Retinal Isomerization Research Articles

Retinal isomerization in bacteriorhodopsin captured by a femtosecond X-ray laser

Retinal isomerization in bacteriorhodopsin captured by a femtosecond X-ray laser

Retinal isomerization in bacteriorhodopsin captured by a femtosecond x-ray laser.

Ultrafast isomerization of retinal is the primary step in photoresponsive biological functions including vision in humans and ion transport across bacterial membranes. We used an x-ray laser to study the subpicosecond structural dynamics of retinal isomerization in the light-driven proton pump bacteriorhodopsin. A series of structural snapshots with near-atomic spatial resolution and temporal resolution in the femtosecond regime show how the excited all-trans retinal samples conformational states within the protein binding pocket before passing through a twisted geometry and emerging in the 13-cis conformation. Our findings suggest ultrafast collective motions of aspartic acid residues and functional water molecules in the proximity of the retinal Schiff base as a key facet of this stereoselective and efficient photochemical reaction.

A distinct abundant group of microbial rhodopsins discovered using functional metagenomics.

Many organisms capture or sense sunlight using rhodopsin pigments1,2, which are integral membrane proteins that bind retinal chromophores. Rhodopsins comprise two distinct protein families 1 , type-1 (microbial rhodopsins) and type-2 (animal rhodopsins). The two families share similar topologies and contain seven transmembrane helices that form a pocket in which retinal is linked covalently as a protonated Schiff base to a lysine at the seventh transmembrane helix2,3. Type-1 and type-2 rhodopsins show little or no sequence similarity to each other, as a consequence of extensive divergence from a common ancestor or convergent evolution of similar structures 1 . Here we report a previously unknown and diverse family of rhodopsins-which we term the heliorhodopsins-that we identified using functional metagenomics and that are distantly related to type-1 rhodopsins. Heliorhodopsins are embedded in the membrane with their N termini facing the cell cytoplasm, an orientation that is opposite to that of type-1 or type-2 rhodopsins. Heliorhodopsins show photocycles that are longer than one second, which is suggestive of light-sensory activity. Heliorhodopsin photocycles accompany retinal isomerization and proton transfer, as in type-1 and type-2 rhodopsins, but protons are never released from the protein, even transiently. Heliorhodopsins are abundant and distributed globally; we detected them in Archaea, Bacteria, Eukarya and their viruses. Our findings reveal a previously unknown family of light-sensing rhodopsins that are widespread in the microbial world.

Spectroscopic Study of Proton-Transfer Mechanism of Inward Proton-Pump Rhodopsin, Parvularcula oceani Xenorhodopsin.

Parvularcula oceani xenorhodopsin is the first light-driven inward proton pump. To understand the mechanism of inward proton transport, comprehensive transient absorption spectroscopy was conducted. Ultrafast pump-probe spectroscopy revealed that the isomerization time of retinal is 1.2 ps, which is considerably slower than those of other microbial rhodopsins (180-770 fs). Following the production of J, the K intermediate was formed at 4 ps. Proton transfer occurred on a slower timescale. Proton release and uptake were observed on the L/M-to-M and M decay, respectively, by monitoring transient absorption changes of pH-indicating dye, pyranine. Although a proton was released from Asp216 into the cytoplasmic medium, no proton-donating residue was identified on the extracellular side in mutation experiments. We revealed that a branched retinal isomerization (from 13-cis-15-anti to 13-cis-15-syn and all-trans-15-anti) occurred simultaneously with proton uptake. Furthermore, although the proton release showed a large kinetic isotope effect (KIE), the KIE of proton uptake was negligible. These results suggest that retinal isomerization is the rate-limiting process in proton uptake and that the regulation of p Ka of the retinal Schiff base by thermal isomerization enables the uptake from extracellular medium. This proton uptake mechanism differs from that of the outward proton pump with an internal proton donor and is important for understanding how the direction of ion transport by membrane proteins is determined.

