Activation of retinal ganglion cells using a biomimetic artificial retina.

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.

Light Harvesting by Equally Contributing Mechanisms in a Photosynthetic Antenna Protein.

We report supramolecular quantum mechanics/molecular mechanics simulations on the peridinin-chlorophyll a protein (PCP) complex from the causative algal species of red tides. These calculations reproduce for the first time quantitatively the distinct peridinin absorptions, identify multichromophoric molecular excitations, and elucidate the mechanisms regulating the strongly allowed S (1A) → S (1B) absorptions of the bound peridinins that span a 58 nm spectral range in the region of maximal solar irradiance. We discovered that protein binding site-imposed conformations, local electrostatics, and electronic coupling contribute equally to the spectral inhomogeneity.

The Forbidden 1B Excited Singlet State in Peridinin and Peridinin Analogues.

Theoretical studies have predicted the presence of a forbidden 1B state in proximity to the strongly allowed 1B excited state in polyenes and carotenoids. The 1B state is invariably predicted to have a very low oscillator strength, which precludes direct optical spectroscopic assignment. We report here a direct UV-vis optical spectroscopic feature assigned to the 1B state of S-2-peridinin, a synthetic analogue of the naturally occurring carotenoid, peridinin.

Expression, purification, and spectral tuning of RhoGC, a retinylidene/guanylyl cyclase fusion protein and optogenetics tool from the aquatic fungus .

RhoGC is a rhodopsin (Rho)-guanylyl cyclase (GC) gene fusion molecule that is central to zoospore phototaxis in the aquatic fungus It has generated considerable excitement because of its demonstrated potential as a tool for optogenetic manipulation of cell-signaling pathways involving cyclic nucleotides. However, a reliable method for expressing and purifying RhoGC is currently lacking. We present here an expression and purification system for isolation of the full-length RhoGC protein expressed in HEK293 cells in detergent solution.

Bacteriochlorins with a Twist: Discovery of a Unique Mechanism to Red-Shift the Optical Spectra of Bacteriochlorins.

Owing to their intense near infrared absorption and emission properties, to the ability to photogenerate singlet oxygen, or to act as photoacoustic imaging agents within the optical window of tissue, bacteriochlorins (2,3,12,13-tetrahydroporphyrins) promise to be of utility in many biomedical and technical applications. The ability to fine-tune the electronic properties of synthetic bacteriochlorins is important for these purposes. In this vein, we report the synthesis, structure determination, optical properties, and theoretical analysis of the electronic structure of a family of expanded bacteriochlorin analogues.

Spectroscopic Investigation of the Carotenoid Deoxyperidinin: Direct Observation of the Forbidden S0 → S1 Transition.

This paper presents a spectroscopic investigation of deoxyperidinin, a synthetic peridinin analogue in which the carbonyl functional group in peridinin was replaced by a nonconjugated methylene group. Steady-state and ultrafast time-resolved absorption and fluorescence spectroscopic experiments are carried out on deoxyperidinin in n-hexane and acetonitrile at room temperature and in 2-methyltetrahydrofuran at 77 K. The spectra of deoxyperidinin have higher vibronic resolution compared to those of peridinin.

Effects of Strong Electronic Coupling in Chlorin and Bacteriochlorin Dyads.

Achieving tunable, intense near-infrared absorption in molecular architectures with properties suitable for solar light harvesting and biomedical studies is of fundamental interest. Herein, we report the photophysical, redox, and molecular-orbital characteristics of nine hydroporphyrin dyads and associated benchmark monomers that have been designed and synthesized to attain enhanced light harvesting. Each dyad contains two identical hydroporphyrins (chlorin or bacteriochlorin) connected by a linker (ethynyl or butadiynyl) at the macrocycle β-pyrrole (3- or 13-) or meso (15-) positions.

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.

High efficiency light harvesting by carotenoids in the LH2 complex from photosynthetic bacteria: unique adaptation to growth under low-light conditions.

Rhodopin, rhodopinal, and their glucoside derivatives are carotenoids that accumulate in different amounts in the photosynthetic bacterium, Rhodoblastus (Rbl.) acidophilus strain 7050, depending on the intensity of the light under which the organism is grown. The different growth conditions also have a profound effect on the spectra of the bacteriochlorophyll (BChl) pigments that assemble in the major LH2 light-harvesting pigment-protein complex.

Excited state properties of a short π-electron conjugated peridinin analogue.

C-peridinin is a synthetic analogue of the important, naturally-occurring carotenoid, peridinin, found in several marine algal species. C-peridinin has five conjugated carbon-carbon double bonds compared to eight possessed by peridinin and also lacks the methyl group functionalities typically present along the polyene chain of carotenoids. These structural modifications lead to unique excited state properties and important insights regarding the factors controlling the photophysics of peridinin and other carbonyl-containing carotenoids, which are critical components of the light-harvesting systems of many photosynthetic organisms.

Photochromic bacteriorhodopsin mutant with high holographic efficiency and enhanced stability via a putative self-repair mechanism.

The Q photoproduct of bacteriorhodopsin (BR) is the basis of several biophotonic technologies that employ BR as the photoactive element. Several blue BR (bBR) mutants, generated by using directed evolution, were investigated with respect to the photochemical formation of the Q state. We report here a new bBR mutant, D85E/D96Q, which is capable of efficiently converting the entire sample to and from the Q photoproduct.

A spectroscopic and theoretical investigation of a free-base meso-trithienylcorrole.

