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Hideki Kandori - One of the best experts on this subject based on the ideXlab platform.
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Biophysics of Rhodopsins and optogenetics
Biophysical Reviews, 2020Co-Authors: Hideki KandoriAbstract:Rhodopsins are photoreceptive proteins and key tools in optogenetics. Although Rhodopsin was originally named as a red-colored pigment for vision, the modern meaning of Rhodopsin encompasses photoactive proteins containing a retinal chromophore in animals and microbes. Animal and microbial Rhodopsins respectively possess 11- cis and all- trans retinal, respectively. As cofactors bound with their animal and microbial Rhodopsin (seven transmembrane α-helices) environments, 11- cis and all- trans retinal undergo photoisomerization into all- trans and 13- cis retinal forms as part of their functional cycle. While animal Rhodopsins are G protein coupled receptors, the function of microbial Rhodopsins is highly divergent. Many of the microbial Rhodopsins are able to transport ions in a passive or an active manner. These light-gated channels or light-driven pumps represent the main tools for respectively effecting neural excitation and silencing in the emerging field of optogenetics. In this article, the biophysics of Rhodopsins and their relationship to optogenetics are reviewed. As history has proven, understanding the molecular mechanism of microbial Rhodopsins is a prerequisite for their rational exploitation as the optogenetics tools of the future.
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Chimeric microbial Rhodopsins for optical activation of Gs-proteins.
Biophysics, 2017Co-Authors: Kazuho Yoshida, Takahiro Yamashita, Yoshinori Shichida, Kengo Sasaki, Keiichi Inoue, Hideki KandoriAbstract:: We previously showed that the chimeric proteins of microbial Rhodopsins, such as light-driven proton pump bacterioRhodopsin (BR) and Gloeobacter Rhodopsin (GR) that contain cytoplasmic loops of bovine Rhodopsin, are able to activate Gt protein upon light absorption. These facts suggest similar protein structural changes in both the light-driven proton pump and animal Rhodopsin. Here we report two trials to engineer chimeric Rhodopsins, one for the inserted loop, and another for the microbial Rhodopsin template. For the former, we successfully activated Gs protein by light through the incorporation of the cytoplasmic loop of β2-adrenergic receptor (β2AR). For the latter, we did not observe any G-protein activation for the light-driven sodium pump from Indibacter alkaliphilus (IndiR2) or a light-driven chloride pump haloRhodopsin from Natronomonas pharaonis (NpHR), whereas the light-driven proton pump GR showed light-dependent G-protein activation. This fact suggests that a helix opening motion is common to G protein coupled receptor (GPCR) and GR, but not to IndiR2 and NpHR. Light-induced difference FTIR spectroscopy revealed similar structural changes between WT and the third loop chimera for each light-driven pump. A helical structural perturbation, which was largest for GR, was further enhanced in the chimera. We conclude that similar structural dynamics that occur on the cytoplasmic side of GPCR are needed to design chimeric microbial Rhodopsins.
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Ion-pumping microbial Rhodopsins
Frontiers in Molecular Biosciences, 2015Co-Authors: Hideki KandoriAbstract:Rhodopsins are light-sensing proteins used in optogenetics. The word “Rhodopsin” originates from the Greek words “rhodo” and “opsis”, indicating rose and sight, respectively. Although the classical meaning of Rhodopsin is the red-colored pigment in our eyes, the modern meaning of Rhodopsin encompasses photoactive proteins containing a retinal chromophore in animals and microbes. Animal and microbial Rhodopsins possess 11-cis and all-trans retinal, respectively, to capture light in seven transmembrane α-helices, and photoisomerizations into all-trans and 13-cis forms, respectively, initiate each function. Ion-transporting proteins can be found in microbial Rhodopsins, such as light-gated channels and light-driven pumps, which are the main tools in optogenetics. Light-driven pumps, such as archaeal H+ pump bacterioRhodopsin (BR) and Cl- pump haloRhodopsin (HR), were discovered in the 1970s, and their mechanism has been extensively studied. On the other hand, different kinds of H+ and Cl- pumps have been found in marine bacteria, such as proteoRhodopsin (PR) and Fulvimarina pelagi Rhodopsin (FR), respectively. In addition, a light-driven Na+ pump was found, Krokinobacter eikastus Rhodopsin 2 (KR2). These light-driven ion-pumping microbial Rhodopsins are classified as DTD, TSA, DTE, NTQ and NDQ Rhodopsins for BR, HR, PR, FR and KR2, respectively. Recent understanding of ion-pumping microbial Rhodopsins is reviewed in this paper.
