Isomerization Of Chromophore Research Articles

Ultrafast Photochemical Reaction of Exiguobacterium sibiricum Rhodopsin (ESR) at Alkaline pH

Rhodopsin from the eubacterium Exiguobacterium sibiricum (ESR) performs the function of light-dependent proton transport. The operation of ESR is based on the ultrafast photochemical reaction of isomerization of the retinal chromophore, which triggers dark processes closed in the photocycle. Many parameters of the photocycle are determined by the degree of protonation of Asp85 – the primary counterion of the chromophore group and the proton acceptor. ESR in detergent micelles pumps protons most efficiently at pH 9, when Asp85 is almost completely deprotonated. In this work, the photochemical reaction of ESR at pH 9.5 was studied by femtosecond laser absorption spectroscopy. It was shown that photoisomerization of the chromophore group occurs in 0.51 ps, and the contribution of the reactive excited state is about 80%. A comparison with the data we obtained at pH 7.4 showed that at pH 9.5 the reaction proceeds much faster and more efficiently. The data obtained confirm the important role of the chromophore group counterion in the photoactivated processes of rhodopsins.

Light-Triggered Reversible Swelling of Azobenzene-Containing Block Copolymer Worms via Confined Deformation Prepared by Polymerization-Induced Self-Assembly.

Stimuli-responsive block copolymer nanoparticles (NPs) have received close attention in recent years owing to their tremendous application potential in smart materials. Azobenzene-containing NPs are widely studied due to the advantages of light as a stimulus and fast reversible trans-cis isomerization of azobenzene chromophores. However, the inefficient preparation process and difficult reversible transformation of morphologies limit their development. Herein it is demonstrated that the light-triggered reversible swelling behavior of wormlike NPs with high azobenzene content could be realized via confined deformation. These worms are prepared in large quantities via polymerization-induced self-assembly based on the copolymerization of 11-(4-(4-butylphenylazo)phenoxy)undecyl methacrylate (MAAz) and N-(methacryloxy)succinimide (NMAS) monomers. Upon UV/visible light irradiation, the reversible deformation of worms is achieved when the feed molar ratio of NMAS/MAAz is relatively high or via crosslinking using diamines, which leads to the reduction of the photoisomerization efficiency. The diameter variation of the worms is influenced by the amount and types of crosslinkers. Moreover, the scalability of this strategy is further proved by the fabrication of photo- and reductant-responsive crosslinked worms. It is expected that this study not only provides a new route to affording reversible photoresponsive NPs but also offers a unique insight into the reversible photodeformation mechanism of azobenzene-containing NPs.

Excited State Vibrational Dynamics Reveals a Photocycle That Enhances the Photostability of the TagRFP-T Fluorescent Protein.

High photostability is a desirable property of fluorescent proteins (FPs) for imaging, yet its molecular basis is poorly understood. We performed ultrafast spectroscopy on TagRFP and its 9-fold more photostable variant TagRFP-T (TagRFP S158T) to characterize their initial photoreactions. We find significant differences in their electronic and vibrational dynamics, including faster excited-state proton transfer and transient changes in the frequency of the v520 mode in the excited electronic state of TagRFP-T. The frequency of v520, which is sensitive to chromophore planarity, downshifts within 0.58 ps and recovers within 0.87 ps. This vibrational mode modulates the distance from the chromophore phenoxy to the side chain of residue N143, which we suggest can trigger cis/trans photoisomerization. In TagRFP, the dynamics of v520 is missing, and this FP therefore lacks an important channel for chromophore isomerization. These dynamics are likely to be a key mechanism differentiating the photostability of the two FPs.

Dimer Interface Destabilization of Photodissociative Dronpa Driven by Asymmetric Monomer Dynamics.

Photoswitchable Dronpa (psDronpa) is a unique member of the fluorescent protein family that can undergo reversible photoinduced switching between fluorescent and dark states and has recently been engineered into a dimer (pdDronpaV) that can dissociate and reassociate as part of its photoswitchable pathway. However, the specific details of the protein structure-function relationship of the dimer interface along with how the dimer proteins interact with each other upon chromophore isomerization are not yet clear. Classical molecular dynamics simulations were performed on psDronpa as monomers and dimers as well as the pdDronpaV dimer and with cis/trans chromophore structures. Analysis of the cis and trans isomers of the chromophore illustrated key differences between their interactions with residues in the protein in both the monomer and dimer forms of psDronpa. Examination of the psDronpa dimer showed nonidentical chromophore interactions between the domains, indicating domain directional favoring. Examination of the trans form of pdDronpaV illuminated the importance of hydrogen bonding between the monomeric domains in maintaining their association, as well as illustrating the motion of dissociation of the domains. This discovery offers important information for possible future mutations of pdDronpaV that might be made to accelerate dissociation.

