Redshifted and Near‐infrared Active Analog Pigments Based upon Archaerhodopsin‐3

Archaerhodopsin‐3 (AR3) is a member of the microbial rhodopsin family of hepta‐helical transmembrane proteins, containing a covalently bound molecule of all‐trans retinal as a chromophore. It displays an absorbance band in the visible region of the solar spectrum (λmax 556 nm) and functions as a light‐driven proton pump in the archaeon Halorubrum sodomense. AR3 and its mutants are widely used in neuroscience as optogenetic neural silencers and in particular as fluorescent indicators of transmembrane potential. In this study, we investigated the effect of analogs of the native ligand all‐trans retinal A1 on the spectral properties and proton‐pumping activity of AR3 and its single mutant AR3 (F229S). While, surprisingly, the 3‐methoxyretinal A2 analog did not redshift the absorbance maximum of AR3, the analogs retinal A2 and 3‐methylamino‐16‐nor‐1,2,3,4‐didehydroretinal (MMAR) did generate active redshifted AR3 pigments. The MMAR analog pigments could even be activated by near‐infrared light. Furthermore, the MMAR pigments showed strongly enhanced fluorescence with an emission band in the near‐infrared peaking around 815 nm. We anticipate that the AR3 pigments generated in this study have widespread potential for near‐infrared exploitation as fluorescent voltage‐gated sensors in optogenetics and artificial leafs and as proton pumps in bioenergy‐based applications.     

Dissection of Environmental Changes at the Cytoplasmic Surface of Light‐activated Bacteriorhodopsin and Visual Rhodopsin: Sequence of Spectrally Silent Steps†

The physico‐chemical properties as well as the conformation of the cytoplasmic surface of the 7‐helix retinal proteins bacteriorhodopsin (bR) and visual rhodopsin change upon light activation. A recent study found evidence for a transient softening of bR in its key intermediate M [Pieper et al. (2008) Phys. Rev. Lett. 100 , 228103] as a direct proof for the functional significance of protein flexibility. In this report we compare environmental and flexibility changes at the cytoplasmic surface of light‐activated bR and rhodopsin detected by time‐resolved fluorescence spectroscopy. The changes in fluorescence of covalently bound fluorescent probes and protein real‐time dynamics were investigated. We found that in fluorescently labeled bR and rhodopsin the intensity of fluorescein and Atto647 increased upon formation of the key intermediates M and metarhodopsin‐II, respectively, suggesting different surface properties compared to the dark state. Furthermore, time‐resolved fluorescence anisotropy experiments reveal an increase in steric restriction of loop flexibility because of changes in the surrounding protein environment in both the M‐intermediate as well as the active metarhodopsin‐II state. The kinetics of the fluorescence changes at the rhodopsin surface uncover multiple transitions, suggesting metarhodopsin‐II substates with different surface properties. Proton uptake from the aqueous bulk phase correlates with the first transition, while late proton release seems to parallel the second transition. The last transition between states of different surface properties correlates with metarhodopsin‐II decay.     

The Photocycle of Channelrhodopsin‐2: Ultrafast Reaction Dynamics and Subsequent Reaction Steps

The photocycle of channelrhodopsin‐2 is investigated in a comprehensive study by ultrafast absorption and fluorescence spectroscopy as well as flash photolysis in the visible spectral range. The ultrafast techniques reveal an excited‐state decay mechanism analogous to that of the archaeal bacteriorhodopsin and sensory rhodopsin II from Natronomonas pharaonis. After a fast vibrational relaxation of the excited‐state population with 150 fs its decay with mainly 400 fs is observed. Hereby, both the initial all‐trans retinal ground state and the 13‐cis‐retinal K photoproduct are populated. The reaction proceeds with a 2.7 ps component assigned to cooling processes. Small spectral shifts are observed on a 200 ps timescale. They are attributed to conformational rearrangements in the retinal binding pocket. The subsequent dynamics progresses with the formation of an M‐like intermediate (7 and 120 μs), which decays into red‐shifted states within 3 ms. Ground‐state recovery including channel closing and reisomerization of the retinal chromophore occurs in a triexponential manner (6 ms, 33 ms, 3.4 s). To learn more about the energy barriers between the different photocycle intermediates, temperature‐dependent flash photolysis measurements are performed between 10 and 30 °C. The first five time constants decrease with increasing temperature. The calculated thermodynamic parameters indicate that the closing mechanism is controlled by large negative entropy changes. The last time constant is temperature independent, which demonstrates that the photocycle is most likely completed by a series of individual steps recovering the initial structure.     

