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A Schiff base in the retinal chromophore of microbial rhodopsin is crucial to its ion transport mechanism. Here, we discovered an unprecedented isotope effect on the CâN stretching frequency of the Schiff base in sodium ion-pumping rhodopsins, showing an unusual interaction of the Schiff base. No amino acid residue attributable to the unprecedented isotope effect was identified, suggesting that the H-O-H bending vibration of a water molecule near the Schiff base was coupled with the CâN stretching vibration. A twist in the polyene chain in the chromophore for the sodium ion-pumping rhodopsins enabled this unusual interaction of the Schiff base. The present discovery provides new insights into the interaction network of the retinal chromophore in microbial rhodopsins.
Assuntos
Bases de Schiff , Sódio , Vibração , Bases de Schiff/química , Sódio/química , Rodopsinas Microbianas/química , Rodopsinas Microbianas/metabolismoRESUMO
Proton (H+) motive force (PMF) serves as the energy source for the flagellar motor rotation, crucial for microbial motility. Here, to control PMF using light, we introduced light-driven inward and outward proton pump rhodopsins, RmXeR and AR3, into Escherichia coli. The motility of E. coli cells expressing RmXeR and AR3 significantly decreased and increased upon illumination, respectively. Tethered cell experiments revealed that, upon illumination, the torque of the flagellar motor decreased to nearly zero (28 pN nm) with RmXeR, while it increased to 1170 pN nm with AR3. These alterations in PMF correspond to +146 mV (RmXeR) and -140 mV (AR3), respectively. Thus, bidirectional optical control of PMF in E. coli was successfully achieved by using proton pump rhodopsins. This system holds a potential for enhancing our understanding of the roles of PMF in various biological functions.
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Escherichia coli , Força Próton-Motriz , Rodopsinas Microbianas , Escherichia coli/metabolismo , Rodopsinas Microbianas/química , Rodopsinas Microbianas/metabolismo , Luz , Bombas de Próton/metabolismo , Bombas de Próton/químicaRESUMO
High sensitivity of scotopic vision (vision in dim light conditions) is achieved by the rods' low background noise, which is attributed to a much lower thermal activation rate (kth) of rhodopsin compared with cone pigments. Frogs and nocturnal geckos uniquely possess atypical rods containing noncanonical cone pigments that exhibit low kth, mimicking rhodopsin. Here, we investigated the convergent mechanism underlying the low kth of rhodopsins and noncanonical cone pigments. Our biochemical analysis revealed that the kth of canonical cone pigments depends on their absorption maximum (λmax). However, rhodopsin and noncanonical cone pigments showed a substantially lower kth than predicted from the λmax dependency. Given that the λmax is inversely proportional to the activation energy of the pigments in the Hinshelwood distribution-based model, our findings suggest that rhodopsin and noncanonical cone pigments have convergently acquired low frequency of spontaneous-activation attempts, including thermal fluctuations of the protein moiety, in the molecular evolutionary processes from canonical cone pigments, which contributes to highly sensitive scotopic vision.
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Evolução Molecular , Visão Noturna , Rodopsina , Animais , Luz , Visão Noturna/fisiologia , Rodopsina/química , Rodopsina/metabolismo , Vertebrados , Opsinas dos Cones/química , Opsinas dos Cones/metabolismoRESUMO
Introduction: To extend individuals' healthy life expectancies, the improvement of subjective health and quality of life (QOL) has been increasingly prioritized, alongside the improvement of their physical functioning. Reports have indicated that intake of medium-chain triglycerides (MCTs) benefits the physical health of older individuals requiring nursing care, and athletes, and healthy individuals. But there are few studies investigating the effects of MCTs on subjective health and QOL. The present study sought to evaluate the combined effects of 12-week MCTs supplements and moderate-intensity walking exercise on the subjective health and QOL of middle-aged and older adults aged 60-74 with low BMIs (< 24 kg/m2) and who had no exercise habits. Methods: A placebo-controlled, double-blind, parallel-group trial was conducted. Three MCTs supplement groups with different doses and fatty acid compositions were compared with a control group. The study used the SF-36v2 questionnaire to assess subjective health and health-related QOL (HRQOL). Results: The result showed significant improvements in the scores on subscales of the physical QOL, such as Physical functioning and General health, and summary scores on the mental QOL, compared to the control. Conclusion: It is estimated that the combination of continuous intake of MCTs and walking exercise may affect HRQOL and improve subjective physical and mental health in sedentary, healthy, middle-aged and older adults. Clinical trial registration: https://rctportal.niph.go.jp/s/detail/um?trial_id=UMIN000046861, UMIN000046861.