Targeting Ultrafast Spectroscopic Insights into Red Fluorescent Proteins.

Red fluorescent proteins (RFPs) represent an increasingly popular class of genetically encodable bioprobes and biomarkers that can advance next-generation breakthroughs across the imaging and life sciences. Since the rational design of RFPs with improved functions or enhanced versatility requires a mechanistic understanding of their working mechanisms, while fluorescence is intrinsically an ultrafast event, a suitable toolset involving steady-state and time-resolved spectroscopic techniques has become powerful in delineating key structural features and dynamic steps which govern irreversible photoconverting or reversible photoswitching RFPs, and large Stokes shift (LSS)RFPs. The pertinent cis-trans isomerization and protonation state change of RFP chromophores in their local environments, involving key residues in protein matrices, lead to rich and complicated spectral features across multiple timescales. In particular, ultrafast excited-state proton transfer in various LSSRFPs showcases the resolving power of wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) in mapping a photocycle with crucial knowledge about the red-emitting species. Moreover, recent progress in noncanonical RFPs with a site-specifically modified chromophore provides an appealing route for efficient engineering of redder and brighter RFPs, highly desirable for bioimaging. Such an effective feedback loop involving physical chemists, protein engineers, and biomedical microscopists will enable future successes to expand fundamental knowledge and improve human health.

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.

Born–Oppenheimer molecular dynamics study on collective protein dynamics invoked by ultrafast photoisomerization of retinal chromophore in bacteriorhodopsin

Large-scale quantum-chemistry-based Born–Oppenheimer molecular dynamics simulations of collective protein dynamics of bacteriorhodopsin initiated by photoinduced all-trans→ 13-cis isomerization of retinal Schiff base chromophore compensate for missing information from time-resolved serial femtosecond X-ray crystallography measurements in terms of electronic states, energy transfer, and details of structural dynamics.

Serial Femtosecond Crystallography Reveals that Photoactivation in a Fluorescent Protein Proceeds via the Hula Twist Mechanism.

Chromophore cis/trans photoisomerization is a fundamental process in chemistry and in the activation of many photosensitive proteins. A major task is understanding the effect of the protein environment on the efficiency and direction of this reaction compared to what is observed in the gas and solution phases. In this study, we set out to visualize the hula twist (HT) mechanism in a fluorescent protein, which is hypothesized to be the preferred mechanism in a spatially constrained binding pocket. We use a chlorine substituent to break the twofold symmetry of the embedded phenolic group of the chromophore and unambiguously identify the HT primary photoproduct. Through serial femtosecond crystallography, we then track the photoreaction from femtoseconds to the microsecond regime. We observe signals for the photoisomerization of the chromophore as early as 300 fs, obtaining the first experimental structural evidence of the HT mechanism in a protein on its femtosecond-to-picosecond timescale. We are then able to follow how chromophore isomerization and twisting lead to secondary structure rearrangements of the protein β-barrel across the time window of our measurements.

The interconnecting hairpin extension "arm": An essential allosteric element of phytochrome activity

In red-light sensing phytochromes, isomerization of the bilin chromophore triggers structural and dynamic changes across multiple domains, ultimately leading to control of the output module (OPM) activity. In between, a hairpin structure, "arm", extends from an interconnecting domain to the chromophore region. Here, by removing this protein segment in a bacteriophytochrome from Deinococcus radiodurans (DrBphP), we show that the arm is crucial for signal transduction. Crystallographic, spectroscopic, and biochemical data indicate that this variant maintains the properties of DrBphP in the resting state. Spectroscopic data also reveal that the armless systems maintain the ability to respond to light. However, there is no subsequent regulation of OPM activity without the arms. Thermal denaturation reveals thatthe arms stabilize the DrBphP structure. Our results underline the importance of the structurally flexible interconnecting hairpin extensions and describe their central role in the allosteric coupling of phytochromes.