Tuning the Protein‐Induced Absorption Shifts of Retinal in Engineered Rhodopsin Mimics

Rational design of light‐capturing properties requires understanding the molecular and electronic structure of chromophores in their native chemical or biological environment. We employ here large‐scale quantum chemical calculations to study the light‐capturing properties of retinal in recently designed human cellular retinol binding protein II (hCRBPII) variants (Wang et al. Science, 2012 , 338, 1340–1343). Our calculations show that these proteins absorb across a large part of the visible spectrum by combined polarization and electrostatic effects. These effects stabilize the ground or excited state energy levels of the retinal by perturbing the Schiff‐base or β‐ionone moieties of the chromophore, which in turn modulates the amount of charge transfer within the molecule. Based on the predicted tuning principles, we design putative in silico mutations that further shift the absorption properties of retinal in hCRBPII towards the ultraviolet and infrared regions of the spectrum.     

Optomechanical Control of Quantum Yield in Trans–Cis Ultrafast Photoisomerization of a Retinal Chromophore Model

The quantum yield of a photochemical reaction is one of the most fundamental quantities in photochemistry, as it measures the efficiency of the transduction of light energy into chemical energy. Nature has evolved photoreceptors in which the reactivity of a chromophore is enhanced by its molecular environment to achieve high quantum yields. The retinal chromophore sterically constrained inside rhodopsin proteins represents an outstanding example of such a control. In a more general framework, mechanical forces acting on a molecular system can strongly modify its reactivity. Herein, we show that the exertion of tensile forces on a simplified retinal chromophore model provokes a substantial and regular increase in the trans ‐to‐cis photoisomerization quantum yield in a counterintuitive way, as these extension forces facilitate the formation of the more compressed cis photoisomer. A rationale for the mechanochemical effect on this photoisomerization mechanism is also proposed.     

RPE65 and the Accumulation of Retinyl Esters in Mouse Retinal Pigment Epithelium

The RPE65 protein of the retinal pigment epithelium (RPE) enables the conversion of retinyl esters to the visual pigment chromophore 11‐cis retinal. Fresh 11‐cis retinal is generated from retinyl esters following photoisomerization of the visual pigment chromophore to all‐trans during light detection. Large amounts of esters accumulate in Rpe65^?/? mice, indicating their continuous formation when 11‐cis retinal generation is blocked. We hypothesized that absence of light, by limiting the conversion of esters to 11‐cis retinal, would also result in the build‐up of retinyl esters in the RPE of wild‐type mice. We used HPLC to quantify ester levels in organic extracts of the RPE from wild‐type and Rpe65^?/? mice. Retinyl ester levels in Sv/129 wild‐type mice that were dark adapted for various intervals over a 4‐week period were similar to those in mice raised in cyclic light. In C57BL/6 mice however, which contain less Rpe65 protein, dark adaptation was accompanied by an increase in ester levels compared to cyclic light controls. Retinyl ester levels were much higher in Rpe65^?/? mice compared to wild type and kept increasing with age. The results suggest that the RPE65 role in retinyl ester homeostasis extends beyond enabling the formation of 11‐cis retinal.     

