A conserved aromatic residue regulating photosensitivity in short-wavelength sensitive cone visual pigments.

Visual pigments have a conserved phenylalanine in transmembrane helix 5 located near the ß-ionone ring of the retinal chromophore. Site-directed mutants of this residue (F207) in a short-wavelength sensitive visual pigment (VCOP) were studied using UV-visible spectroscopy to investigate its role in photosensitivity and formation of the light-activated state. The side chain is important for pigment formation: VCOP(F207A), VCOP(F207L), VCOP(F207M), and VCOP(F207W) substitutions all bound 11-cis-retinal and formed a stable visual pigment, while VCOP(F207V), VCOP(F207S), VCOP(F207T), and VCOP(F207Y) substitutions do not. The extinction coefficients of all pigments are close, ranging between 35800 and 45600 M⁻¹ cm⁻¹. Remarkably, the mutants exhibit an up to 5-fold reduction in photosensitivity and also abnormal photobleaching behavior. One mutant, VCOP(F207A), forms an isomeric composition of the retinal chromophore after illumination comparable to that of wild-type VCOP yet does not release the all-trans-retinal chromophore. These findings suggest that the conserved F207 residue is important for a normal photoactivation pathway, formation of the active conformation and the exit of all-trans-retinal from the chromophore-binding pocket.

meso-arylporpholactones and their reduction products.

The rational syntheses of meso-tetraaryl-3-oxo-2-oxaporphyrins 5, known as porpholactones, via MnO(4)(-)-mediated oxidations of the corresponding meso-tetraaryl-2,3-dihydroxychlorins (7) is detailed. Since chlorin 7 is prepared from the parent porphyrin 1, this amounts to a 2-step replacement of a pyrrole moiety in 1 by an oxazolone moiety. The stepwise reduction of the porpholactone 5 results in the formation of chlorin analogues, meso-tetraaryl-3-hydroxy-2-oxachlorin (11) and meso-tetraaryl-2-oxachlorins (12). The reactivity of 11 with respect to nucleophilic substitution by O-, N-, and S-nucleophiles is described. The profound photophysical consequences of the formal replacement of a pyrrole with an oxazolone (porphyrin-like chromophore) or (substituted) oxazole moiety (chlorin-like chromophore with, for the parent oxazolochlorin 12, red-shifted Q(x) band with enhanced oscillator strengths) are detailed and rationalized on the basis of SAC-CI and MNDO-PSDCI molecular orbital theory calculations. The single crystal X-ray structures of the porpholactones point at a minor steric interaction between the carbonyl oxygen and the flanking phenyl group. The essentially planar structures of all chromophores in all oxidation states prove that the observed optical properties originate from the intrinsic electronic properties of the chromophores and are not subject to conformational modulation.

Conserved residues in the extracellular loops of short-wavelength cone visual pigments.

The role of the extracellular loop region of a short-wavelength sensitive pigment, Xenopus violet cone opsin, is investigated via computational modeling, mutagenesis, and spectroscopy. The computational models predict a complex H-bonding network that stabilizes and connects the EC2-EC3 loop and the N-terminus. Mutations that are predicted to disrupt the H-bonding network are shown to produce visual pigments that do not stably bind chromophore and exhibit properties of a misfolded protein. The potential role of a disulfide bond between two conserved Cys residues, Cys(105) in TM3 and Cys(182) in EC2, is necessary for proper folding and trafficking in VCOP. Lastly, certain residues in the EC2 loop are predicted to stabilize the formation of two antiparallel ß-strands joined by a hairpin turn, which interact with the chromophore via H-bonding or van der Waals interactions. Mutations of conserved residues result in a decrease in the level of chromophore binding. These results demonstrate that the extracellular loops are crucial for the formation of this cone visual pigment. Moreover, there are significant differences in the structure and function of this region in VCOP compared to that in rhodopsin.

Glutamic acid 181 is negatively charged in the bathorhodopsin photointermediate of visual rhodopsin.