Ultrafast protein response in the Pfr state of Cph1 phytochrome

Photoisomerization is a fundamental process in several classes of photoreceptors. Phytochromes sense red and far-red light in their Pr and Pfr states, respectively. Upon light absorption, these states react via individual photoreactions to the other state. Cph1 phytochrome shows a photoisomerization of its phycocyanobilin (PCB) chromophore in the Pfr state with a time constant of 0.7 ps. The dynamics of the PCB chromophore has been described, but whether or not the apoprotein exhibits an ultrafast response too, is not known. Here, we compare the photoreaction of 13C/15N labeled apoprotein with unlabeled apoprotein to unravel ultrafast apoprotein dynamics in Cph1. In the spectral range from 1750 to 1620 cm−1 we assigned several signals due to ultrafast apoprotein dynamics. A bleaching signal at 1724 cm−1 is tentatively assigned to deprotonation of a carboxylic acid, probably Asp207, and signals around 1670 cm−1 are assigned to amide I vibrations of the capping helix close to the chromophore. These signals remain after photoisomerization. The apoprotein dynamics appear upon photoexcitation or concomitant with chromophore isomerization. Thus, apoprotein dynamics occur prior to and after photoisomerization on an ultrafast time-scale. We discuss the origin of the ultrafast apoprotein response with the ‘Coulomb hammer’ mechanism, i.e. an impulsive change of electric field and Coulombic force around the chromophore upon excitation.Graphical abstract

Early Proton Transfer Reaction in a Primate Blue-Sensitive Visual Pigment.

The proton transfer reaction belongs to one of the key triggers for the functional expression of membrane proteins. Rod and cone opsins are light-sensitive G-protein-coupled receptors (GPCRs) that undergo the cis-trans isomerization of the retinal chromophore in response to light. The isomerization event initiates a conformational change in the opsin protein moiety, which propagates the downstream effector signaling. The final step of receptor activation is the deprotonation of the retinal Schiff base, a proton transfer reaction which has been believed to be identical among the cone opsins. Here, we report an unexpected proton transfer reaction occurring in the early photoreaction process of primate blue-sensitive pigment (MB). By using low-temperature UV-visible spectroscopy, we found that the Lumi intermediate of MB formed in transition from the BL intermediate shows an absorption maximum in the UV region, indicating the deprotonation of the retinal Schiff base. Comparison of the light-induced difference FTIR spectra of Batho, BL, and Lumi showed significant α-helical backbone C=O stretching and protonated carboxylate C=O stretching vibrations only in the Lumi intermediate. The transition from BL to Lumi thus involves dramatic changes in protein environment with a proton transfer reaction between the Schiff base and the counterion resulting in an absorption maximum in the UV region.

The structural effect between the output module and chromophore-binding domain is a two-way street via the hairpin extension

Signal transduction typically starts with either ligand binding or cofactor activation, eventually affecting biological activities in the cell. In red light-sensing phytochromes, isomerization of the bilin chromophore results in regulation of the activity of diverse output modules. During this process, several structural elements and chemical events influence signal propagation. In our study, we have studied the full-length bacteriophytochrome from Deinococcus radiodurans as well as a previously generated optogenetic tool where the native histidine kinase output module has been replaced with an adenylate cyclase. We show that the composition of the output module influences the stability of the hairpin extension. The hairpin, often referred as the PHY tongue, is one of the central structural elements for signal transduction. It extends from a distinct domain establishing close contacts with the chromophore binding site. If the coupling between these interactions is disrupted, the dynamic range of the enzymatic regulation is reduced. Our study highlights the complex conformational properties of the hairpin extension as a bidirectional link between the chromophore-binding site and the output module, as well as functional properties of diverse output modules.

Reisomerization of retinal represents a molecular switch mediating Na+ uptake and release by a bacterial sodium-pumping rhodopsin

Sodium-pumping rhodopsins (NaRs) are membrane transporters that utilize light energy to pump Na+ across the cellular membrane. Within the NaRs, the retinal Schiff base chromophore absorbs light, and a photochemically induced transient state, referred to as the “O intermediate”, performs both the uptake and release of Na+. However, the structure of the O intermediate remains unclear. Here, we used time-resolved cryo-Raman spectroscopy under preresonance conditions to study the structure of the retinal chromophore in the O intermediate of an NaR from the bacterium Indibacter alkaliphilus. We observed two O intermediates, termed O1 and O2, having distinct chromophore structures. We show O1 displays a distorted 13-cis chromophore, while O2 contains a distorted all-trans structure. This finding indicated that the uptake and release of Na+ are achieved not by a single O intermediate but by two sequential O intermediates that are toggled via isomerization of the retinal chromophore. These results provide crucial structural insight into the unidirectional Na+ transport mediated by the chromophore-binding pocket of NaRs.