The unique optical properties of free-base meso-tris(5-methylthien-2-yl)corrole were compared to those of the widely investigated meso-triphenyl-substituted analogue. A combination of spectroscopic and computational experiments was undertaken to elucidate the relationship between structural features of the neutral, mono-anionic and mono-cationic forms of the corroles and their corresponding optical properties. A general bathochromic shift was measured for the thienyl-substituted corrole.

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 β-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.

Directed evolution of bacteriorhodopsin for applications in bioelectronics.

In nature, biological systems gradually evolve through complex, algorithmic processes involving mutation and differential selection. Evolution has optimized biological macromolecules for a variety of functions to provide a comparative advantage. However, nature does not optimize molecules for use in human-made devices, as it would gain no survival advantage in such cooperation.

The nature of the intramolecular charge transfer state in peridinin.

Experimental and theoretical evidence is presented that supports the theory that the intramolecular charge transfer (ICT) state of peridinin is an evolved state formed via excited-state bond-order reversal and solvent reorganization in polar media. The ICT state evolves in <100 fs and is characterized by a large dipole moment (~35 D). The charge transfer character involves a shift of electron density within the polyene chain, and it does not involve participation of molecular orbitals localized in either of the β-rings.

Energetics and dynamics of the low-lying electronic states of constrained polyenes: implications for infinite polyenes.

Steady-state and ultrafast transient absorption spectra were obtained for a series of conformationally constrained, isomerically pure polyenes with 5-23 conjugated double bonds (N). These data and fluorescence spectra of the shorter polyenes reveal the N dependence of the energies of six (1)B(u)(+) and two (1)A(g)(-) excited states. The (1)B(u)(+) states converge to a common infinite polyene limit of 15,900 ± 100 cm(-1).

Effect of Molecular Symmetry on the Spectra and Dynamics of the Intramolecular Charge Transfer (ICT) state of peridinin.

The spectroscopic properties and dynamics of the excited states of two different synthetic analogues of peridinin were investigated as a function of solvent polarity using steady-state absorption, fluorescence, and ultrafast time-resolved optical spectroscopy. The analogues are denoted S-1- and S-2-peridinin and differ from naturally occurring peridinin in the location of the lactone ring and its associated carbonyl group, known to be obligatory for the observation of a solvent dependence of the lifetime of the S(1) state of carotenoids. Relative to peridinin, S-1- and S-2-peridinin have their lactone rings two and four carbons more toward the center of the π-electron system of conjugated carbon-carbon double bonds, respectively.

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).

Rapid release of retinal from a cone visual pigment following photoactivation.

As part of the visual cycle, the retinal chromophore in both rod and cone visual pigments undergoes reversible Schiff base hydrolysis and dissociation following photobleaching. We characterized light-activated release of retinal from a short-wavelength-sensitive cone pigment (VCOP) in 0.1% dodecyl maltoside using fluorescence spectroscopy.

Green proteorhodopsin reconstituted into nanoscale phospholipid bilayers (nanodiscs) as photoactive monomers.

Over 4000 putative proteorhodopsins (PRs) have been identified throughout the oceans and seas of the Earth. The first of these eubacterial rhodopsins was discovered in 2000 and has expanded the family of microbial proton pumps to all three domains of life. With photophysical properties similar to those of bacteriorhodopsin, an archaeal proton pump, PRs are also generating interest for their potential use in various photonic applications.

Femtosecond transient absorption spectroscopic study of a carbonyl-containing carotenoid analogue, 2-(all-trans-retinylidene)-indan-1,3-dione.

The photophysical properties of a carbonyl-containing carotenoid analogue in an s-cis configuration, relative to the conjugated π system, 2-(all-trans-retinylidene)-indan-1,3-dione (C20Ind), were investigated by femtosecond time-resolved spectroscopy in various solvents. The lifetime of the optically forbidden S(1) state of C20Ind becomes long as solvent polarity increases. This trend is completely opposite to the situation of S(1-ICT) dynamics of carbonyl-containing carotenoids, such as peridinin and fucoxanthin.

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.

Photochemical and thermal stability of green and blue proteorhodopsins: implications for protein-based bioelectronic devices.

The photochemical and thermal stability of the detergent-solubilized blue- and green-absorbing proteorhodpsins, BPR and GPR, respectively, are investigated to determine the viability of these proteins for photonic device applications. Photochemical stability is studied by using pulsed laser excitation and differential UV-vis spectroscopy to assign the photocyclicity. GPR, with a cyclicity of 7 × 10(4) photocycles protein(-1), is 4-5 times more stable than BPR (9 × 10(3) photocycles protein(-1)), but is less stable than native bacteriorhodopsin (9 × 10(5) photocycles protein(-1)) or the 4-keto-bacteriorhodopsin analogue (1 × 10(5) photocycles protein(-1)).

The intramolecular charge transfer state in carbonyl-containing polyenes and carotenoids.

Numerous femtosecond time-resolved optical spectroscopic experiments have reported that the lifetime of the low-lying S(1) state of carbonyl-containing polyenes and carotenoids decreases with increasing solvent polarity. The effect becomes even more pronounced as the number of double bonds in the conjugated π-electron system decreases. The effect has been attributed to an intramolecular charge transfer (ICT) state coupled to S(1), but it is still not clear what the precise molecular nature of this state is, and how it is able to modulate the spectral and dynamic properties of polyenes and carotenoids.