Retinal Isomerization Research Articles

Theoretical study on the structure, spectroscopic, and current–voltage behavior of 11-Cis and Trans retinal isomers in rhodopsin

In this study, the electronic transport properties of 11-Cis and Trans retinal, components of rhodopsin, were investigated as optical molecular switches using the nonequilibrium Green’s function (NEGF) formalism combined with first-principles density functional theory (DFT). These isomers, which can be reversibly converted into each other, were examined in detail. The structural and spectroscopic properties, including infrared (IR), Raman, nuclear magnetic resonance (NMR), and ultraviolet (UV) spectra, were analyzed using the hybrid B3LYP/6–311 + + G** level of theory. Complete vibrational assignments were performed for both forms utilizing the scaled quantum mechanical force field (SQMFF) methodology. To evaluate the conductivity of these molecules, we utilized current–voltage (I-V) characteristics, transmission spectra, molecular projected self-consistent Hamiltonian (MPSH), HOMO–LUMO gap, and second-order interaction energies (E2). The trendline extrapolation of the current–voltage plots confirmed our findings. We investigated the effect of different electrodes (Ag, Au, Pt) and various connection sites (hollow, top, bridge) on conductivity. The Ag electrode with the hollow site exhibited the highest efficiency. Our results indicate that the Cis form has higher conductivity than the Trans form.

Uncommon opsin’s retinal isomer is involved in mammalian sperm thermotaxis

In recent years it became apparent that, in mammals, rhodopsin and other opsins, known to act as photosensors in the visual system, are also present in spermatozoa, where they function as highly sensitive thermosensors for thermotaxis. The intriguing question how a well-conserved protein functions as a photosensor in one type of cells and as a thermosensor in another type of cells is unresolved. Since the moiety that confers photosensitivity on opsins is the chromophore retinal, we examined whether retinal is substituted in spermatozoa with a thermosensitive molecule. We found by both functional assays and mass spectrometry that retinal is present in spermatozoa and required for thermotaxis. Thus, starvation of mice for vitamin A (a precursor of retinal) resulted in loss of sperm thermotaxis, without affecting motility and the physiological state of the spermatozoa. Thermotaxis was restored after replenishment of vitamin A. Using reversed-phase ultra-performance liquid chromatography mass spectrometry, we detected the presence of retinal in extracts of mouse and human spermatozoa. By employing UltraPerformance convergence chromatography, we identified a unique retinal isomer in the sperm extracts—tri-cis retinal, different from the photosensitive 11-cis isomer in the visual system. The facts (a) that opsins are thermosensors for sperm thermotaxis, (b) that retinal is essential for thermotaxis, and (c) that tri-cis retinal isomer uniquely resides in spermatozoa and is relatively thermally unstable, suggest that tri-cis retinal is involved in the thermosensing activity of spermatozoa.

Assessing the Role of R120 in the Gating of CrChR2 by Time-Resolved Spectroscopy from Femtoseconds to Seconds.

The light-gated ion channel channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) is the most frequently used optogenetic tool in neurosciences. However, the precise molecular mechanism of the channel opening and the correlation among retinal isomerization, the photocycle, and the channel activity of the protein are missing. Here, we present electrophysiological and spectroscopic investigations on the R120H variant of CrChR2. R120 is a key residue in an extended network linking the retinal chromophore to several gates of the ion channel. We show that despite the deficient channel activity, the photocycle of the variant is intact. In a comparative study for R120H and the wild type, we resolve the vibrational changes in the spectral range of the retinal and amide I bands across the time range from femtoseconds to seconds. Analysis of the amide I mode reveals a significant impairment of the ultrafast protein response after retinal excitation. We conclude that channel opening in CrChR2 is prepared immediately after retinal excitation. Additionally, chromophore isomerization is essential for both photocycle and channel activities, although both processes can occur independently.

Effects of the Unique Chromophore-Protein Interactions on the Primary Photoreaction of Schizorhodopsin.

Schizorhodopsin (SzR) is a newly discovered microbial rhodopsin subfamily, functioning as an unusual inward-proton (H+) pump upon absorbing light. Two major protein structural differences around the chromophore have been found, resulting in unique chromophore-protein interactions that may be responsible for its unusual function. Therefore, it is important to elucidate how such a difference affects the primary photoreaction dynamics. We study the primary dynamics of SzR and its C75S mutant by femtosecond time-resolved absorption (TA) spectroscopy. The obtained TA data revealed that the photoisomerization in SzR proceeds more slowly and less efficiently than typical outward H+-pumping rhodopsins and that it further slows in the C75S mutant. We performed impulsive stimulated Raman measurements to clarify the effect of the cysteine residue on the retinal chromophore and found that interactions with Cys75 flatten the retinal chromophore of wild-type SzR. We discuss the effect of the unique chromophore-cysteine interaction on the retinal isomerization dynamics and structure of SzR.

