Retinal Photoisomerization Research Articles

Exploring the Global Reaction Coordinate for Retinal Photoisomerization: A Graph Theory-Based Machine Learning Approach.

Unraveling the reaction pathway of photoinduced reactions poses a great challenge owing to its complexity. Recently, graph theory-based machine learning combined with nonadiabatic molecular dynamics (NAMD) has been applied to obtain the global reaction coordinate of the photoisomerization of azobenzene. However, NAMD simulations are computationally expensive as they require calculating the nonadiabatic coupling vectors at each time step. Here, we showed that ab initio molecular dynamics (AIMD) can be used as an alternative to NAMD by choosing an appropriate initial condition for the simulation. We applied our methodology to determine a plausible global reaction coordinate of retinal photoisomerization, which is essential for human vision. On rank-ordering the internal coordinates, based on the mutual information (MI) between the internal coordinates and the HOMO energy, NAMD and AIMD give a similar trend. Our results demonstrate that our AIMD-based machine learning protocol for retinal is 1.5 times faster than that of NAMD to study reaction coordinates.

Retinal photoisomerization versus counterion protonation in light and dark-adapted bacteriorhodopsin and its primary photoproduct

Discovered over 50 years ago, bacteriorhodopsin is the first recognized and most widely studied microbial retinal protein. Serving as a light-activated proton pump, it represents the archetypal ion-pumping system. Here we compare the photochemical dynamics of bacteriorhodopsin light and dark-adapted forms with that of the first metastable photocycle intermediate known as “K”. We observe that following thermal double isomerization of retinal in the dark from bio-active all-trans 15-anti to 13-cis, 15-syn, photochemistry proceeds even faster than the ~0.5 ps decay of the former, exhibiting ballistic wave packet curve crossing to the ground state. In contrast, photoexcitation of K containing a 13-cis, 15-anti chromophore leads to markedly multi-exponential excited state decay including much slower stages. QM/MM calculations, aimed to interpret these results, highlight the crucial role of protonation, showing that the classic quadrupole counterion model poorly reproduces spectral data and dynamics. Single protonation of ASP212 rectifies discrepancies and predicts triple ground state structural heterogeneity aligning with experimental observations. These findings prompt a reevaluation of counter ion protonation in bacteriorhodopsin and contribute to the broader understanding of its photochemical dynamics.

Modeling pH-Dependent Biomolecular Photochemistry.

The tuning mechanism of pH can be extremely challenging to model computationally in complex biological systems, especially with respect to the photochemical properties. This article reports a protocol aimed at modeling pH-dependent photodynamics using a combination of constant-pH molecular dynamics and semiclassical nonadiabatic molecular dynamics simulations. With retinal photoisomerization in Anabaena sensory rhodopsin (ASR) as a testbed, we show that our protocol produces pH-dependent photochemical properties, such as the isomerization quantum yield or decay rates. We decompose our results into single-titrated residue contributions, identifying some key tuning amino acids. Additionally, we assess the validity of the single protonation state picture to represent the system at a given pH and propose the most populated protein charge state as a compromise between cost and accuracy.

Multi-objective optimization for retinal photoisomerization models with respect to experimental observables

The fitting of physical models is often done only using a single target observable. However, when multiple targets are considered, the fitting procedure becomes cumbersome, there being no easy way to quantify the robustness of the model for all different observables. Here, we illustrate that one can jointly search for the best model for each desired observable through multi-objective optimization. To do so, we construct the Pareto front to study if there exists a set of parameters of the model that can jointly describe multiple, or all, observables. To alleviate the computational cost, the predicted error for each targeted objective is approximated with a Gaussian process model as it is commonly done in the Bayesian optimization framework. We applied this methodology to improve three different models used in the simulation of stationary state cis-trans photoisomerization of retinal in rhodopsin, a significant biophysical process. Optimization was done with respect to different experimental measurements, including emission spectra, peak absorption frequencies for the cis and trans conformers, and energy storage. Advantages and disadvantages of previously proposed models are exposed.

Inverse Hydrogen-Bonding Change Between the Protonated Retinal Schiff Base and Water Molecules upon Photoisomerization in Heliorhodopsin 48C12.