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Chimeric proton-pumping Rhodopsins containing the cytoplasmic loop of bovine Rhodopsin.
PLOS ONE, 2014Co-Authors: Kengo Sasaki, Kazuho Yoshida, Takahiro Yamashita, Yoshinori Shichida, Keiichi Inoue, Hideki KandoriAbstract:G-protein-coupled receptors (GPCRs) transmit stimuli to intracellular signaling systems. Rhodopsin (Rh), which is a prototypical GPCR, possesses an 11-cis retinal. Photoisomerization of 11-cis to all-trans leads to structural changes in the protein of cytoplasmic loops, activating G-protein. Microbial Rhodopsins are similar heptahelical membrane proteins that function as bacterial sensors, light-driven ion-pumps, or light-gated channels. They possess an all-trans retinal, and photoisomerization to 13-cis triggers structural changes in protein. Despite these similarities, there is no sequence homology between visual and microbial Rhodopsins, and microbial Rhodopsins do not activate G-proteins. In this study, new chimeric proton-pumping Rhodopsins, proteoRhodopsin (PR) and Gloeobacter Rhodopsin (GR) were designed by replacing cytoplasmic loops with bovine Rh loops. Although G-protein was not activated by the PR chimeras, all 12 GR chimeras activated G-protein. The GR chimera containing the second cytoplasmic loop of bovine Rh did not activate G-protein. However, the chimera with a second and third double-loop further enhanced G-protein activation. Introduction of an E132Q mutation slowed the photocycle 30-fold and enhanced activation. The highest catalytic activity of the GR chimera was still 3,200 times lower than bovine Rh but only 64 times lower than amphioxus Go-Rhodopsin. This GR chimera showed a strong absorption change of the amide-I band on a light-minus-dark difference FTIR spectrum which could represent a larger helical opening, important for G-protein activation. The light-dependent catalytic activity of this GR chimera makes it a potential optogenetic tool for enzymatic activation by light.
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A light-driven sodium ion pump in marine bacteria
Nature Communications, 2013Co-Authors: Keiichi Inoue, Hikaru Ono, Rei Abe-yoshizumi, Hiroyasu Ito, Susumu Yoshizawa, Kazuhiro Kogure, Hideki KandoriAbstract:Light-driven proton-pumping Rhodopsins are widely distributed in many microorganisms. They convert sunlight energy into proton gradients that serve as energy source of the cell. Here we report a new functional class of a microbial Rhodopsin, a light-driven sodium ion pump. We discover that the marine flavobacterium Krokinobacter eikastus possesses two Rhodopsins, the first, KR1, being a prototypical proton pump, while the second, KR2, pumps sodium ions outward. Rhodopsin KR2 can also pump lithium ions, but converts to a proton pump when presented with potassium chloride or salts of larger cations. These data indicate that KR2 is a compatible sodium ion-proton pump, and spectroscopic analysis showed it binds sodium ions in its extracellular domain. These findings suggest that light-driven sodium pumps may be as important in situ as their proton-pumping counterparts.
John L Spudich - One of the best experts on this subject based on the ideXlab platform.
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Science, 2015Co-Authors: Elena G. Govorunova, Roger Janz, Xiaoqin Liu, Oleg A Sineshchekov, John L SpudichAbstract:Light-gated Rhodopsin cation channels from chlorophyte algae have transformed neuroscience research through their use as membrane-depolarizing optogenetic tools for targeted photoactivation of neuron firing. Photosuppression of neuronal action potentials has been limited by the lack of equally efficient tools for membrane hyperpolarization.We describe anion channel Rhodopsins (ACRs), a family of light-gated anion channels from cryptophyte algae that provide highly sensitive and efficient membrane hyperpolarization and neuronal silencing through light-gated chloride conduction. ACRs strictly conducted anions, completely excluding protons and larger cations, and hyperpolarized the membrane of cultured animal cells with much faster kinetics at less than one-thousandth of the light intensity required by the most efficient currently available optogenetic proteins. Natural ACRs provide optogenetic inhibition tools with unprecedented light sensitivity and temporal precision.