Retinal isomerization and water-pore formation in channelrhodopsin-2

Channelrhodopsin-2 (ChR2) is a light-sensitive ion channel widely used in optogenetics. Photoactivation triggers a trans-to-cis isomerization of a covalently bound retinal. Ensuing conformational changes open a cation-selective channel. We explore the structural dynamics in the early photocycle leading to channel opening by classical (MM) and quantum mechanical (QM) molecular simulations. With QM/MM simulations, we generated a protein-adapted force field for the retinal chromophore, which we validated against absorption spectra. In a 4-µs MM simulation of a dark-adapted ChR2 dimer, water entered the vestibules of the closed channel. Retinal all-trans to 13-cis isomerization, simulated with metadynamics, triggered a major restructuring of the charge cluster forming the channel gate. On a microsecond time scale, water penetrated the gate to form a membrane-spanning preopen pore between helices H1, H2, H3, and H7. This influx of water into an ion-impermeable preopen pore is consistent with time-resolved infrared spectroscopy and electrophysiology experiments. In the retinal 13-cis state, D253 emerged as the proton acceptor of the Schiff base. Upon proton transfer from the Schiff base to D253, modeled by QM/MM simulations, we obtained an early-M/P2390-like intermediate. Rapid rotation of the unprotonated Schiff base toward the cytosolic side effectively prevents its reprotonation from the extracellular side. From MM and QM simulations, we gained detailed insight into the mechanism of ChR2 photoactivation and early events in pore formation. By rearranging the network of charges and hydrogen bonds forming the gate, water emerges as a key player in light-driven ChR2 channel opening.

Catalysis of Ground State cis[Formula: see text] trans Isomerization of Bacteriorhodopsin's Retinal Chromophore by a Hydrogen-Bond Network.

For the photocycle of the membrane protein bacteriorhodopsin to proceed efficiently, the thermal 13-cis to all-trans back-isomerization of the retinal chromophore must return the protein to its resting state on a time-scale of milliseconds. Here, we report on quantum mechanical/molecular mechanical energy calculations examining the structural and energetic determinants of the retinal cis-trans isomerization in the protein environment. The results suggest that a hydrogen-bonded network consisting of the retinal Schiff base, active site amino acid residues, and water molecules can stabilize the twisted retinal, thus reducing the intrinsic energy cost of the cis-trans thermal isomerization barrier.

Human Blue Cone Opsin Regeneration Involves Secondary Retinal Binding with Analog Specificity

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigments.

Interfering resonance as an underlying mechanism in the adaptive feedback control of radiationless transitions: Retinal isomerization.

Control of molecular processes via adaptive feedback often yields highly structured laser pulses that have eluded physical explanation. By contrast, coherent control approaches propose physically transparent mechanisms but are not readily visible in experimental results. Here, an analysis of a condensed phase adaptive feedback control experiment on retinal isomerization shows that it manifests a quantum interference based coherent control mechanism: control via interfering resonances. The result promises deep insight into the physical basis for the adaptive feedback control of a broad class of bound state processes.

Photocatalytic E → Z Isomerization of Polarized Alkenes Inspired by the Visual Cycle: Mechanistic Dichotomy and Origin of Selectivity.

Iteratively executed with exquisite spatial and temporal control, the selective isomerization of polarized alkenes underpins a plethora of complex biological processes ranging from natural product biosynthesis through to the mammalian visual cycle. However, nature's proficiency conceals the inherent difficulties in replicating this contra-thermodynamic transformation in the laboratory. Recently, we disclosed the first highly Z-selective isomerization of polarized alkenes, employing the cinnamoyl chromophore as a retinal surrogate under UV-irradiation (402 nm) with (-)-riboflavin (vitamin B2) as an inexpensive, organic photocatalyst (J. Am. Chem. Soc. 2015, 137, 11254-11257). This study was inspired by the propensity of crystalline (-)-riboflavin in the eyes of vertebrates to invert the intrinsic directionality of retinal isomerization. Herein, we extend this methodology to include a bioinspired, catalytic E → Z isomerization of α,β-unsaturated nitriles, thereby mimicking the intermediate Opsin-derived, protonated Schiff base in the visual cycle with simple polarized alkenes. Replacement of the iminium motif by a cyano group is well tolerated and gives an additional degree of versatility for postisomerization functionalization. Broad substrate scope is demonstrated (up to 99:1 Z:E) together with evidence of mechanistic dichotomy via both singlet and triplet energy transfer mechanisms. Kinetic studies, temperature dependent photostationary state correlations and investigation of substituent-based electronic perturbation of the alkene identified polarization combined with increased Z-isomer activation barriers as the selectivity governing factors in catalysis. This investigation demonstrates the importance of internal structural preorganization on photostationary composition and explicates the augmented Z-selectivity upon hydrogen-alkyl exchange at the β-position of the alkene.