Heliorhodopsin (HeR) is a new class of the rhodopsin family discovered in 2018 through functional metagenomic analysis (named 48C12). Similar to typical microbial rhodopsins, HeR possesses seven transmembrane (TM) α-helices and an all-trans-retinal covalently bonded to the lysine residue on TM7 via a protonated Schiff base. Remarkably, the HeR membrane topology is inverted compared with that of typical microbial rhodopsins. The X-ray crystal structure of HeR 48C12 was elucidated after the first report on a HeR variant from Thermoplasmatales archaeon SG8-52-1, which revealed the water-mediated hydrogen-bonding network connected to the Schiff base region in the cytoplasmic side. Herein, low-temperature light-induced FTIR spectroscopic analyses of HeR 48C12 and 15N isotopically labeled proteins were used to elucidate the structural changes during retinal photoisomerization. N-D stretching vibrations of the protonated retinal Schiff base (PRSB) at 2286 and 2302 cm-1 in the dark state, and 2239 and 2252 cm-1 in the K intermediate were observed. The frequency changes indicated that the hydrogen bond of PRSB strengthens upon photoisomerization in HeR. Moreover, O-D stretching vibration frequencies of the internal water molecules indicate that the hydrogen-bonding strength decreases concomitantly. Therefore, the PRSB hydrogen bond responds to photoisomerization in an opposite way to the hydrogen-bonding network involving water molecules. No frequency changes of the indole N-H or N-D stretching vibrations of tryptophan residues were observed upon photoisomerization, suggesting that all tryptophan residues in the HeR 48C12 maintained the hydrogen-bonding strengths in the K intermediate. These results provide insights into the molecular mechanism of the energy storage and propagation upon retinal photoisomerization in HeR.

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.

Light-induced difference FTIR spectroscopy of primate blue-sensitive visual pigment at 163 K.

Structural studies of color visual pigments lag far behind those of rhodopsin for scotopic vision. Using difference FTIR spectroscopy at 77 K, we report the first structural data of three primate color visual pig­ments, monkey red (MR), green (MG), and blue (MB), where the batho-intermediate (Batho) exhibits photo­equilibrium with the unphotolyzed state. This photo­chromic property is highly advantageous for limited samples since the signal-to-noise ratio is improved, but may not be applicable to late intermediates, because of large structural changes to proteins. Here we report the photochromic property of MB at 163 K, where the BL intermediate, formed by the relaxation of Batho, is in photoequilibrium with the initial MB state. A comparison of the difference FTIR spectra at 77 and 163 K provided information on what happens in the process of transition from Batho to BL in MB. The coupled C11=C12 HOOP vibration in the planer structure in MB is decoupled by distortion in Batho after retinal photoisomerization, but returns to the coupled C11=C12 HOOP vibration in the all-trans chromophore in BL. The Batho formation accompanies helical structural perturbation, which is relaxed in BL. Protein-bound water molecules that form an extended water cluster near the retinal chromophore change hydrogen bonds differently for Batho and BL, being stronger in the latter than in the initial state. In addition to structural dynamics, the present FTIR spectra show no signals of protonated carboxylic acids at 77 and 163 K, suggesting that E181 is deprotonated in MB, Batho and BL.

Formation Mechanism of Ion Channel in Channelrhodopsin-2: Molecular Dynamics Simulation and Steering Molecular Dynamics Simulations.

Channelrhodopsin-2 (ChR2) is a light-activated and non-selective cationic channel protein that can be easily expressed in specific neurons to control neuronal activity by light. Although ChR2 has been extensively used as an optogenetic tool in neuroscience research, the molecular mechanism of cation channel formation following retinal photoisomerization in ChR2 is not well understood. In this paper, studies of the closed and opened state ChR2 structures are presented. The formation of the cationic channel is elucidated in atomic detail using molecular dynamics simulations on the all-trans-retinal (ChR2-trans) configuration of ChR2 and its isomerization products, 13-cis-retinal (ChR2-cis) configuration, respectively. Photoisomerization of the retinal-chromophore causes the destruction of interactions among the crucial residues (e.g., E90, E82, N258, and R268) around the channel and the extended H-bond network mediated by numerous water molecules, which opens the pore. Steering molecular dynamics (SMD) simulations show that the electrostatic interactions at the binding sites in intracellular gate (ICG) and central gate (CG) can influence the transmembrane transport of Na+ in ChR2-cis obviously. Potential of mean force (PMF) constructed by SMD and umbrella sampling also found the existing energy wells at these two binding sites during the transportation of Na+. These wells partly hinder the penetration of Na+ into cytoplasm through the ion channel. This investigation provides a theoretical insight on the formation mechanism of ion channels and the mechanism of ion permeation.