English

Bacteriophytochromes (BphPs) are light-regulated biliprotein photoreceptors widely occurring in bacteria. BphPs exists in two spectral distinct states: the red-light absorbing state (Pr) and the far-red light-absorbing state (Pfr). BphPs are composed of an N-terminal photosensory core module and a light-controlled C-terminal histidine kinase module. The detailed mechanism on how photoconversion, initiated by a light-triggered isomerization of the biliverdin chromophore, is conducted from the N-terminal to the C-terminal domain and how the histidine kinase is modulated is still not clear. In this work Agp1 from Agrobacterium tumefaciens is used as a model phytochrome. The method of labeling full-length Agp1 mutants with single accessible Cys residue using fluorescence probes is described. Different absorption spectra showed that neither mutagenesis nor fluorescence labeling exerts significant effects on the photoconversion behavior of various Agp1 mutants. The histidine kinase activity assay revealed that the labeled holoproteins exhibit wild-types like autophosphorylation activity that depends on the Pr and the Pfr state. Fluorescence spectroscopic data suggested that fluorescence resonance energy transfer occurs from the donor Atto495 to the acceptor Atto565 in double-labeled Agp1-A362C, K514C, H554C, R603C, I653C, and V674C mutants. Red light irradiation gave rise to small changes on fluorescence emission intensity in some mutants but in some no remarkable inter-subunit distance changes occurred during the photo-conversion from Pr to Pfr in full-length Agp1. In contrast to double-labeled samples, significant changes in fluorescence intensity were observed in single-labeled samples.   Key words: Bacteriophytochromes (BphPs), C-terminal domain, photosensory, phytochrome. 

Isorhodopsin: An Undervalued Visual Pigment Analog

Rhodopsin, the first visual pigment identified in the animal retina, was shown to be a photosensitive membrane protein containing covalently bound retinal in the 11-cis configuration, as a chromophore. Upon photoexcitation the chromophore isomerizes in femtoseconds to all-trans, which drives the protein into the active state. Soon thereafter, another geometric isomer—9-cis retinal—was also shown to stably incorporate into the binding pocket, generating a slightly blue-shifted photosensitive protein. This pigment, coined isorhodopsin, was less photosensitive, but could also reach the active state. However, 9-cis retinal was not detected as a chromophore in any of the many animal visual pigments studied, and isorhodopsin was passed over as an exotic and little-relevant rhodopsin analog. Consequently, few in-depth studies of its photochemistry and activation mechanism have been performed. In this review, we aim to illustrate that it is unfortunate that isorhodopsin has received little attention in the visual research and literature. Elementary differences in photoexcitation of rhodopsin and isorhodopsin have already been reported. Further in-depth studies of the photochemical properties and pathways of isorhodopsin would be quite enlightening for the initial steps in vision, as well as being beneficial for biotechnological applications of retinal proteins.

Femtosecond-to-millisecond mid-IR spectroscopy of photoactive yellow protein uncovers structural micro-transitions of the chromophore's protonation mechanism.

Protein structural dynamics can span many orders of magnitude in time. Photoactive yellow protein's (PYP) reversible photocycle encompasses picosecond isomerization of the light-absorbing chromophore as well as large scale protein backbone motions occurring on a millisecond timescale. Femtosecond-to-millisecond time-resolved mid-infrared spectroscopy is employed here to uncover structural details of photocycle intermediates up to chromophore protonation and the first structural changes leading to the formation of the partially unfolded signaling state pB. The data show that a commonly thought stable transient photocycle intermediate is actually formed after a sequence of several smaller structural changes. We provide residue-specific spectroscopic evidence that protonation of the chromophore on a few hundreds of microseconds timescale is delayed with respect to deprotonation of the nearby E46 residue. That implies that the direct proton donor is not E46 but most likely a water molecule. Such details may assist the ongoing photocycle and protein folding simulation efforts on the complex and wide time-spanning photocycle of the model system PYP.

The Photocycle of Bacteriophytochrome Is Initiated by Counterclockwise Chromophore Isomerization.