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Microbial Sensory Rhodopsins: Photochemistry and Function
Israel Journal of Chemistry, 2013Co-Authors: John L Spudich, David N. Zacks, Roberto A. BogomolniAbstract:The review covers recent progress on microbial sensory Rhodopsins, visual pigment-like retinylidene photoreceptors that function in phototaxis by archaeons, such as Halobacterium salinarium, and by unicellular eukaryotic algae, such as Chlamydomonas reinhardtii. Six demonstrably different sensory Rhodopsins are known in halophilic archaea. The best characterized is sensory Rhodopsin I (SR-I), a color-sensitive receptor that relays attractant and repellent photosignals to a tightly bound transducer protein HtrI (halobacterial transducer for sensory Rhodopsin I). New advances in the mechanism of signal transduction by the SR-I/HtrI complex from molecular-biological and biophysical approaches are summarized. Effects of HtrI on light-induced proton transfers in the receptor are discussed for their possible role in signaling. Current knowledge concerning the growing family of related archaeal sensory Rhodopsins is presented. The evidence for a sensory Rhodopsin in phototaxis by C. reinhardtii and other unicellular eukaryotic algae is reviewed. The molecular information is more limited than for the archaeal organisms, but the physiological information is rich and complex. Compelling data exist for a single retinal-containing receptor mediating both phototaxis and photophobic responses in C. reinhardtii. From retinal analog studies, the isomeric configuration and ring/chain conformation of the retinal in the receptor appear to be identical to those of the archaeal sensory Rhodopsins. Also, photoisomerization from all-trans- to 13-cis-retinal appears to be the trigger for signaling, as in the archaeal pigments. Conflicting early studies suggesting an 11-cis-retinal chromophore and signaling without photoisomerization are analyzed and possible explanations for those reports are suggested. As a general conclusion, the microbial sensory Rhodopsins provide an opportunity to explore photochemistry and protein/protein interaction in photosensory transduction in genetically tractable organisms.
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conformational changes in the photocycle of anabaena sensory Rhodopsin absence of the schiff base counterion protonation signal
Journal of Biological Chemistry, 2006Co-Authors: Vladislav B Bergo, Vishwa D Trivedi, Jason J Amsden, Maria Ntefidou, Kenneth J. Rothschild, Joel M Kralj, John L SpudichAbstract:Abstract Anabaena sensory Rhodopsin (ASR) is a novel microbial Rhodopsin recently discovered in the freshwater cyanobacterium Anabaena sp. PCC7120. This protein most likely functions as a photosensory receptor as do the related haloarchaeal sensory Rhodopsins. However, unlike the archaeal pigments, which are tightly bound to their cognate membrane-embedded transducers, ASR interacts with a soluble cytoplasmic protein analogous to transducers of animal vertebrate Rhodopsins. In this study, infrared spectroscopy was used to examine the molecular mechanism of photoactivation in ASR. Light adaptation of the pigment leads to a phototransformation of an all-trans/15-anti to 13-cis/15-syn retinylidene-containing species very similar in chromophore structural changes to those caused by dark adaptation in bacterioRhodopsin. Following 532 nm laser-pulsed excitation, the protein exhibits predominantly an all-trans retinylidene photocycle containing a deprotonated Schiff base species similar to those of other microbial Rhodopsins such as bacterioRhodopsin, sensory Rhodopsin II, and Neurospora Rhodopsin. However, no changes are observed in the Schiff base counterion Asp-75, which remains unprotonated throughout the photocycle. This result along with other evidence indicates that the Schiff base proton release mechanism differs significantly from that of other known microbial Rhodopsins, possibly because of the absence of a second carboxylate group at the ASR photoactive site. Several conformational changes are detected during the ASR photocycle including in the transmembrane helices E and G as indicated by hydrogen-bonding alterations of their native cysteine residues. In addition, similarly to animal vertebrate Rhodopsin, perturbations of the polar head groups of lipid molecules are detected.