Detection of bacteriorhodopsin trimeric rotation at thermal phase transitions of purple membrane in suspension

Bacteriorhodopsin (bR) of purple membrane (PM) is a retinal protein that forms aggregates in the form of trimers constituting, together with archaeal lipids, the crystalline structure of PM. The rotary motion of bR inside PM may be pertinent in understanding the essence of the crystalline lattice. An attempt has been made to determine the rotation of bR trimers which has been found to be detected solely at thermal phase transitions of PM, namely lipid, crystalline lattice and protein melting phase transitions. The temperature dependences of dielectric versus electronic absorption spectra of bR have been determined. The results suggest that the rotation of bR trimers, together with concomitant bending of PM, are most likely brought by structural changes in bR which might be driven by retinal isomerization and mediated by lipid. The rupturing of the lipid-protein contact might consequently lead to rotation of trimers associated with bending, curling or vesicle formation of PM. So the retinal reorientation may underlie the concomitant rotation of trimers. Most importantly, rotation of trimers might play a role, in terms of the essence of the crystalline lattice, in the functional activity of bR and may serve physiological relevance.

Natural selection of carotenoid binding in Gloeobacter rhodopsin

Energy transfer from the 4-ketocarotenoid antenna (salinixanthin) and echinenone to xanthorhodopsin has been confirmed in Salinibacter ruber and Gloeobacter violaceus, respectively. Hundreds of carotenoids and numerous rhodopsin homologs are spreading across the life domain. Here, we found that ketocarotenoid binds to Gloeobacter rhodopsin (GR), suggesting an increased likelihood of the existence of the carotenoid-rhodopsin complex. Additionally, the recruitment of the secondary chromophore antenna is precisely controlled by rhodopsin. Therefore, comparing the binding of multiple carotenoids is of interest to GR. Particular attention was paid to the structural interaction of ligands within the protein binding pocket. While different degrees of carotenoid association are evident in the GR-carotenoid experimental data and molecular docking results, the retinal isomerization dynamics defined the relative orientation of the β-ionone ring, and structural changes in the protein acted as modest barriers for the best carotenoid for GR. Notably, the selection of carotenoid binding can be summarized in three points: (1) structural matching of a ligand molecule with the binding pocket; (2) strong binding affinity and rapid binding kinetics; (3) dynamic flexibility that is versatile to rhodopsin activation. The selected carotenoids showed significant enhancement in light-driven proton pumping as extra light-harvesting antennae and provided intrinsic protection from stress.

Ultrafast structural changes direct the first molecular events of retinal isomerisation and rhodopsin activation.

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs). A photon is absorbed by the 11-cis retinal chromophore of rhodopsin which isomerises within 200 femtoseconds to the all-trans conformation. In this work, we use ultrafast time-resolved crystallography at room temperature to determine how an isomerised twisted all-trans retinal stores the photon energy required to initiate retinal isomerization. The distorted retinal at 1 ps time-delay of photoactivation has reduced its numerous interactions with the amino acids in its binding pocket. The excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Strikingly, the very early structural motions in the protein side chains of rhodopsin appear in regions involved in later stages of the conserved Class A GPCR activation mechanism. This indicates a conserved energy dissipation pathway and a dual function of the internal hydrogen bond networks of the GPCR linked to its dynamic conformational landscape. Our ultrafast free electron laser measurements shed light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of photo activation of retinal proteins.

Impact of protein-chromophore interaction on the retinal excited state and photocycle of Gloeobacter rhodopsin: role of conserved tryptophan residues.

The function of microbial as well as mammalian retinal proteins (aka rhodopsins) is associated with a photocycle initiated by light excitation of the retinal chromophore of the protein, covalently bound through a protonated Schiff base linkage. Although electrostatics controls chemical reactions of many organic molecules, attempt to understand its role in controlling excited state reactivity of rhodopsins and, thereby, their photocycle is scarce. Here, we investigate the effect of highly conserved tryptophan residues, between which the all-trans retinal chromophore of the protein is sandwiched in microbial rhodopsins, on the charge distribution along the retinal excited state, quantum yield and nature of the light-induced photocycle and absorption properties of Gloeobacter rhodopsin (GR). Replacement of these tryptophan residues by non-aromatic leucine (W222L and W122L) or phenylalanine (W222F) does not significantly affect the absorption maximum of the protein, while all the mutants showed higher sensitivity to photobleaching, compared to wild-type GR. Flash photolysis studies revealed lower quantum yield of trans-cis photoisomerization in W222L as well as W222F mutants relative to wild-type. The photocycle kinetics are also controlled by these tryptophan residues, resulting in altered accumulation and lifetime of the intermediates in the W222L and W222F mutants. We propose that protein-retinal interactions facilitated by conserved tryptophan residues are crucial for achieving high quantum yield of the light-induced retinal isomerization, and affect the thermal retinal re-isomerization to the resting state.