FTIR Analysis of a Light-driven Inward Proton-pumping Rhodopsin at 77 K.

Parvularcula oceani xenorhodopsin (PoXeR) is a light-driven inward proton pump that was discovered from deep ocean marine bacteria. PoXeR is categorized into the same family of Anabaena sensory rhodopsin (ASR) that functions as a photochromic sensor. In this study, we applied light-induced difference Fourier-transform infrared (FTIR) spectroscopy to PoXeR at 77 K, and the obtained spectra were compared with those of ASR. The structure and structural changes in the primary processes of PoXeR are common to all microbial rhodopsins. The red-shifted K formation (PoXeRK ) was accompanied by retinal photoisomerization from the all-trans to 13-cis form resulting in a distorted structure of the retinal. The observed hydrogen out-of-plane (HOOP) vibrations were sensitive to H/D exchange, indicating that the chromophore distortion by retinal isomerization is located near the Schiff base region in PoXeR. The hydrogen-bonding strength of the protonated Schiff base is similar between PoXeR and ASR, whose acceptor is presumably a water molecule. Unlike ASR, however, PoXeR contains strongly hydrogen-bonded water (O-D stretch at 2277 cm-1 in D2 O), which is also the case for outward proton pumps. The detailed structure, structural changes upon retinal photoisomerization, as well as functional correlation in PoXeR are discussed based on the present FTIR study.

Modulation of Energy Conversion Through Manipulation of the Retinal Thermal Equilibrium by an Aromatic Residue in the Seven-Transmembrane Receptor Bacteriorhodopsin

Light-driven proton pumps, a number of the microbial rhodopsin family seven-transmembrane receptors, distribute widely among Archaea, Eubacteria, and Eukaryota for harvesting and converting light energy in a wider spectral range. The common feature of those proteins is that the retinal chromophore maintains a cis-trans thermal equilibrium in the dark-adapted state. Absorption of a photon causes photo isomerization of the chromophore from the all-trans to the 13-cis, 15-anti configuration and triggers a series of structural rearrangements in the protein that initiates the vectorial translocation of a proton out of the cell. However, how the cis-trans thermal equilibrium impacts on the proton translocation photocycle, and further impacts on the ATP formation is still poorly understood. Here, as a novel study to address this challenge, Y185F mutant, an aromatic residue at the retinal binding pocket of bacteriorhodopsin, is identified to shift the cis-trans isomerization thermal equilibrium to a cis dominated state and causes a weakening of the M state, loss of the O state and elongation of the proton pumping cycle, and further decreases the ATP formation rate. Different interactions of Tyrosine 185 with the retinal chromophore are observed in the dark-adapted state and the M state, indicating that Tyrosine 185 may serve as a rotamer switch to maintain the proton translocation kinetics and further the energy conversion in bacteriorhodopsin.Reference: Ding, X., et al. 2016. Mediation mechanism of tyrosine 185 on the retinal isomerization equilibrium and the proton release channel in the seven-transmembrane receptor bacteriorhodopsin. Biochim. Biophys. Acta 1857:1786-1795.