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sensory Rhodopsin signaling in green flagellate algae
2005Co-Authors: Oleg A Sineshchekov, John L SpudichAbstract:The sensory Rhodopsins of the green f lagellate alga Chlamydomonas reinhardtii are recent additions to the large and diverse family of microbial Rhodopsins, proteins that are characterized by their visual pigment-like domain, consisting of 7-transmembrane helices forming an internal pocket for the chromophore retinal (see Spudich and Jung, Chapter 1). The first members of this family were observed in halophilic Archaea (reviewed in Hoff et al., 1997; Schafer et al., 1999; Spudich et al., 2000). In haloarchaea, two are light-driven ion pumps [bacterioRhodopsin (BR) and haloRhodopsin (HR)], and two are sensory receptors for phototaxis [sensory Rhodopsins I and II (SRI and SRII)]. Over the past four years, cloning, heterologous expression, and functional analysis of homologous genes from cultivated-microorganism genome projects as well as environmental genomics of uncultivated microbes have revealed photoactive archaeal-Rhodopsin homologs in the other two domains of life as well, i.e. Bacteria (Beja et al., 2001; Jung et al., 2003) and Eucarya (Bieszke et al., 1999a; Sineshchekov et al., 2002). Both transport and sensory Rhodopsins exist in Archaea and both functional classes are also found in Bacteria (e.g. proton-pumping marine proteoRhodopsins and Anabaena sensory Rhodopsin); so far only sensory members have been demonstrated in microbial Eucarya, namely the C. reinhardtii sensory Rhodopsins reviewed here. The prokaryotic Rhodopsins rank among the best-understood membrane proteins in terms of structure and function at the atomic level. Atomic resolution structures, which exist for <60 membrane proteins, have been obtained from electron and X-ray crystallography of BR (Grigorieff et al., 1996; Pebay-Peyroula et al., 1997; Essen et al., 1998; Luecke et al., 1999), HR (Kolbe et al., 2000), SRII (Luecke et al., 2001; Royant et al., 2001), and Anabaena sensory Rhodopsin (Vogeley et al., 2004). This knowledge and the fact that they can be activated by light, which permits precise temporal reso-
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Handbook of Photosensory Receptors - Sensory Rhodopsin Signaling in Green Flagellate Algae
Handbook of Photosensory Receptors, 2005Co-Authors: Oleg A Sineshchekov, John L SpudichAbstract:The sensory Rhodopsins of the green f lagellate alga Chlamydomonas reinhardtii are recent additions to the large and diverse family of microbial Rhodopsins, proteins that are characterized by their visual pigment-like domain, consisting of 7-transmembrane helices forming an internal pocket for the chromophore retinal (see Spudich and Jung, Chapter 1). The first members of this family were observed in halophilic Archaea (reviewed in Hoff et al., 1997; Schafer et al., 1999; Spudich et al., 2000). In haloarchaea, two are light-driven ion pumps [bacterioRhodopsin (BR) and haloRhodopsin (HR)], and two are sensory receptors for phototaxis [sensory Rhodopsins I and II (SRI and SRII)]. Over the past four years, cloning, heterologous expression, and functional analysis of homologous genes from cultivated-microorganism genome projects as well as environmental genomics of uncultivated microbes have revealed photoactive archaeal-Rhodopsin homologs in the other two domains of life as well, i.e. Bacteria (Beja et al., 2001; Jung et al., 2003) and Eucarya (Bieszke et al., 1999a; Sineshchekov et al., 2002). Both transport and sensory Rhodopsins exist in Archaea and both functional classes are also found in Bacteria (e.g. proton-pumping marine proteoRhodopsins and Anabaena sensory Rhodopsin); so far only sensory members have been demonstrated in microbial Eucarya, namely the C. reinhardtii sensory Rhodopsins reviewed here. The prokaryotic Rhodopsins rank among the best-understood membrane proteins in terms of structure and function at the atomic level. Atomic resolution structures, which exist for
Kwang-hwan Jung - One of the best experts on this subject based on the ideXlab platform.