Multiple time scale open systems: Reaction rates and quantum coherence in model retinal photoisomerization under incoherent excitation.

Many important open quantum systems, such as light harvesting systems irradiated with natural incoherent light, present challenging computational problems. Specifically, such systems are characterized by multiple time scales over many orders of magnitude. We describe and apply an efficient approach to determine rates and dynamics in such systems. As an example, we present a theoretical and computational analysis of retinal isomerization under incoherent solar excitation using a minimal retinal model. Solar- and bath-induced Fano coherences are shown to have a small but non-negligible effect on the reaction dynamics, and the effect of Fano coherences on the reaction rate is shown to depend strongly upon the form and strength of the system-bath coupling. Using the isomerization probability to obtain the time-dependent cellular hyperpolarization, we show that the effect of coherence on hyperpolarization dynamics is small compared to the effect of natural variations in the solar intensity.

Photosensitized Retinal Isomerization in Rhodopsin Mediated by a Triplet State

AbstractDirect 11‐cis to all‐trans retinal photoisomerization within rhodopsin is well known to be the initial chemical reaction triggering the process of vision in mammalians, such as bovine. Nevertheless, deep‐sea fish are known to use chlorophyll derivatives as photosensitizers in order to see deep‐red light, at wavelengths where retinal does not absorb. Also, some photodynamic therapy treatments were shown to enhance the vision of patients in dim light conditions. Energy transfer from the photosensitizer to rhodopsin was therefore proposed as a mechanism to populate the triplet state of the retinal chromophore. Herein, by means of hybrid quantum mechanics‐coupled‐molecular mechanics modeling techniques, we give insights into the possible energy mechanism and describe the retinal isomerization mediated by the lowest‐lying triplet state. Especially, we show how a few kcal/mol energy barrier separates a T1 minimum from a S0/T1 intersection region, hence proposing an equilibrium between phosphorescence and isomerization processes. Moreover, the eventual self‐production of singlet oxygen, constituting a potential danger for the integrity of rhodopsin, is discussed.

Bacteriorhodopsin: Structural Insights Revealed Using X-Ray Lasers and Synchrotron Radiation.

Directional transport of protons across an energy transducing membrane-proton pumping-is ubiquitous in biology. Bacteriorhodopsin (bR) is a light-driven proton pump that is activated by a buried all-trans retinal chromophore being photoisomerized to a 13-cis conformation. The mechanism by which photoisomerization initiates directional proton transport against a proton concentration gradient has been studied by a myriad of biochemical, biophysical, and structural techniques. X-ray free electron lasers (XFELs) have created new opportunities to probe the structural dynamics of bR at room temperature on timescales from femtoseconds to milliseconds using time-resolved serial femtosecond crystallography (TR-SFX). Wereview these recent developments and highlight where XFEL studies reveal new details concerning the structural mechanism of retinal photoisomerization and proton pumping. We also discuss the extent to which these insights were anticipated by earlier intermediate trapping studies using synchrotron radiation. TR-SFX will open up the field for dynamical studies of other proteins that are not naturally light-sensitive.

Photochemistry of Bacteriorhodopsin with Various Oligomeric Statuses in Controlled Membrane Mimicking Environments: A Spectroscopic Study from Femtoseconds to Milliseconds.