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Hydrogen bond formation and deformation are crucial for the structural construction and functional expression of biomolecules. However, direct observation of exchangeable hydrogens, especially for oxygen-bound hydrogens, relevant to hydrogen bonds is challenging for current structural analysis approaches. Using solution-state NMR spectroscopy, this study detected the functionally important exchangeable hydrogens (i.e., Y49-ηOH and Y178-ηOH) involved in the pentagonal hydrogen bond network in the active site of R. xylanophilus rhodopsin (RxR), which functions as a light-driven proton pump. Moreover, utilization of the original light-irradiation NMR approach allowed us to detect and characterize the late photointermediate state (i.e., O-state) of RxR and revealed that hydrogen bonds relevant to Y49 and Y178 are still maintained during the photointermediate state. In contrast, the hydrogen bond between W75-εNH and D205-γCOO- is strengthened and stabilizes the O-state.
Assuntos
Bombas de Próton , Rodopsina , Rodopsina/química , Bombas de Próton/química , Ligação de Hidrogênio , Espectroscopia de Ressonância MagnéticaRESUMO
An adequate nutritional intake is recommended for the prevention of physical frailty and sarcopenia. In particular, medium-chain fatty acids (MCFAs) are reportedly important for muscle strength in nursing home residents. However, the effects of MCFAs on healthy adults at risk for frailty remain unknown. Hence, a randomized, placebo-controlled study was conducted to investigate the effects of 12 weeks of medium-chain triglycerides (MCTs) intake and walking on muscle mass and function in healthy, sedentary, middle-aged and older adults with a low body mass index. Three MCT intake groups with different amounts of octanoic and decanoic acid intake were compared with a control group. After 12 weeks, knee extension strength increased in all groups, with the increases in all MCT intake groups being significantly higher than those in the control group (p < 0.05). Grip strength significantly increased from baseline in the MCT 6 g/day intake group (p < 0.05). The combination of aerobic exercise and MCT intake may be effective in preventing decline in muscle strength and promoting increase in muscle strength as they can improve muscle energy production, thereby contributing to the maintenance of good health for middle-aged and older adults at high risk for frailty and sarcopenia.
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Fragilidade , Sarcopenia , Pessoa de Meia-Idade , Humanos , Idoso , Sarcopenia/prevenção & controle , Suplementos Nutricionais , Força Muscular , Força da Mão , Exercício Físico , Triglicerídeos/farmacologia , Músculo Esquelético , Método Duplo-CegoRESUMO
Upon white light illumination, the growth of the budding yeast Saccharomyces cerevisiae was extremely impaired only in the presence of iodide ions, but not fluoride, chloride and bromide ions. Action spectroscopy revealed that the maximum wavelength of the light is around at 373 nm, corresponding to the UVA region. Using a genetic approach, several genes, including OPY1, HEM1, and PAU11, were identified as suppressors of this growth inhibition. This iodide-dependent UVA-triggered growth inhibition method, along with its suppressive molecules, would be beneficial for understanding cell growth processes in eukaryotes and can be utilized for medium sterilization using UVA light.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Iodetos , Raios Ultravioleta , Luz , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Diatoms are a major phytoplankton group responsible for approximately 20% of carbon fixation on Earth. They perform photosynthesis using light-harvesting chlo-rophylls located in plastids, an organelle obtained through eukaryote-eukaryote endosymbiosis. Microbial rhodopsin, a photoreceptor distinct from chlo-rophyll-based photosystems, was recently identified in some diatoms. However, the physiological function of diatom rhodopsin remains unclear. Heterologous expression techniques were herein used to investigate the protein function and subcellular localization of diatom rhodopsin. We demonstrated that diatom rhodopsin acts as a light-driven proton pump and localizes primarily to the outermost membrane of four membrane-bound complex plastids. Using model simulations, we also examined the effects of pH changes inside the plastid due to rhodopsin-mediated proton transport on photosynthesis. The results obtained suggested the involvement of rhodopsin-mediated local pH changes in a photosynthetic CO2-concentrating mechanism in rhodopsin-possessing diatoms.