All-trans retinal levels and formation of lipofuscin precursors after bleaching in rod photoreceptors from wild type and Abca4-/- mice

The accumulation of lipofuscin in the cells of the retinal pigment epithelium (RPE) is thought to play an important role in the development and progression of degenerative diseases of the retina. The bulk of RPE lipofuscin originates in reactions of the rhodopsin chromophore, retinal, with components of the photoreceptor outer segment. The 11-cis retinal isomer is generated in the RPE and supplied to rod photoreceptor outer segments where it is incorporated as the chromophore of rhodopsin. It is photoisomerized during light detection to all-trans and subsequently released by photoactivated rhodopsin as all-trans retinal, which is removed through reduction to all-trans retinol in a reaction requiring metabolic input in the form of NADPH. Both 11-cis and all-trans retinal can form lipofuscin precursor fluorophores in rod photoreceptor outer segments. Increased accumulation of lipofuscin has been suggested to result from excess formation of lipofuscin precursors due to buildup of all-trans retinal released by light exposure. In connection with this suggestion, the Abca4 transporter protein, an outer segment protein defects in which result in recessive Stargardt disease, has been proposed to promote the removal of all-trans retinal by facilitating its availability for reduction. To examine this possibility, we have measured the outer segment levels of all-trans retinal, all-trans retinol, and of lipofuscin precursors after bleaching by imaging the fluorescence of single rod photoreceptors isolated from wild type and Abca4-/- mice. We found that all-trans retinol and all-trans retinal levels increased after bleaching in both wild type and Abca4-/- rods. At all times after bleaching, there was no significant difference in all-trans retinal levels between the two strains. All-trans retinol levels were not significantly different between the two strains at early times, but were lower in Abca4-/- rods at times longer than 20 min after bleaching. Bleaching in the presence of lower metabolic substrate concentrations resulted in higher all-trans retinal levels and increased formation of lipofuscin precursors in both wild type and Abca4-/- rods. The results show that conditions that result in buildup of all-trans retinal levels result in increased generation of lipofuscin precursors in both wild type and Abca4-/- rods. The results are consistent with the proposal that Abca4 facilitates the reduction of all-trans retinal to retinol; absence of Abca4 however does not appear to be associated with higher all-trans retinal levels compared to wild type.

The Study and Application of Photoreceptive Membrane Protein, Rhodopsin

Abstract Microbial rhodopsins are photoreceptive seven-transmembrane proteins diversely found in unicellular microorganisms. The chromophore of microbial rhodopsin is all-trans retinal. The retinal isomerizes to 13-cis form upon light-absorption and it triggers the expression of various biological functions by light. We studied the mechanism of various types of microbial rhodopsins (H+ pump, Cl− pump and sensors) by physicochemical spectroscopic methods. On the basis of these studies, we found the importance of the residues on the third helix (helix C) for the function of microbial rhodopsin, and it led to the discovery of a new functional class of microbial rhodopsin, light-driven outward Na+ pump. The mechanism of Na+ pump rhodopsin was studied by several spectroscopic methods and X-ray crystallography. These studies provided new insights very informative for the development of novel functional artificial rhodopsins (K+ and Cs+ pumps). These new natural and artificial rhodopsins are expected to have high potential for applications such as optogenetics.

Mediation mechanism of tyrosine 185 on the retinal isomerization equilibrium and the proton release channel in the seven-transmembrane receptor bacteriorhodopsin

Electrostatic coupling leading to conformational changes in proteins is challenging to demonstrate directly, it requires that both the local, discrete electronic details and dynamic information relevant to the functional descriptions are probed. Here, as a novel study to address this challenge, the roles of an aromatic residue in influencing the functional conformational changes of a membrane receptor in its natural membrane environment are reported. Previously intractable discrete electronic details have been obtained using 2D solid-state NMR of specifically labelled receptor, reinforced with molecular dynamic simulations, mutational analysis and functional assays, supported by and compared with rigid-atom crystal structural models. Hydrogen bonding and hydrophobic interactions are identified as the mechanistic origin for direct electromechanical coupling to the dynamics of conformational changes within the receptor.

Unraveling the mechanism of proton translocation in the extracellular half-channel of bacteriorhodopsin.