Proteorhodopsin Photocycle Kinetics Between pH 5 and pH 9

The retinal protein proteorhodopsin is a homolog of the well‐characterized light‐driven proton pump bacteriorhodopsin. Basic mechanisms of proton transport seem to be conserved, but there are noticeable differences in the pH ranges of proton transport. Proton transport and protonation state of a carboxylic acid side chain, the primary proton acceptor, are correlated. In case of proteorhodopsin, the pK_a of the primary proton acceptor Asp‐97 (pK_a ≈ 7.5) is unexpectedly close to environmental pH (pH ≈ 8). A significant fraction of proteorhodopsin is possibly inactive at natural pH, in contrast to bacteriorhodopsin. We investigated photoinduced kinetics of proteorhodopsin between pH 5 and pH 9 by time resolved UV/vis absorption spectroscopy. Kinetics is inhomogeneous within that pH region and can be considered as a superposition of two fractions. These fractions are correlated with the Asp‐97 titration curve. Beside Asp‐97, protonation equilibria of other groups influence kinetics, but the observations do not point toward major differences of primary proton acceptor function in proteorhodopsin and bacteriorhodopsin. The pK_a of proteorhodopsin and some of its variants is suspected to be an example of molecular adaptation to the physiology of the original organisms.     

UV‐Vis Spectroscopy Reveals a Correlation Between Y263 and BV Protonation States in Bacteriophytochromes

Red‐light photosensory proteins, phytochromes, link light activation to biological functions by interconverting between two conformational states. For this, they undergo large‐scale secondary and tertiary changes which follow small‐scale Z to E bond photoisomerization of the covalently bound bilin chromophore. The complex network of amino acid interactions in the chromophore‐binding pocket plays a central role in this process. Highly conserved Y263 and H290 have been found to be important for the photoconversion yield, while H260 has been identified as important for bilin protonation and proton transfer steps. Here, we focus on the roles these amino acids are playing in preserving the chemical properties of bilin in the resting Pr state of the photosensory unit of a bacteriophytochrome from Deinococcus radiodurans. By using pH‐dependent UV‐Vis spectroscopy and spectral decomposition modeling, we confirm the importance of H260 for biliverdin protonation. Further, we demonstrate that in the canonical bacteriophytochromes, the pK_a value of the phenol group of the Y263 is uncommonly low. This directly influences the protonation of the bilin molecule and likely the functional properties of the protein. Our study expands the understanding of the tight interplay between the nearby amino acids and bilin in the phytochrome family.     

Melanopsin and the Non‐visual Photochemistry in the Inner Retina of Vertebrates

Melanopsin (Opn4), a member of the G‐protein‐coupled receptor family, is a vitamin A‐based opsin in the vertebrate retina that has been shown to be involved in the synchronization of circadian rhythms, pupillary light reflexes, melatonin suppression and other light‐regulated tasks. In nonmammalian vertebrates there are two Opn4 genes, Opn4m and Opn4x, the mammalian and Xenopus orthologs respectively. Opn4x is only expressed in nonmammalian vertebrates including reptiles, fish and birds, while Opn4m is found in a subset of retinal ganglion cells (RGCs), the intrinsically photosensitive (ip) RGCs of the inner retina of both mammals and nonmammalian vertebrates. All opsins described utilize retinaldehyde as chromophore, photoisomerized from 11‐cis‐ to all‐trans‐retinal upon light exposure. Visual retinal photoreceptor cones and rods, responsible for day and night vision respectively, recycle retinoids through a process called the visual cycle that involves the retinal pigment epithelium or glial Müller cells. Although Opn4 has been characterized as a bistable photopigment, little is known about the mechanism/s involved in its chromophore regeneration. In this review, we will attempt to shed light on the visual cycle taking place in the inner retina and discuss the state of the art in the nonvisual photochemistry of vertebrates.     

The influence of water on the photochemical reaction cycle of proteorhodopsin at low and high pH

Dried samples were prepared from suspension of proteorhodopsin. With HCl and NaOH the pH of the samples was adjusted below and above the pKa of the proton acceptor Asp-97, which was established earlier to be 7.1. Both types of samples were photoactive, and exhibited a truncated photocycle, compared to that measured in suspension. The photocycle of the low pH sample had a K like red shifted intermediate, decaying through an energized PR' intermediate to the ground state protein. The high pH sample had a more complex photocycle in which beside of the red shifted intermediate an M like intermediate could be identified, having a deprotonated Schiff-base. This blue shifted intermediate decays through an intermediate we designated PR', which is spectrally identical to the unphotolysed ground state. The humidity and temperature dependence of the photocycle in both cases was studied to understand the role of water in the function of the proteorhodopsin. The effects measured on proteorhodopsin were very similar to that measured earlier on bacteriorhodopsin.     