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Diatomáceas , Bombas de Próton/genética , Bombas de Próton/metabolismo , Rodopsina/genética , Fitoplâncton/metabolismo , Fotossíntese , Ciclo do Carbono , Carbono/metabolismoRESUMO
Light-driven inward proton pump rhodopsin RmXeR was embedded in pH-sensitive liposomes. Substance release from the proteoliposomes was observed following light illumination both in vitro and in cells, indicating the successful production of light-induced disruptive liposomes (LiDL). Thus, LiDL is a photoswitchable carrier utilized for intracellular substance delivery.
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Lipossomos , Rodopsina , Concentração de Íons de Hidrogênio , Transporte de Íons , Rodopsina/metabolismoRESUMO
An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source, thereby reducing the risk of kidney stone formation in host animals. The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations, occluded and outward-facing states. The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate. The occluded pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the pocket are completely blocked by extensive interdomain interactions, which can be opened solely by a flip of a single side chain neighbouring the substrate. This study shows the structural basis underlying metabolic interactions enabling favourable symbiosis.
Assuntos
Microbioma Gastrointestinal , Oxalatos , Animais , Oxalatos/química , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transporte Biológico , Bactérias/metabolismoRESUMO
Microbial rhodopsins, a family of photoreceptive membrane proteins containing the chromophore retinal, show a variety of light-dependent molecular functions. Channelrhodopsins work as light-gated ion channels and are widely utilized for optogenetics, which is a method for controlling neural activities by light. Since two cation channelrhodopsins were identified from the chlorophyte alga Chlamydomonas reinhardtii, recent advances in genomic research have revealed a wide variety of channelrhodopsins including anion channelrhodopsins (ACRs), describing their highly diversified molecular properties (e.g., spectral sensitivity, kinetics and ion selectivity). Here, we report two channelrhodopsin-like rhodopsins from the Colpodellida alga Vitrella brassicaformis, which are phylogenetically distinct from the known channelrhodopsins. Spectroscopic and electrophysiological analyses indicated that these rhodopsins are green- and blue-sensitive pigments (λmax = ~ 550 and ~ 440 nm) that exhibit light-dependent ion channeling activities. Detailed electrophysiological analysis revealed that one of them works as a monovalent anion (Cl-, Br- and NO3-) channel and we named it V. brassicaformis anion channelrhodopsin-2, VbACR2. Importantly, the absorption maximum of VbACR2 (~ 440 nm) is blue-shifted among the known ACRs. Thus, we identified the new blue-shifted ACR, which leads to the expansion of the molecular diversity of ACRs.
Assuntos
Fenômenos Eletrofisiológicos , Canais Iônicos , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Canais Iônicos/metabolismo , Ânions/metabolismo , Transporte de Íons/fisiologiaRESUMO
[This corrects the article DOI: 10.1039/D2RA07073A.].
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The phototransduction cascade in vertebrate rod visual cells is initiated by the photoactivation of rhodopsin, which enables the activation of the visual G protein transducin. It is terminated by the phosphorylation of rhodopsin, followed by the binding of arrestin. Here we measured the solution X-ray scattering of nanodiscs containing rhodopsin in the presence of rod arrestin to directly observe the formation of the rhodopsin/arrestin complex. Although arrestin self-associates to form a tetramer at physiological concentrations, it was found that arrestin binds to phosphorylated and photoactivated rhodopsin at 1:1 stoichiometry. In contrast, no complex formation was observed for unphosphorylated rhodopsin upon photoactivation, even at physiological arrestin concentrations, suggesting that the constitutive activity of rod arrestin is sufficiently low. UV-visible spectroscopy demonstrated that the rate of the formation of the rhodopsin/arrestin complex well correlates with the concentration of arrestin monomer rather than the tetramer. These findings indicate that arrestin monomer, whose concentration is almost constant due to the equilibrium with the tetramer, binds to phosphorylated rhodopsin. The arrestin tetramer would act as a reservoir of monomer to compensate for the large changes in arrestin concentration in rod cells caused by intense light or adaptation.