High-performance optical control of GPCR signaling by bistable animal opsins MosOpn3 and LamPP in a molecular property–dependent manner

Optical control of G protein-coupled receptor (GPCR) signaling is a highly valuable approach for comprehensive understanding of GPCR-based physiologies and controlling them precisely. However, optogenetics for GPCR signaling is still developing and requires effective and versatile tools with performance evaluation from their molecular properties. Here, we systematically investigated performance of two bistable opsins that activate Gi/Go-type G protein (mosquito Opn3 (MosOpn3) and lamprey parapinopsin (LamPP)) in optical control in vivo using Caenorhabditis elegans. Transgenic worms expressing MosOpn3, which binds 13-cis retinal to form photopigments, in nociceptor neurons showed light-induced avoidance responses in the presence of all-trans retinal, a retinal isomer ubiquitously present in every tissue, like microbial rhodopsins and unlike canonical vertebrate opsins. Remarkably, transgenic worms expressing MosOpn3 were ~7,000 times more sensitive to light than transgenic worms expressing ChR2 in this light-induced behavior, demonstrating the advantage of MosOpn3 as a light switch. LamPP is a UV-sensitive bistable opsin having complete photoregenerative ability by green light. Accordingly, transgenic worms expressing LamPP in cholinergic motor neurons stopped moving upon violet light illumination and restored coordinate movement upon green light illumination, demonstrating color-dependent control of behavior using LamPP. Furthermore, we applied molecular engineering to produce MosOpn3-based tools enabling light-dependent upregulation of cAMP or Ca2+ levels and LamPP-based tool enabling clamping cAMP levels color dependently and context independently, extending their usability. These findings define the capacity of two bistable opsins with similar retinal requirement as ChR2, providing numerous strategies for optical control of various GPCR-based physiologies as well as GPCR signaling itself.

Bidirectional Photochemistry of Antarctic Microbial Rhodopsin: Emerging Trend of Ballistic Photoisomerization from the 13-cis Resting State.

The decades-long ultrafast examination of nearly a dozen microbial retinal proteins, ion pumps, and sensory photoreceptors has not identified structure–function indicators which predict photoisomerization dynamics, whether it will be sub-picosecond and ballistic or drawn out with complex curve-crossing kinetics. Herein, we report the emergence of such an indicator. Using pH control over retinal isomer ratios, photoinduced transient absorption is recorded in an inward proton pumping Antarctic microbial rhodopsin (AntR) for 13-cis and all-trans retinal resting states. The all-trans fluorescent state decays with 1 ps exponential kinetics. In contrast, in 13-cis it decays within ∼300 fs accompanied by continuous spectral evolution, indicating ballistic internal conversion. The coherent wave packet nature of 13-cis isomerization in AntR matches published results for bacteriorhodopsin (BR) and Anabaena sensory rhodopsin (ASR), which also accommodate both all-trans and 13-cis retinal resting states, marking the emergence of a first structure–photodynamics indicator which holds for all three tested pigments.

Capturing a rhodopsin receptor signalling cascade across a native membrane

G protein-coupled receptors (GPCRs) are cell-surface receptors that respond to various stimuli to induce signalling pathways across cell membranes. Recent progress has yielded atomic structures of key intermediates1,2 and roles for lipids in signalling3,4. However, capturing signalling events of a wild-type receptor in real time, across a native membrane to its downstream effectors, has remained elusive. Here we probe the archetypal class A GPCR, rhodopsin, directly from fragments of native disc membranes using mass spectrometry. We monitor real-time photoconversion of dark-adapted rhodopsin to opsin, delineating retinal isomerization and hydrolysis steps, and further showing that the reaction is significantly slower in its native membrane than in detergent micelles. Considering the lipids ejected with rhodopsin, we demonstrate that opsin can be regenerated in membranes through photoisomerized retinal–lipid conjugates, and we provide evidence for increased association of rhodopsin with unsaturated long-chain phosphatidylcholine during signalling. Capturing the secondary steps of the signalling cascade, we monitor light activation of transducin (Gt) through loss of GDP to generate an intermediate apo-trimeric G protein, and observe Gαt•GTP subunits interacting with PDE6 to hydrolyse cyclic GMP. We also show how rhodopsin-targeting compounds either stimulate or dampen signalling through rhodopsin–opsin and transducin signalling pathways. Our results not only reveal the effect of native lipids on rhodopsin signalling and regeneration but also enable us to propose a paradigm for GPCR drug discovery in native membrane environments.