Bacteriorhodopsin, a light activated protein that creates a proton gradient in halobacteria, has long served as a simple model of proton pumps. Within bacteriorhodopsin, several key sites undergo protonation changes during the photocycle, moving protons from the higher pH cytoplasm to the lower pH extracellular side. The mechanism underlying the long-range proton translocation between the central (the retinal Schiff base SB216, D85, and D212) and exit clusters (E194 and E204) remains elusive. To obtain a dynamic view of the key factors controlling proton translocation, a systematic study using molecular dynamics simulation was performed for eight bacteriorhodopsin models varying in retinal isomer and protonation states of the SB216, D85, D212, and E204. The side-chain orientation of R82 is determined primarily by the protonation states of the residues in the EC. The side-chain reorientation of R82 modulates the hydrogen-bond network and consequently possible pathways of proton transfer. Quantum mechanical intrinsic reaction coordinate calculations of proton-transfer in the methyl guanidinium-hydronium-hydroxide model system show that proton transfer via a guanidinium group requires an initial geometry permitting proton donation and acceptance by the same amine. In all the bacteriorhodopsin models, R82 can form proton wires with both the CC and the EC connected by the same amine. Alternatively, rare proton wires for proton transfer from the CC to the EC without involving R82 were found in an O' state where the proton on D85 is transferred to D212.

Proteorhodopsin Activation Captured by Molecular Dynamics Simulations

Proteorhodopsin, a light-activated proton pump, is widely distributed among marine bacteria and has promising applications in areas such as optogenetics. Proteorhodopsin activation and proton pumping is triggered by the photoisomerization of the covalently bond ligand retinal. Similar to other retinal proteins, protein structural changes along with the rearrangement of internal water molecules are expected to take place during the proton pumping photocycle. However, little experimental information regarding the structures of the photointermediates is known and the underpinning mechanism of proton pumping remains largely elusive. We have conducted molecular dynamics simulations to investigate the initial stages of proteorhodopsin activation at the atomistic level of detail. A series of µs-long simulations of the K state of proteorhodopsin were performed with in silico isomerization of all-trans to 13-cis retinal and were compared with simulations of the inactive state starting from the X-ray crystal structure of blue proteorhodopsin. Each of the amino acid residues known to be implicated in proton pumping were found to be well-hydrated. Our simulations revealed formation of a hydrogen bond between the color tuning switch Q105 and the protonated Schiff base after retinal isomerization, supporting a previous FTIR difference spectrum study of K state structural changes [1]. Another significant phenomenon in the K state was that an increase in water flux occurred connecting the proton release group, a conserved arginine residue, and the retinal binding pocket. Transient water channel formation in the interior of the protein was also discovered, potentially facilitating distal proton transfer among active sites. Altogether these results provide suggestions for the molecular mechanism of protonation switches and proton relay which are key to our understanding of proton pumping in PR and activation of retinal proteins in general. [1] Amdsden, J.J. (2008) Biochemistry 47:11490.

Function of Tyr185 in Stabilizing the Isomerization Equilibrium of the Retinal Chromophore in the Bacteriorhodopsin Ground State

Bacteriorhodopsin (bR), a member of the microbial rhodopsin family's seven trans-membrane receptors, functions as a light-driven proton pump for light energy capture and conversion in Halobacterium Salinarum. In the dark-adapted (DA) state, bR contains two thermally interconvertible retinal isomers: 13-cis, 15-syn retinal (bRcis), and all-trans, 15-anti retinal (bRtrans) with a molar ratio of 6:4 in the purple membrane. Absorption of a photon causes photoisomerization of the chromophore from the all-trans to the 13-cis, 15-anti configuration and triggers a series of structural rearrangements of the protein that initiates a vectorial translocation of a proton out of the cell.The function of Tyrosine 185 (Tyr185) in bR photoreaction mechanism has long been a topic of discussion, and been suggested to play an important role in stabilizing the pentagonal hydrogen-bonded network on the extracellular side of the protonated Schiff base during the photoreaction. However, little is to know on coupling of Tyr185 with the protonated Schiff base with respect to the thermal isomerization of the retinal chromophore at the bR ground state. Heteronuclear single molecular manipulation, combined with homonuclear correlation experiments, UV spectroscopy, light-induced transient absorption change measurements, molecular dynamic simulations and mutagenesis, has been established as a powerful tool for providing insight into the conformation and coupling of Tyr185 with the retinal chromophore at the bR ground state in the purple membrane. Our results show that the retinal chromophore in 13-cis and 15-syn configurations has more accumulated charge and, is more twisted and rigid than that in the all-trans configuration. Moreover, the cis-trans thermal isomerization equilibrium with the molar ratio of 6:4 at the ground state is stabilized by coupling with Tyr185.