Assuntos
Células Fotorreceptoras Retinianas Bastonetes , Rodopsina , Rodopsina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Arrestina/metabolismo , Fosforilação , Proteínas de Ligação ao GTP/metabolismoRESUMO
Rhodopsins are transmembrane proteins with retinal chromophores that are involved in photo-energy conversion and photo-signal transduction in diverse organisms. In this study, we newly identified and characterized a rhodopsin from a thermophilic bacterium, Bellilinea sp. Recombinant Escherichia coli cells expressing the rhodopsin showed light-induced alkalization of the medium only in the presence of sodium ions (Na+), and the alkalization signal was enhanced by addition of a protonophore, indicating an outward Na+ pump function across the cellular membrane. Thus, we named the protein Bellilinea Na+-pumping rhodopsin, BeNaR. Of note, its Na+-pumping activity is significantly greater than that of the known Na+-pumping rhodopsin, KR2. We further characterized its photochemical properties as follows: (i) Visible spectroscopy and HPLC revealed that BeNaR has an absorption maximum at 524 nm with predominantly (>96%) the all-trans retinal conformer. (ii) Time-dependent thermal denaturation experiments revealed that BeNaR showed high thermal stability. (iii) The time-resolved flash-photolysis in the nanosecond to millisecond time domains revealed the presence of four kinetically distinctive photointermediates, K, L, M and O. (iv) Mutational analysis revealed that Asp101, which acts as a counterion, and Asp230 around the retinal were essential for the Na+-pumping activity. From the results, we propose a model for the outward Na+-pumping mechanism of BeNaR. The efficient Na+-pumping activity of BeNaR and its high stability make it a useful model both for ion transporters and optogenetics tools.
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Rodopsina , ATPase Trocadora de Sódio-Potássio , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Transporte de Íons , Bactérias/metabolismo , Íons , Sódio/química , Sódio/metabolismo , LuzRESUMO
Microbial rhodopsin is a family of photoreceptive membrane proteins that commonly consist of a seven-transmembrane domain and a derivative of vitamin-A, retinal, as a chromophore. In 2011, archaeorhodopsin-3 (AR3) was shown to exhibit voltage-dependent fluorescence changes in mammalian cells. Since then, AR3 and its variants have been used as genetically encoded voltage indicators, in which mostly intense laser stimulation (1-1000 W/cm2) is used for the detection of dim fluorescence of rhodopsin, leading to high spatiotemporal resolution. However, intense laser stimulation potentially causes serious cell damage, particularly during long-term imaging over minutes. In this study, we present the successful detection of voltage-sensitive fluorescence of AR3 and its high fluorescence mutant Archon1 in a variety of mammalian cell lines using low-intensity light emitting diode stimulation (0.15 W/cm2) with long exposure time (500 ms). The detection system enables real-time imaging of drug-induced slow changes in voltage within the cells for minutes harmlessly and without fluorescence bleaching. Therefore, we demonstrate a method to quantitatively understand the dynamics of slow changes in membrane voltage on long time scales.
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Rhodopsins, a family of photoreceptive membrane proteins, contain retinal as a chromophore and were firstly identified as reddish pigments from frog retina in 1876. Since then, rhodopsin-like proteins have been identified mainly from animal eyes. In 1971, a rhodopsin-like pigment was discovered from the archaeon Halobacterium salinarum and named bacteriorhodopsin. While it was believed that rhodopsin- and bacteriorhodopsin-like proteins were expressed only in animal eyes and archaea, respectively, before the 1990s, a variety of rhodopsin-like proteins (called animal rhodopsins or opsins) and bacteriorhodopsin-like proteins (called microbial rhodopsins) have been progressively identified from various tissues of animals and microorganisms, respectively. Here, we comprehensively introduce the research conducted on animal and microbial rhodopsins. Recent analysis has revealed that the two rhodopsin families have common molecular properties, such as the protein structure (i.e., 7-transmembrane structure), retinal structure (i.e., binding ability to cis- and trans-retinal), color sensitivity (i.e., UV- and visible-light sensitivities), and photoreaction (i.e., triggering structural changes by light and heat), more than what was expected at the early stages of rhodopsin research. Contrastingly, their molecular functions are distinctively different (e.g., G protein-coupled receptors and photoisomerases for animal rhodopsins and ion transporters and phototaxis sensors for microbial rhodopsins). Therefore, based on their similarities and dissimilarities, we propose that animal and microbial rhodopsins have convergently evolved from their distinctive origins as multi-colored retinal-binding membrane proteins whose activities are regulated by light and heat but independently evolved for different molecular and physiological functions in the cognate organism.