Ruthenium(II)–Polyimine–Coumarin Light‐Harvesting Molecular Arrays: Design Rationale and Application for Triplet–Triplet‐Annihilation‐Based Upconversion

Ru^II–bis‐pyridine complexes typically absorb below 450 nm in the UV spectrum and their molar extinction coefficients are only moderate (ε<16 000 M ^?1 cm^?1). Thus, Ru^II–polyimine complexes that show intense visible‐light absorptions are of great interest. However, no effective light‐harvesting ruthenium(II)/organic chromophore arrays have been reported. Herein, we report the first visible‐light‐harvesting Ru^II–coumarin arrays, which absorb at 475 nm (ε up to 63 300 M ^?1 cm^?1, 4‐fold higher than typical Ru^II–polyimine complexes). The donor excited state in these arrays is efficiently converted into an acceptor excited state (i.e., efficient energy‐transfer) without losses in the phosphorescence quantum yield of the acceptor. Based on steady‐state and time‐resolved spectroscopy and DFT calculations, we proposed a general rule for the design of Ru^II–polypyridine–chromophore light‐harvesting arrays, which states that the ¹IL energy level of the ligand must be close to the respective energy level of the metal‐to‐ligand charge‐transfer (M LCT) states. Lower energy levels of ¹IL/³IL than the corresponding ¹M LCT/³M LCT states frustrate the cascade energy‐transfer process and, as a result, the harvested light energy cannot be efficiently transferred to the acceptor. We have also demonstrated that the light‐harvesting effect can be used to improve the upconversion quantum yield to 15.2 % (with 9,10‐diphenylanthracene as a triplet‐acceptor/annihilator), compared to the parent complex without the coumarin subunit, which showed an upconversion quantum yield of only 0.95 %.     

Engineered Functional Recovery of Microbial Rhodopsin Without Retinal‐Binding Lysine

Definition of rhodopsin is the retinal‐binding membrane protein with the Schiff base linkage at a lysine on the 7^th transmembrane helix. However, ~ 600 microbial rhodopsins lack retinal‐binding lysine at the corresponding position (Rh‐noK) among ~ 5500 known microbial rhodopsins, suggesting that Rh‐noK has each functional role without chromophore. Here, we report successful functional recovery of Rh‐noK. Two Rh‐noKs from bacteria were heterologously expressed in Escherichia coli, which exhibited no color. When retinal‐binding lysine was introduced, one of them gained visible color. Additional mutation of the Schiff base counterion further gained proton‐pumping activity. Successful engineered functional recovery such as visible color and proton‐pump activity suggests that the Rh‐noK protein forms a characteristic structure of microbial rhodopsins.     

Origins of the Intermediate Spectral Form in M100 Mutants of Photoactive Yellow Protein

Numerous single‐site mutants of photoactive yellow protein (PYP) from Halorhodospira halophila and as well as PYP homologs from other species exhibit a shoulder on the short wavelength side of the absorbance maximum in their dark‐adapted states. The structural basis for the occurrence of this shoulder, called the “intermediate spectral form,” has only been investigated in detail for the Y42F mutation. Here we explore the structural basis for occurrence of the intermediate spectral form in a M121E derivative of a circularly permuted H. halophila PYP (M121E‐cPYP). The M121 site in M121E‐cPYP corresponds to the M100 site in wild‐type H. halophila PYP. High‐resolution NMR measurements with a salt‐tolerant cryoprobe enabled identification of those residues directly affected by increasing concentrations of ammonium chloride, a salt that greatly enhances the fraction of the intermediate spectra form. Residues in the surface loop containing the M121E (M100E) mutation were found to be affected by ammonium chloride as well as a discrete set of residues that link this surface loop to the buried hydroxyl group of the chromophore via a hydrogen bond network. Localized changes in the conformational dynamics of a surface loop can thereby produce structural rearrangements near the buried hydroxyl group chromophore while leaving the large majority of residues in the protein unaffected.     