Crystallographic Studies of Rhodopsins: Structure and Dynamics.

Crystal structures have provided detailed insight in the architecture of rhodopsin photoreceptors. Of particular interest are the protein-chromophore interactions that govern the light-induced retinal isomerization and ultimately induce the large structural changes important for the various biological functions of the family. The reaction intermediates occurring along the rhodopsin photocycle have vastly differing lifetimes, from hundreds of femtoseconds to milliseconds. Detailed insight at high spatial and temporal resolution can be obtained by time-resolved crystallography using pump-probe approaches at X-ray free-electron lasers. Alternatively, cryotrapping approaches can be used. Both the approaches are described, including illumination and sample delivery. The importance of appropriate photoexcitation avoiding multiphoton absorption is stressed.

Light-Induced Conformational Alterations in Heliorhodopsin Triggered by the Retinal Excited State.

Heliorhodopsins are a recently discovered diverse retinal protein family with an inverted topology of the opsin where the retinal protonated Schiff base proton is facing the cell cytoplasmic side in contrast to type 1 rhodopsins. To explore whether light-induced retinal double-bond isomerization is a prerequisite for triggering protein conformational alterations, we utilized the retinal oxime formation reaction and thermal denaturation of a native heliorhodopsin of Thermoplasmatales archaeon SG8-52-1 (TaHeR) as well as a trans-locked retinal analogue (TaHeRL) in which the critical C13=C14 double-bond isomerization is prevented. We found that both reactions are light-accelerated not only in the native but also in the “locked” pigment despite lacking any isomerization. It is suggested that light-induced charge redistribution in the retinal excited state polarizes the protein and triggers protein conformational perturbations that thermally decay in microseconds. The extracted activation energy and the frequency factor for both the reactions reveal that the light enhancement of TaHeR differs distinctly from the earlier studied type 1 microbial rhodopsins.

Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin.

Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore.

Early-stage dynamics of chloride ion–pumping rhodopsin revealed by a femtosecond X-ray laser

Chloride ion-pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl- into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation-diffusion process upon light-triggered retinal isomerization.

Femtochemistry of Rhodopsins

The review considers the spectral kinetic data obtained by us by femtosecond absorption laser spectroscopy for the photochromic reaction of retinal isomerization in animal rhodopsin (type II), namely, bovine visual rhodopsin and microbial rhodopsins (type I), such as Exiguobacterium sibiricum rhodopsin and Halobacterium salinarum bacteriorhodopsin. It is shown that the elementary act of the photoreaction of retinal isomerization in type I and type II rhodopsins can be interpreted as a transition through a conical intersection with retention of the coherence of the vibrational wave packets generated during excitation. The coherent nature of the reaction is most pronounced in visual rhodopsin as a result of the barrier-free movement along the excited surface of potential energy, which also leads to an extremely high rate of retinal isomerization compared to microbial rhodopsins. Differences in the dynamics of photochemical reactions of type I and type II rhodopsins can be related to both differences in the initial isomeric forms of their chromophores (all-trans and 11-cis retinal, respectively), as well as with the effect of the protein environment on the chromophore. Despite the practically identical values of the quantum yields of the direct photoreaction of visual rhodopsin and bacteriorhodopsin, the reverse photoreaction of visual rhodopsin is much less effective (φ = 0.15) than in the case of bacteriorhodopsin (φ = 0.81). It can be assumed that the photobiological mechanism for converting light into an information process in the evolutionarily younger visual rhodopsins (type II rhodopsins) should be more reliable than the mechanism for converting light into a photoenergetic process in the evolutionarily more ancient microbial rhodopsins (type I rhodopsins). The low value of the quantum yield of the reverse reaction of visual rhodopsin can be considered as an increase in the reliability of the forward reaction, which triggers the process of phototransduction.