Preparing transmembrane protein in controllable lipid bilayers is essential for unravelling the coupling of the environments and its dynamic functions. Monomerized bacteriorhodopsin (mbR) embedded in covalently circularized nanodiscs was prepared with dimyristoylphosphatidylglycerol (DMPG) lipid and circular membrane scaffold proteins of two different sizes, cE3D1 and cΔ H5, respectively. The retinal photoisomerization kinetics and thermodynamic photocycle were examined by femtosecond and nanosecond transient absorption, respectively, covering the time scale from femtoseconds to hundreds of milliseconds. The kinetics of the retinal isomerization and proton migration from the protonated Schiff base to Asp-85 were not significantly different for monomeric bR solubilized in Triton X-100 or embedded in circularized nanodiscs. This can be ascribed to the local tertiary structures at the retinal pocket vicinity being similar among monomeric bR in various membrane mimicking environments. However, the aforementioned processes are intrinsically different for trimeric bR in purple membrane (PM) and delipidated PM. The reprotonation of the deprotonated Schiff base from Asp-96 in association with the decay of intermediate M, which involved wide-ranged structural alteration, manifested a difference in terms of the oligomeric statuses, as well as a slight dependence on the size of the nanodisc. In summary, bR oligomeric statuses, rather than the environmental factors, such as membrane mimicking systems and nanodisc size, play a significant role in bR photocycle associated with short-range processes, such as the retinal isomerization and deprotonation of protonated Schiff base at the retinal pocket. On the other hand, the environmental factors, such as the types of membrane mimicking systems and the size of nanodiscs, affect those dynamic processes involving wider structural alterations during the photocycle.

Ultrafast Dynamics of Heliorhodopsins.

Heliorhodopsins (HeR) constitute a new rhodopsin family and show only <15% sequence identities with type-1 and type-2 rhodopsins. The large difference in amino acid sequence between HeRs and other rhodopsins raises a question whether their biological function is triggered by efficient and rapid photoisomerization of the retinal chromophore as in the case of other rhodopsins. We performed femtosecond time-resolved absorption measurements of two HeRs, HeR 48C12 and HeR from Thermoplasmatales archaeon SG8-52-1. Both HeRs exhibit excited-state absorption around 480 nm and stimulated emission in the >650 nm region, and these transient signals decay concomitantly with appearance of photoproduct absorption on a subpicosecond time scale. The observed spectral change indicates that ultrafast retinal photoisomerization proceeds in the femtosecond time region. The transient spectra and dynamics of HeRs are surprisingly similar to those of type-1 rhodopsins, despite remarkable differences in amino acid arrangement in the hydrophobic region of the retinal binding site.

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.

Membrane Independence of Ultrafast Photochemistry in Pharaonis Halorhodopsin: Testing the Role of Bacterioruberin.

Ultrafast photochemistry of pharaonis halorhodopsin (p-HR) in the intact membrane of Natronomonas pharaonis has been studied by photoselective femtosecond pump-hyperspectral probe spectroscopy with high time resolution. Two variants of this sample were studied, one with wild-type retinal prosthetic groups and another after shifting the retinal absorption deep into the blue range by reducing the Schiff base linkage, and the results were compared to a previous study on detergent-solubilized p-HR. This comparison shows that retinal photoisomerization dynamics is identical in the membrane and in the solubilized sample. Selective photoexcitation of bacterioruberin, which is associated with the protein in the native membrane, in wild-type and reduced samples, demonstrates conclusively that unlike the carotenoids associated with some bacterial retinal proteins the carrotenoid in p-HR does not act as a light-harvesting antenna.

Quantum-classical model of retinal photoisomerization reaction in visual pigment rhodopsin.

A quantum-classical model of photoisomerization of the visual pigment rhodopsin chromophore is proposed. At certain (and more realistic) parameter value combinations, the model is shown to accurately reproduce a number of independent experimental data on the photoreaction dynamics: the quantum yield, the time to reach the point of conical intersection of potential energy surfaces, the termination time of the evolution of quantum subsystem, as well as the characteristic low frequencies of retinal molecular lattice fluctuations during photoisomerization. In addition, the model behavior is in good accordance with experimental data about coherence and local character of quantum transition.