Dual Illumination Enhances Transformation of an Engineered Green‐to‐Red Photoconvertible Fluorescent Protein

The molecular mechanisms for the photoconversion of fluorescent proteins remain elusive owing to the challenges of monitoring chromophore structural dynamics during the light‐induced processes. We implemented time‐resolved electronic and stimulated Raman spectroscopies to reveal two hidden species of an engineered ancestral GFP‐like protein LEA, involving semi‐trapped protonated and trapped deprotonated chromophores en route to photoconversion in pH 7.9 buffer. A new dual‐illumination approach was examined, using 400 and 505 nm light simultaneously to achieve faster conversion and higher color contrast. Substitution of UV irradiation with visible light benefits bioimaging, while the spectral benchmark of a trapped chromophore with characteristic ring twisting and bridge‐H bending motions enables rational design of functional proteins. With the improved H‐bonding network and structural motions, the photoexcited chromophore could increase the photoswitching‐aided photoconversion while reducing trapped species.     

pH-dependent transitions in xanthorhodopsin

Xanthorhodopsin (XR), the light-driven proton pump of the halophilic eubacterium Salinibacter ruber, exhibits substantial homology to bacteriorhodopsin (BR) of archaea and proteorhodopsin (PR) of marine bacteria, but unlike them contains a light-harvesting carotenoid antenna, salinixanthin, as well as retinal. We report here the pH-dependent properties of XR. The pKa of the retinal Schiff base is as high as in BR, i.e. > or =12.4. Deprotonation of the Schiff base and the ensuing alkaline denaturation cause large changes in the absorption bands of the carotenoid antenna, which lose intensity and become broader, making the spectrum similar to that of salinixanthin not bound to XR. A small redshift of the retinal chromophore band and increase of its extinction, as well as the pH-dependent amplitude of the M intermediate indicate that in detergent-solubilized XR the pKa of the Schiff base counterion and proton acceptor is about 6 (compared to 2.6 in BR, and 7.5 in PR). The protonation of the counterion is accompanied by a small blueshift of the carotenoid absorption bands. The pigment is stable in the dark upon acidification to pH 2. At pH < 2 a transition to a blueshifted species absorbing around 440 nm occurs, accompanied by loss of resolution of the carotenoid absorption bands. At pH < 3 illumination of XR with continuous light causes accumulation of long-lived photoproduct(s) with an absorption maximum around 400 nm. The photocycle of XR was examined between pH 4 and 10 in solubilized samples. The pH dependence of recovery of the initial state slows at both acid and alkaline pH, with pKas of 6.0 and 9.3. The decrease in the rates with pKa 6.0 is apparently caused by protonation of the counterion and proton acceptor, and that at high pH reflects the pKa of the internal proton donor, Glu94, at the times in the photocycle when this group equilibrates with the bulk.     

Electron density deformations provide new insights into the spectral shift of rhodopsins

Spectral shifts of rhodopsin, which are related to variations of the electron distribution in 11‐cis‐retinal, are investigated here using the method of deformed atoms in molecules. We found that systems carrying the M207R and S186W mutations display large perturbations of the π‐conjugated system with respect to wild‐type rhodopsins. These changes agree with the predicted behavior of the bond length alternation (BLA) and the blue shifts of vertical excitation energies of these systems. The effect of the planarity of the central and Schiff‐base regions of retinal chain on the electronic structure of the chromophore is also investigated. By establishing nonlinear polynomial relations between BLA, chain distortions, and vertical excitation energies, we are also able to provide a semiquantitative approach for the understanding of the mechanisms regulating spectral shifts in rhodopsin and its mutants. © 2013 Wiley Periodicals, Inc.     