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Biosynthetic Production of an isotopically Labelled Retinal in E. Coli
Biophysical Journal, 2017Co-Authors: Rachel A. Munro, Kwang-hwan Jung, Meaghan E. Ward, Vladimir Ladizhansky, Leonid S BrownAbstract:Solid-state Nuclear Magnetic Resonance (ssNMR) is an emerging biophysical technique which has been useful in studying the structure of integral membrane proteins such as microbial Rhodopsins. However, this technique requires the incorporation of isotopically-labelled atoms into the protein. This is usually accomplished through over-expression of Rhodopsins in E. coli, in minimal media wherein all carbon and nitrogen sources are isotopically labeled. The isomerization of a covalently bound retinal is an integral part of both microbial and animal Rhodopsin function. As such, the retinal binding pocket is of significant interest for ssNMR assignments. Unfortunately, the de novo organic synthesis of an isotopically-labelled retinal is cost-prohibitive in large scale expression. Previously, the biosynthesis of a retinal precursor, beta-carotene, has been introduced into many different organisms. This system can be extended to the E. coli expression strains UT5600 and BL21. We have shown that the novel biosynthetic production of an isotopically labelled retinal ligand concurrently with its apoprotein proteoRhodopsin in E. coli presents a cost effective alternative to de novo organic synthesis. By using alternately labeled carbon sources (glycerol) we were able to verify the biosynthetic pathway for retinal and assign several new carbon resonances for proteoRhodopsin-bound retinal.
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ph dependence of anabaena sensory Rhodopsin retinal isomer composition rate of dark adaptation and photochemistry
Journal of Physical Chemistry B, 2014Co-Authors: Rinat Rozin, Kwang-hwan Jung, Sanford Ruhman, Amir Wand, Mordechai ShevesAbstract:Microbial Rhodopsins are photoactive proteins, and their binding site can accommodate either all-trans or 13-cis retinal chromophore. The pH dependence of isomeric composition, dark-adaptation rate, and primary events of Anabaena sensory Rhodopsin (ASR), a microbial Rhodopsin discovered a decade ago, are presented. The main findings are: (a) Two pKa values of 6.5 and 4.0 assigned to two different protein residues are observed using spectroscopic titration experiments for both ground-state retinal isomers: all-trans, 15-anti (AT) and 13-cis, 15-syn (13C). The protonation states of these protein residues affect the absorption spectrum of the pigment and most probably the isomerization process of the retinal chromophore. An additional pKa value of 8.5 is observed only for 13C-ASR. (b) The isomeric composition of ASR is determined over a wide pH range and found to be almost pH-independent in the dark (>96% AT isomer) but highly pH-dependent in the light-adapted form. (c) The kinetics of dark adaptation is rec...
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Application of a sensitive near-field microwave microprobe to the nondestructive characterization of microbial Rhodopsin.
Journal of Biophotonics, 2012Co-Authors: Youngwoon Yoon, Ahreum Choi, Kwang-hwan Jung, Arsen Babajanyan, Tigran AbrahamyanAbstract:We study the opto-electrical properties of Natronomonas pharaonis sensory Rhodopsin II (NpSRII) by using a near-field microwave microprobe (NFMM) under external light illumination. To investigate the possibility of application of NFMM to biological macromolecules, we used time dependent properties of NPSRII before/after light activation which has three distinct states – ground-state, M-state, and O-state. The diagnostic ability of NFMM is demonstrated by measuring the microwave reflection coefficient (S11) spectrum of NpSRII under steady-state illumination in the wavelength range of 350–650 nm. Moreover, we present microwave reflection coefficient S11 spectra in the same wavelength range for two fast-photocycling Rhodopsins: green light-absorbing proteoRhodopsin (GPR) and Gloeobacter Rhodopsin (GR). In addition the frequency sweep shift can be detected completely even for tiny amounts of sample (∼10–3 OD of Rhodopsin). Based on these results NFMM shows both very high sensitivity for detecting conformational changes and produces a good time-resolved spectrum. (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
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asymmetric toggling of a natural photoswitch ultrafast spectroscopy of anabaena sensory Rhodopsin
Journal of the American Chemical Society, 2011Co-Authors: Amir Wand, Kwang-hwan Jung, Rinat Rozin, Tamar Eliash, Mordechai Sheves, Sanford RuhmanAbstract:Photochemistry in retinal proteins (RPs) is determined both by the properties of the retinal chromophore and by its interactions with the surrounding protein. The initial retinal configuration, and the isomerization coordinates active in any specific protein, must be important factors influencing the course of photochemistry. This is illustrated by the vast differences between the photoisomerization dynamics in visual pigments which start 11-cis and end all-trans, and those observed in microbial ion pumps and sensory Rhodopsins which start all-trans and end in a 13-cis configuration. However, isolating these factors is difficult since most RPs accommodate only one active stable ground-state configuration. Anabaena sensory Rhodopsin, allegedly functioning in cyanobacteria as a wavelength sensor, exists in two stable photoswitchable forms, containing all-trans and 13-cis retinal isomers, at a wavelength-dependent ratio. Using femtosecond spectroscopy, and aided by extraction of coherent vibrational signatur...