Light-Dark Adaptation of Channelrhodopsin Involves Photoconversion between the all-trans and 13-cis Retinal Isomers.

Channelrhodopsins (ChR) are light-gated ion channels of green algae that are widely used to probe the function of neuronal cells with light. Most ChRs show a substantial reduction in photocurrents during illumination, a process named "light adaptation". The main objective of this spectroscopic study was to elucidate the molecular processes associated with light-dark adaptation. Here we show by liquid and solid-state nuclear magnetic resonance spectroscopy that the retinal chromophore of fully dark-adapted ChR is exclusively in an all-trans configuration. Resonance Raman (RR) spectroscopy, however, revealed that already low light intensities establish a photostationary equilibrium between all-trans,15-anti and 13-cis,15-syn configurations at a ratio of 3:1. The underlying photoreactions involve simultaneous isomerization of the C(13)═C(14) and C(15)═N bonds. Both isomers of this DAapp state may run through photoinduced reaction cycles initiated by photoisomerization of only the C(13)═C(14) bond. RR spectroscopic experiments further demonstrated that photoinduced conversion of the apparent dark-adapted (DAapp) state to the photocycle intermediates P500 and P390 is distinctly more efficient for the all-trans isomer than for the 13-cis isomer, possibly because of different chromophore-water interactions. Our data demonstrating two complementary photocycles of the DAapp isomers are fully consistent with the existence of two conducting states that vary in quantitative relation during light-dark adaptation, as suggested previously by electrical measurements.

Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy

Channelrhodopsin-2 from Chlamydomonas reinhardtii is a light-gated ion channel. Over recent years, this ion channel has attracted considerable interest because of its unparalleled role in optogenetic applications. However, despite considerable efforts, an understanding of how molecular events during the photocycle, including the retinal trans-cis isomerization and the deprotonation/reprotonation of the Schiff base, are coupled to the channel-opening mechanism remains elusive. To elucidate this question, changes of conformation and configuration of several photocycle and conducting/nonconducting states need to be determined at atomic resolution. Here, we show that such data can be obtained by solid-state NMR enhanced by dynamic nuclear polarization applied to (15)N-labeled channelrhodopsin-2 carrying 14,15-(13)C2 retinal reconstituted into lipid bilayers. In its dark state, a pure all-trans retinal conformation with a stretched C14-C15 bond and a significant out-of-plane twist of the H-C14-C15-H dihedral angle could be observed. Using a combination of illumination, freezing, and thermal relaxation procedures, a number of intermediate states was generated and analyzed by DNP-enhanced solid-state NMR. Three distinct intermediates could be analyzed with high structural resolution: the early [Formula: see text] K-like state, the slowly decaying late intermediate [Formula: see text], and a third intermediate populated only under continuous illumination conditions. Our data provide novel insight into the photoactive site of channelrhodopsin-2 during the photocycle. They further show that DNP-enhanced solid-state NMR fills the gap for challenging membrane proteins between functional studies and X-ray-based structure analysis, which is required for resolving molecular mechanisms.

Time-resolved infrared spectroscopic techniques as applied to channelrhodopsin.

Among optogenetic tools, channelrhodopsins, the light gated ion channels of the plasma membrane from green algae, play the most important role. Properties like channel selectivity, timing parameters or color can be influenced by the exchange of selected amino acids. Although widely used, in the field of neurosciences for example, there is still little known about their photocycles and the mechanism of ion channel gating and conductance. One of the preferred methods for these studies is infrared spectroscopy since it allows observation of proteins and their function at a molecular level and in near-native environment. The absorption of a photon in channelrhodopsin leads to retinal isomerization within femtoseconds, the conductive states are reached in the microsecond time scale and the return into the fully dark-adapted state may take more than minutes. To be able to cover all these time regimes, a range of different spectroscopical approaches are necessary. This mini-review focuses on time-resolved applications of the infrared technique to study channelrhodopsins and other light triggered proteins. We will discuss the approaches with respect to their suitability to the investigation of channelrhodopsin and related proteins.