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Apoptosis is a type of programmed cell death that commonly occurs in multicellular organisms including humans and that is essential to eliminate unnecessary cells to keep organisms healthy. Indeed, inappropriate apoptosis leads to various diseases such as cancer and autoimmune disease. Here, we developed an optical method to regulate apoptotic cell death by controlling the intracellular pH with outward or inward proton pump rhodopsins, Archaerhodopsin-3 (AR3) or Rubricoccus marinas xenorhodopsin (RmXeR), respectively. The alkalization-induced shrinking of human HeLa cells cultured at pH 9.0 was significantly accelerated or decelerated by light-activated AR3 or RmXeR, respectively, implying the contribution of intracellular alkalization to the cell death. The light-activated AR3 induced cell shrinking at a physiologically neutral pH 7.4 and biochemical analysis revealed that the intracellular alkalization caused by AR3 triggered the mitochondrial apoptotic signaling pathway, which resulted in cell death accompanied by morphological changes. Phototriggered apoptosis (PTA) was also observed for other human cell lines, SH-SY5Y and A549 cells, implying its general applicability. We then used the PTA method with the nematode Caenorhabditis elegans as a model for living animals. Irradiation of transgenic worms expressing AR3 in chemosensing amphid sensory neurons significantly decreased their chemotaxis responses, which suggests that AR3 induced the cell death of amphid sensory neurons and the depression of chemotaxis responses. Thus, the PTA method has a high applicability both in vivo and in vitro, which suggests its potential as an optogenetic tool to selectively eliminate target cells with a high spatiotemporal resolution.
Assuntos
Bombas de Próton , Rodopsina , Animais , Apoptose , Células HeLa , Humanos , Transporte de Íons , Bombas de Próton/química , Rodopsina/químicaRESUMO
Microbial rhodopsins are photoreceptive membrane proteins showing various light-dependent biological activities. Styrene-maleic acid (SMA) copolymers spontaneously form nanoscale lipid particles containing membrane proteins and associated lipids without detergent, and can be used to characterize membrane molecules. Here, we provide a protocol to functionally express a thermally stable rhodopsin, Rubrobacter xylanophilus rhodopsin, and an unstable rhodopsin, Halobacterium salinarum sensory rhodopsin I, in Escherichia coli. We then describe the preparation of SMA and the extraction and purification of rhodopsin molecules using SMA. For complete details on the use and execution of this protocol, please refer to Ueta et al. (2020).
Assuntos
Escherichia coli , Rodopsinas Microbianas , Escherichia coli/genética , Maleatos , Proteínas de Membrana , Rodopsina , Rodopsinas Microbianas/genéticaRESUMO
We have developed a methodology for identifying further thermostabilizing mutations for an intrinsically thermostable membrane protein. The methodology comprises the following steps: (1) identifying thermostabilizing single mutations (TSSMs) for residues in the transmembrane region using our physics-based method; (2) identifying TSSMs for residues in the extracellular and intracellular regions, which are in aqueous environment, using an empirical force field FoldX; and (3) combining the TSSMs identified in steps (1) and (2) to construct multiple mutations. The methodology is illustrated for thermophilic rhodopsin whose apparent midpoint temperature of thermal denaturation Tm is â¼91.8 °C. The TSSMs previously identified in step (1) were F90K, F90R, and Y91I with ΔTm â¼5.6, â¼5.5, and â¼2.9 °C, respectively, and those in step (2) were V79K, T114D, A115P, and A116E with ΔTm â¼2.7, â¼4.2, â¼2.6, and â¼2.3 °C, respectively (ΔTm denotes the increase in Tm). In this study, we construct triple and quadruple mutants, F90K+Y91I+T114D and F90K+Y91I+V79K+T114D. The values of ΔTm for these multiple mutants are â¼11.4 and â¼13.5 °C, respectively. Tm of the quadruple mutant (â¼105.3 °C) establishes a new record in a class of outward proton pumping rhodopsins. It is higher than Tm of Rubrobacter xylanophilus rhodopsin (â¼100.8 °C) that was the most thermostable in the class before this study.