Assignment of the protonation state of the residue Glu-181 is important to our understanding of the primary event, activation processes and wavelength selection in rhodopsin. Despite extensive study, there is no general agreement on the protonation state of this residue in the literature. Electronic assignment is complicated by the location of Glu-181 near the nodal point in the electrostatic charge shift that accompanies excitation of the chromophore into the low-lying, strongly allowed ππ* state. Thus, the charge on this residue is effectively hidden from electronic spectroscopy. This situation is resolved in bathorhodopsin, because photoisomerization of the chromophore places Glu-181 well within the region of negative charge shift following excitation. We demonstrate that Glu-181 is negatively charged in bathorhodopsin on the basis of the shift in the batho absorption maxima at 10 K [λ(max) band (native) = 544 ± 2 nm, λ(max) band (E181Q) = 556 ± 3 nm] and the decrease in the λ(max) band oscillator strength (0.069 ± 0.004) of E181Q relative to that of the native protein. Because the primary event in rhodopsin does not include a proton translocation or disruption of the hydrogen-bonding network within the binding pocket, we may conclude that the Glu-181 residue in rhodopsin is also charged.

Activation of retinal ganglion cells using a biomimetic artificial retina.

Objective.Biomimetic protein-based artificial retinas offer a new paradigm for restoring vision for patients blinded by retinal degeneration. Artificial retinas, comprised of an ion-permeable membrane and alternating layers of bacteriorhodopsin (BR) and a polycation binder, are assembled using layer-by-layer electrostatic adsorption. Upon light absorption, the oriented BR layers generate a unidirectional proton gradient. The main objective of this investigation is to demonstrate the ability of the ion-mediated subretinal artificial retina to activate retinal ganglion cells (RGCs) of degenerated retinal tissue.Approach. Ex vivoextracellular recording experiments with P23H line 1 rats are used to measure the response of RGCs following selective stimulation of our artificial retina using a pulsed light source. Single-unit recording is used to evaluate the efficiency and latency of activation, while a multielectrode array (MEA) is used to assess the spatial sensitivity of the artificial retina films.Main results.The activation efficiency of the artificial retina increases with increased incident light intensity and demonstrates an activation latency of ∼150 ms. The results suggest that the implant is most efficient with 200 BR layers and can stimulate the retina using light intensities comparable to indoor ambient light. Results from using an MEA show that activation is limited to the targeted receptive field.Significance.The results of this study establish potential effectiveness of using an ion-mediated artificial retina to restore vision for those with degenerative retinal diseases, including retinitis pigmentosa.

Ultrafast time-resolved absorption spectroscopy of geometric isomers of carotenoids.

The structures of a number of stereoisomers of carotenoids have been revealed in three-dimensional X-ray crystallographic investigations of pigment-protein complexes from photosynthetic organisms. Despite these structural elucidations, the reason for the presence of stereoisomers in these systems is not well understood. An important unresolved issue is whether the natural selection of geometric isomers of carotenoids in photosynthetic pigment-protein complexes is determined by the structure of the protein binding site or by the need for the organism to accomplish a specific physiological task. The association of cis isomers of a carotenoid with reaction centers and trans isomers of the same carotenoid with light-harvesting pigment-protein complexes has led to the hypothesis that the stereoisomers play distinctly different physiological roles. A systematic investigation of the photophysics and photochemistry of purified, stable geometric isomers of carotenoids is needed to understand if a relationship between stereochemistry and biological function exists. In this work we present a comparative study of the spectroscopy and excited state dynamics of cis and trans isomers of three different open-chain carotenoids in solution. The molecules are neurosporene (n = 9), spheroidene (n = 10), and spirilloxanthin (n = 13), where n is the number of conjugated pi-electron double bonds. The spectroscopic experiments were carried out on geometric isomers of the carotenoids purified by high performance liquid chromatography (HPLC) and then frozen to 77 K to inhibit isomerization. The spectral data taken at 77 K provide a high resolution view of the spectroscopic differences between geometric isomers. The kinetic data reveal that the lifetime of the lowest excited singlet state of a cis-isomer is consistently shorter than that of its corresponding all-trans counterpart despite the fact that the excited state energy of the cis molecule is typically higher than that of the trans molecule. Quantum theoretical calculations on an n = 9 linear polyene were carried out to examine this process. The calculations indicate that the electronic coupling terms are significantly higher for the cis isomer, and when combined with the Franck-Condon factors, predict internal conversion rates roughly double those of the all-trans species. The electronic effects more than offset the decrease in coupling efficiencies associated with the higher system origin energies and explain the observed shorter cis isomer lifetimes.