Femtosecond spectroscopic study of photochromic reactions of bacteriorhodopsin and visual rhodopsin

Photochromic ultrafast reactions of bacteriorhodopsin (H. salinarum) and bovine rhodopsin were conducted with a femtosecond two-pump probe pulse setup with the time resolution of 20–25fs. The dynamics of the forward and reverse photochemical reactions for both retinal-containing proteins was compared. It is demonstrated that when retinal-containing proteins are excited by femtosecond pulses, dynamics pattern of the vibrational coherent wave packets in the course of the reaction is different for bacteriorhodopsin and visual rhodopsin. As shown in these studies, the low-frequencies that form a wave packets experimentally observed in the dynamics of primary products formation as a result of retinal photoisomerization have different intensities and are clearer for bovine rhodopsin. Photo-reversible reactions for both retinal proteins were performed from the stage of the relatively stable photointermediates that appear within 3–5ps after the light pulse impact. It is demonstrated that the efficiency of the reverse phototransition K-form→bacteriorhodopsin is almost five-fold higher than that of the Batho-intermediate→visual rhodopsin phototransition. The results obtained indicate that in the course of evolution the intramolecular mechanism of the chromophore-protein interaction in visual rhodopsin becomes more perfect and specific. The decrease in the probability of the reverse chromophore photoisomerization (all-trans→11-cis retinal) in primary photo-induced rhodopsin products causes an increase in the efficiency of the photoreception process.

Lack of evidence for phase-only control of retinal photoisomerization in the strict one-photon limit.

The concept of shaping electric fields to steer light-induced processes coherently has fascinated scientists for decades. Despite early theoretical considerations that ruled out one-photon coherent control (CC), several experimental studies reported that molecular responses are sensitive to the shape of the excitation field in the weak-field limit. These observations were largely attributed to the presence of rapid-decay channels, but experimental verification is lacking. Here, we test this hypothesis by investigating the degree of achievable control over the photoisomerization of the retinal protonated Schiff-base in bacteriorhodopsin, isorhodopsin and rhodopsin, all of which exhibit similar chromophores but different isomerization yields and excited-state lifetimes. Irrespective of the system studied, we find no evidence for dissipation-dependent behaviour, nor for any CC in the strict one-photon limit. Our results question the extent to which a photochemical process at ambient conditions can be controlled at the amplitude level, and how the underlying molecular potential-energy surfaces and dynamics may influence this controllability.

Molecular Dynamics of Channelrhodopsin at the Early Stages of Channel Opening.

Channelrhodopsin (ChR) is a light-gated cation channel that responds to blue light. Since ChR can be readily expressed in specific neurons to precisely control their activities by light, it has become a powerful tool in neuroscience. Although the recently solved crystal structure of a chimeric ChR, C1C2, provided the structural basis for ChR, our understanding of the molecular mechanism of ChR still remains limited. Here we performed electrophysiological analyses and all-atom molecular dynamics (MD) simulations, to investigate the importance of the intracellular and central constrictions of the ion conducting pore observed in the crystal structure of C1C2. Our electrophysiological analysis revealed that two glutamate residues, Glu122 and Glu129, in the intracellular and central constrictions, respectively, should be deprotonated in the photocycle. The simulation results suggested that the deprotonation of Glu129 in the central constriction leads to ion leakage in the ground state, and implied that the protonation of Glu129 is important for preventing ion leakage in the ground state. Moreover, we modeled the 13-cis retinal bound; i.e., activated C1C2, and performed MD simulations to investigate the conformational changes in the early stage of the photocycle. Our simulations suggested that retinal photoisomerization induces the conformational change toward channel opening, including the movements of TM6, TM7 and TM2. These insights into the dynamics of the ground states and the early photocycle stages enhance our understanding of the channel function of ChR.

Incoherent control of the retinal isomerization in rhodopsin.

We propose to control the retinal photoisomerization yield through the backaction dynamics imparted by a nonselective optical measurement of the molecular electronic state. This incoherent effect is easier to implement than comparable coherent pulse shaping techniques, and is also robust to environmental noise. A numerical simulation of the quantum dynamics shows that the isomerization yield of this important biomolecule can be substantially increased above the natural limit.