tested and confirmed in this report.
Animals↗
Large-scale morphological survey of mouse retinal ganglion cells.
Five hundred twenty ganglion cells in an isolated whole-mount preparation of the mouse retina were labeled using the "DiOlistic" method (Gan et al. [2000] Neuron 27:219-225) and were classified according to their morphological properties. Tungsten particles coated with a lipophilic dye (DiI) were propelled into the whole-mount retina using a gene gun. When a dye-coated particle contacted the cell membrane, the entire cell was labeled. The ganglion cells were classified into four groups based on their soma size, dendritic field size, and pattern and level of stratification. Broadly monostratified cells were classified into three groups: RG(A) cells (large soma, large dendritic field), RG(B) cells (small to medium-sized soma, small to medium-sized dendritic field), and RG(C) cells (small to medium-sized size soma, medium-sized to large dendritic field). Bistratified cells were classified as RG(D). This study represents the most complete morphological classification of mouse retinal ganglion cells available to date and provides a foundation for further understanding of the correlation of physiology and morphology and ganglion cell function with genetically manipulated animals.
Animals↗
Excited-state properties and environmental effects for protonated schiff bases: a theoretical study.
Complete active space self-consistent field (CASSCF), multireference configuration interaction (MRCI), density functional theory (DFT), time dependent DFT (TDDFT) and the singles and doubles coupled-cluster (CC2) methodologies have been used to study the ground state and excited states of protonated and neutral Schiff bases (PSB and SB) as models for the retinal chromophore. Systems with two to four conjugated double bonds are investigated. Geometry relaxation effects are studied in the excited pipi* state using the aforementioned methods. Taking the MRCI results as reference we find that CASSCF results are quite reliable even though overshooting of geometry changes is observed. TDDFT does not reproduce bond alternation well in the pipi* state. CC2 takes an intermediate position. Environmental effects due to solvent or protein surroundings have been studied in the excited states of the PSBs and SBs using a water molecule and solvated formate as model cases. Particular emphasis is given to the proton transfer process from the PSB to its solvent partner in the excited state. It is found that its feasibility is significantly enhanced in the excited state as compared to the ground state, which means that a proton transfer could be initiated already at an early step in the photodynamics of PSBs.
Computer Simulation↗
Micellar electrokinetic capillary chromatography of three natural vitamin A derivatives.
Micellar electrokinetic chromatography (MEKC) using bile salts has been employed to separate retinoids differing in structure and charge; bile salts in MEKC allows the separation of liposoluble molecules but, to the best of our knowledge, there are only few data on the above-mentioned technique for the separation of highly hydrophobic compounds. The three natural vitamin A derivatives, retinal, retinol and retinoic acid, were successfully separated by MEKC using sodium cholate within a relatively short time (ca. 25 min), whereas the separation of these compounds was not successful using sodium dodecyl sulfate or sodium deoxycholate. Several parameters (pH and organic modifiers, in addition to bile salts concentration) have been tested to provide a system that can be extended to synthetic retinoids, which are often used in treating several diseases, including cancer prevention and therapy.
Buffers↗