Ultrafast Time-resolved Absorption Spectroscopy of Geometric Isomers of Carotenoids.

The structures of a number of stereoisomers of carotenoids have been revealed in three-dimensional X-ray crystallographic investigations of pigment-protein complexes from photosynthetic organisms. Despite these structural elucidations, the reason for the presence of stereoisomers in these systems is not well understood. An important unresolved issue is whether the natural selection of geometric isomers of carotenoids in photosynthetic pigment-protein complexes is determined by the structure of the protein binding site or by the need for the organism to accomplish a specific physiological task. The association of cis isomers of a carotenoid with reaction centers and trans isomers of the same carotenoid with light-harvesting pigment-protein complexes has led to the hypothesis that the stereoisomers play distinctly different physiological roles. A systematic investigation of the photophysics and photochemistry of purified, stable geometric isomers of carotenoids is needed to understand if a relationship between stereochemistry and biological function exists. In this work we present a comparative study of the spectroscopy and excited state dynamics of cis and trans isomers of three different open-chain carotenoids in solution. The molecules are neurosporene (n=9), spheroidene (n=10), and spirilloxanthin (n=13), where n is the number of conjugated pi-electron double bonds. The spectroscopic experiments were carried out on geometric isomers of the carotenoids purified by high performance liquid chromatography (HPLC) and then frozen to 77 K to inhibit isomerization. The spectral data taken at 77 K provide a high resolution view of the spectroscopic differences between geometric isomers. The kinetic data reveal that the lifetime of the lowest excited singlet state of a cis-isomer is consistently shorter than that of its corresponding all-trans counterpart despite the fact that the excited state energy of the cis molecule is typically higher than that of the trans molecule. Quantum theoretical calculations on an n=9 linear polyene were carried out to examine this process. The calculations indicate that the electronic coupling terms are significantly higher for the cis isomer, and when combined with the Franck-Condon factors, predict internal conversion rates roughly double those of the all-trans species. The electronic effects more than offset the decrease in coupling efficiencies associated with the higher system origin energies and explain the observed shorter cis isomer lifetimes.

Low-Temperature Trapping of Photointermediates of the Rhodopsin E181Q Mutant.

Three active-site components in rhodopsin play a key role in the stability and function of the protein: 1) the counter-ion residues which stabilize the protonated Schiff base, 2) water molecules, and 3) the hydrogen-bonding network. The ionizable residue Glu-181, which is involved in an extended hydrogen-bonding network with Ser-186, Tyr-268, Tyr-192, and key water molecules within the active site of rhodopsin, has been shown to be involved in a complex counter-ion switch mechanism with Glu-113 during the photobleaching sequence of the protein. Herein, we examine the photobleaching sequence of the E181Q rhodopsin mutant by using cryogenic UV-visible spectroscopy to further elucidate the role of Glu-181 during photoactivation of the protein. We find that lower temperatures are required to trap the early photostationary states of the E181Q mutant compared to native rhodopsin. Additionally, a Blue Shifted Intermediate (BSI, λmax = 498 nm, 100 K) is observed after the formation of E181Q Bathorhodopsin (Batho, λmax = 556 nm, 10 K) but prior to formation of E181Q Lumirhodopsin (Lumi, λmax = 506 nm, 220 K). A potential energy diagram of the observed photointermediates suggests the E181Q Batho intermediate has an enthalpy value 7.99 KJ/mol higher than E181Q BSI, whereas in rhodopsin, the BSI is 10.02 KJ/mol higher in enthalpy than Batho. Thus, the Batho to BSI transition is enthalpically driven in E181Q and entropically driven in native rhodopsin. We conclude that the substitution of Glu-181 with Gln-181 results in a significant perturbation of the hydrogen-bonding network within the active site of rhodopsin. In addition, the removal of a key electrostatic interaction between the chromophore and the protein destabilizes the protein in both the dark state and Batho intermediate conformations while having a stabilizing effect on the BSI conformation. The observed destabilization upon this substitution further supports that Glu-181 is negatively charged in the early intermediates of the photobleaching sequence of rhodopsin.