Studies on pyrylretinal analogues of bacteriorhodopsin

The retinal analogues 3-methyl-5-(1-pyryl)-2E,4E-pentadienal (1) and 3,7-dimethyl-9-(1-pyryl)-2E,4E,6E,8E-nonatetr aenal (2), which contain the tetra aromatic pyryl system, have been synthesized and characterized in order to examine the effect of the extended ring system on the binding capabilities and the function of bacteriorhodopsin (bR). The two bR mutants, E194Q and E204Q, known to have distinct proton-pumping patterns, were also examined so that the effect of the bulky ring system on the proton-pumping mechanism could be studied. Both retinals formed pigments with all three bacterioopsins, and these pigments were found to have absorption maxima in the range 498-516 nm. All the analogue pigments showed activity as proton pumps. The pigment formed from wild-type apoprotein bR with 1 (with the shortened polyene side chain) showed an M intermediate at 400 nm and exhibited fast proton release followed by proton uptake. Extending the polyene side chain to the length identical with retinal, analogue 2 with wild-type apoprotein gave a pigment that shows M and O intermediates at 435 nm and 650 nm, respectively. This pigment shows both fast and slow proton release at pH 7, suggesting that the pKa of the proton release group (in the M-state) is higher in this pigment compared to native bR. Hydrogen azide ions were found to accelerate the rise and decay of the O intermediate at neutral pH in pyryl 2 pigment. The pigments formed between 2 and E194Q and E204Q showed proton-pumping behavior similar to pigments formed with the native retinal, suggesting that the size of the chromophore ring does not alter the protein conformation at these sites.     

Characterizing the Structure and Photocycle of PR 2D Crystals with CD and FTIR Spectroscopy†

We present here a study on proteorhodopsin (PR) 2D crystals with analytical ultracentrifugation, circular dichroism and Fourier transform infrared (FTIR) spectroscopy. The aim of our experiments was to test the activity of 2D crystal sample preparations and to gain further insight in PR structure, stability and function with these techniques. Our results demonstrate higher stability compared to detergent‐solubilized or reconstituted samples. For different pH values, low pH 2D crystals tend to form bigger aggregates and are less stable than at basic pH. The pH 9 sample shows a sharp phase transition during heat denaturation and there is also evidence for protein–protein interaction due to the close proximity of the proteins in the 2D crystals. In the FTIR measurements at cryogenic temperatures (77 K), we characterized the first step in the PR photocycle. At pH 9, the K intermediate could be observed and the samples showed no orientation effects. At pH 5, we could trap the K/L intermediate, characterized by its negative IR signal at 1741 cm^?1. In rapid‐scan FTIR experiments, we could also identify the M intermediate of the photocycle at basic pH. We conclude that the PR 2D crystals exhibit a fully functional photocycle and are therefore well suited for further studies on the proton transport mechanism of PR.     

A Tin Oxide Transparent Electrode Provides the Means for Rapid Time‐resolved pH Measurements: Application to Photoinduced Proton Transfer of Bacteriorhodopsin and Proteorhodopsin†

An electrochemical cell was previously reported in which bacteriorhodopsin (BR, purple membrane) was adsorbed on the surface of a transparent SnO₂ electrode, and illumination resulted in potential or current changes (Koyama et al., Science 265:762–765, 1994; Robertson and Lukashev, Biophys. J. 68:1507–1517, 1995; Koyama et al., Photochem. Photobiol. 68:400–406, 1998). In this paper, we concluded that pH changes caused by proton transfer by the deposited BR or proteorhodopsin (PR) films lead to the flash‐induced potential change in the SnO₂ electrode. Thus, the signals originate from BR and PR acting as light‐driven proton pumps. This conclusion was drawn from the following observations. (1) The relation between the potential of a bare electrode and pH is linear for a wide pH range. (2) The flash‐induced potential changes decrease with an increase in the buffer concentration. (3) The action spectrum of PR agrees well with the absorption spectrum. (4) The present electrode can monitor the pH change in the time range from 10 ms to several hundred milliseconds, as deduced by comparing the SnO₂ signal with the signals of pH‐sensitive dyes. Using this electrode system, flash‐induced proton transfer by BR was measured for a wide pH range from 2 to 10. From these data, we reconfirmed various pK_a values reported previously, indicating that the present method can give the correct pK_a values. This is the first report to estimate these pK_a values directly from the proton transfer. We then applied this method to flash‐induced proton transfer of PR. We observed proton uptake followed by release for the pH range from 4 to 9.5, and in other pH ranges, proton release followed by uptake was observed.