Structures of the archaerhodopsin-3 transporter reveal that disordering of internal water networks underpins receptor sensitization

Many transmembrane receptors have a desensitized state, in which they are unable to respond to external stimuli. The family of microbial rhodopsin proteins includes one such group of receptors, whose inactive or dark-adapted (DA) state is established in the prolonged absence of light. Here, we present high-resolution crystal structures of the ground (light-adapted) and DA states of Archaerhodopsin-3 (AR3), solved to 1.1 Å and 1.3 Å resolution respectively. We observe significant differences between the two states in the dynamics of water molecules that are coupled via H-bonds to the retinal Schiff Base. Supporting QM/MM calculations reveal how the DA state permits a thermodynamic equilibrium between retinal isomers to be established, and how this same change is prevented in the ground state in the absence of light. We suggest that the different arrangement of internal water networks in AR3 is responsible for the faster photocycle kinetics compared to homologs.

Comparative Femtosecond Spectroscopy of Primary Photoreactions of Exiguobacterium sibiricum Rhodopsin and Halobacterium salinarum Bacteriorhodopsin.

The primary stages of the Exiguobacterium sibiricum rhodopsin (ESR) photocycle were investigated by femtosecond absorption laser spectroscopy in the spectral range of 400-900 nm with a time resolution of 25 fs. The dynamics of the ESR photoreaction were compared with the reactions of bacteriorhodopsin (bR) in purple membranes (bRPM) and in recombinant form (bRrec). The primary intermediates of the ESR photocycle were similar to intermediates I, J, and K in bacteriorhodopsin photoconversion. The CONTIN program was applied to analyze the characteristic times of the observed processes and to clarify the reaction scheme. A similar photoreaction pattern was observed for all studied retinal proteins, including two consecutive dynamic Stokes shift phases lasting ∼0.05 and ∼0.15 ps. The excited state decays through a femtosecond reactive pathway, leading to retinal isomerization and formation of product J, and a picosecond nonreactive pathway that leads only to the initial state. Retinal photoisomerization in ESR takes 0.69 ps, compared with 0.48 ps in bRPM and 0.74 ps in bRrec. The nonreactive excited state decay takes 5 ps in ESR and ∼3 ps in bR. We discuss the similarity of the primary reactions of ESR and other retinal proteins.

The effect on ion channel of different protonation states of E90 in channelrhodopsin-2: a molecular dynamics simulation.

Channelrhodopsin-2 (ChR2) is a cationic channel protein that has been extensively studied in optogenetics. The ion channel is opened via a series of proton transfers and H-bond changes during the photocycle but the detailed mechanism is still unknown. Molecular dynamics (MD) simulations with enhanced sampling were performed on the dark-adapted state (i.e., D470) and two photocycle intermediates (P1500 and P2390) to study the proton transfer path of the Schiff base and the subsequent conformational changes. The results suggest there are two possible proton transfer pathways from the Schiff base to proton acceptors (i.e., E123 or D253), depending on the protonation of E90. If E90 is protonated in the P1500 state, the proton on the Schiff base will transfer to E123. The polyene chain of 13-cis retinal tilts and opens the channel that detours the blocking central gate (CG) and forms a narrow channel through the transmembrane helices (TM) 2, 3, 6 and 7. In contrast, if E90 deprotonates after retinal isomerization, the primary proton acceptor is D253, and an almost-open channel through TM1, 2, 3 and 7 is generated. The channel diameter is very close to the experimental value. The potential mean force (PMF) suggests that the free energy is extremely low for ions passing through this channel.

Absorption and Emission Spectroscopic Investigation of the Thermal Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor Archon2.

Archon2 is a fluorescent voltage sensor derived from Archaerhodopsin 3 (Arch) of Halorubrum sodomense using robotic multidimensional directed evolution approach. Here we report absorption and emission spectroscopic studies of Archon2 in Tris buffer at pH 8. Absorption cross-section spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation spectra were determined. The thermal stability of Archon2 was studied by long-time attenuation coefficient measurements at room temperature (21 ± 1 °C) and at refrigerator temperature (3 ± 1 °C). The apparent melting temperature was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 63 ± 3 °C). In the protein melting process protonated retinal Schiff base (PRSB) with absorption maximum at 586 nm converted to de-protonated retinal Schiff base (RSB) with absorption maximum at 380 nm. Storage of Archon2 at room temperature and refrigerator temperature caused absorption coefficient decrease because of partial protein clustering to aggregates at condensation nuclei and sedimentation. At room temperature an onset of light scattering was observed after two days because of the beginning of protein unfolding. During the period of observation (18 days at 21 °C, 22 days at 3 °C) no change of retinal isomer composition was observed indicating a high potential energy barrier of S0 ground-state isomerization.