Photoactivation of bacteriophytochrome involves a cis–trans photoisomerization of a biliverdin chromophore, but neither the precise sequence of events nor the direction of the isomerization is known. Here, we used nonadiabatic molecular dynamics simulations on the photosensory protein dimer to resolve the isomerization mechanism in atomic detail. In our simulations the photoisomerization of the D ring occurs in the counterclockwise direction. On a subpicosecond time scale, the photoexcited chromophore adopts a short-lived intermediate with a highly twisted configuration stabilized by an extended hydrogen-bonding network. Within tens of picoseconds, these hydrogen bonds break, allowing the chromophore to adopt a more planar configuration, which we assign to the early Lumi-R state. The isomerization process is completed via helix inversion of the biliverdin chromophore to form the late Lumi-R state. The mechanistic insights into the photoisomerization process are essential to understand how bacteriophytochrome has evolved to mediate photoactivation and to engineer this protein for new applications.

Reversible Controlling the Supramolecular Chirality of Side Chain Azobenzene Polymers: Chiral Induction and Modulation.

Chirality represents a fundamental structure in nature and the induction and reversible modulation of supramolecular chirality with feasible techniques is of great value in the design of new chiroptical smart materials. Herein, two kinds of azobenzene side-chain polymers (without spacer: Azo-PMA0; with 6 spacers: Azo-PMA6) are synthesized, the length of spacer and azobenzene chromophores play a vital role in chirality transfer and modulation. The supramolecular chiral arrangement of Azo-PMA0 (amorphous phase) can be completely controlled and reversibly modulated over multiple cycles by 450nm circularly polarized light driven by the supramolecular interaction between azo groups of polymer chains, with an absorption dissymmetry factor (g) value of 0.0019. The chiroptical properties of Azo-PMA6 (liquid crystal state) can also be reversibly modulated by UV light and thermal annealing treatment during trans-cis isomerization of azo chromophore, with the g-value changes from 0-0.038. The successful construction of reversible chiral induction and modulation based on side chain azobenzene polymers may pave the way for designing photo-switchable functional materials.

Temperature-modulated photomechanical actuation of photoactive liquid crystal elastomers

Photoactive liquid crystal elastomers are polymer networks of liquid crystal mesogens embedded with chromophores like azobenzene. They undergo large deformation when illuminated by light of a certain wavelength through photochemical reaction, inspiring exciting new applications. However, despite the recent progresses in both the experiment and theory of these materials, the fundamental understanding of the temperature effect on their photomechanical actuation through various molecular-to-mesoscale processes have remained largely unexplored. This paper constructs a theoretical model to investigate this temperature-modulated photomechanical actuation, by integrating different temperature-dependent processes into a continuum framework. The model studies a special working condition where the material is subjected to a uniaxial tensile load, a prescribed temperature, and a polarized light illumination. We explore the free energy landscape of the system and the uniaxial stress–stretch responses under various conditions. We exploit the coupling between individual controls of temperature and light in a single photomechanical actuation for several working scenarios, including the temperature-modulated photomechanical snap-through instability, specific work, and blocking stress. We study the effect of the temperature-dependent backward isomerization of chromophores on the photomechanical actuation. These results are hoped to motivate future fundamental studies and new applications of various photomechanical material systems.

Light Driven Ultrafast Bioinspired Molecular Motors: Steering and Accelerating Photoisomerization Dynamics of Retinal.

Photoisomerization of retinal protonated Schiff base in microbial and animal rhodopsins are strikingly ultrafast and highly specific. Both protein environments provide conditions for fine-tuning the photochemistry of their chromophores. Here, by combining time-resolved action absorption spectroscopy and high-level electronic structure theory, we show that similar control can be gained in a synthetically engineered retinal chromophore. By locking the dimethylated retinal Schiff base at the C11═C12 double bond in its trans configuration (L-RSB), the excited-state decay is rendered from a slow picosecond to an ultrafast subpicosecond regime in the gas phase. Steric hindrance and pretwisting of L-RSB are found to be important for a significant reduction in the excited-state energy barriers, where isomerization of the locked chromophore proceeds along C9═C10 rather than the preferred C11═C12 isomerization path. Remarkably, the accelerated excited-state dynamics also becomes steered. We show that L-RSB is capable of unidirectional 360° rotation from all-trans to 9-cis and from 9-cis to all-trans in only two distinct steps induced by consecutive absorption of two 600 nm photons. This opens a way for the rational design of red-light-driven ultrafast molecular rotary motors based on locked retinal chromophores.