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An inward proton transport using anabaena sensory Rhodopsin
The Journal of Microbiology, 2011Co-Authors: Akira Kawanabe, Yuji Furutani, Kwang-hwan Jung, Hideki KandoriAbstract:ATP is synthesized by an enzyme that utilizes proton motive force and thus nature creates various proton pumps. The best understood proton pump is bacterioRhodopsin (BR), an outward-directed light-driven proton pump in Halobacterium salinarum . Many archaeal and eubacterial Rhodopsins are now known to show similar proton transport activity. Proton pumps must have a specific mechanism to exclude transport in the reverse direction to maintain a proton gradient, and in the case of BR, a highly hydrophobic cytoplasmic domain may constitute such machinery. Although an inward proton pump has neither been created naturally nor artificially, we recently reported that an inward-directed proton transport can be engineered from a bacterial Rhodopsin by a single amino acid replacement Anabaena sensory Rhodopsin (ASR) is a photochromic sensor in freshwater cyanobacteria, possessing little proton transport activity. When we replace Asp217 at the cytoplasmic domain (distance ∼15 Å from the retinal chromophore) to Glu, ASR is converted into an inward proton transport, driven by absorption of a single photon. FTIR spectra clearly show an increased proton affinity for Glu217, which presumably controls the unusual directionality opposite to normal proton pumps.
Yuki Sudo - One of the best experts on this subject based on the ideXlab platform.
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presence of a haloarchaeal haloRhodopsin like cl pump in marine bacteria
Microbes and Environments, 2018Co-Authors: Yu Nakajima, Takashi Kikukawa, Yohei Kumagai, Jaeho Song, Yoshitoshi Ogura, Kazuhiro Kogure, Takashi Tsukamoto, Tetsuya Hayashi, Makoto Demura, Yuki SudoAbstract:: Light-driven ion-pumping Rhodopsins are widely distributed among bacteria, archaea, and eukaryotes in the euphotic zone of the aquatic environment. H+-pumping Rhodopsin (proteoRhodopsin: PR), Na+-pumping Rhodopsin (NaR), and Cl--pumping Rhodopsin (ClR) have been found in marine bacteria, which suggests that these genes evolved independently in the ocean. Putative microbial Rhodopsin genes were identified in the genome sequences of marine Cytophagia. In the present study, one of these genes was heterologously expressed in Escherichia coli cells and the Rhodopsin protein named Rubricoccus marinus haloRhodopsin (RmHR) was identified as a light-driven inward Cl- pump. Spectroscopic assays showed that the estimated dissociation constant (Kd,int.) of this Rhodopsin was similar to that of haloarchaeal haloRhodopsin (HR), while the Cl--transporting photoreaction mechanism of this Rhodopsin was similar to that of HR, but different to that of the already-known marine bacterial ClR. This amino acid sequence similarity also suggested that this Rhodopsin is similar to haloarchaeal HR and cyanobacterial HRs (e.g., SyHR and MrHR). Additionally, a phylogenetic analysis revealed that retinal biosynthesis pathway genes (blh and crtY) belong to a phylogenetic lineage of haloarchaea, indicating that these marine Cytophagia acquired Rhodopsin-related genes from haloarchaea by lateral gene transfer. Based on these results, we concluded that inward Cl--pumping Rhodopsin is present in genera of the class Cytophagia and may have the same evolutionary origins as haloarchaeal HR.
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the early steps in the photocycle of a photosensor protein sensory Rhodopsin i from salinibacter ruber
Journal of Physical Chemistry B, 2014Co-Authors: Yuki Sudo, Misao Mizuno, Satoshi Takeuchi, Tahei Tahara, Yasuhisa MizutaniAbstract:Light absorption by the photoreceptor microbial Rhodopsin triggers trans–cis isomerization of the retinal chromophore surrounded by seven transmembrane α-helices. Sensory Rhodopsin I (SRI) is a dual functional photosensory Rhodopsin both for positive and negative phototaxis in microbes. By making use of the highly stable SRI protein from Salinibacter ruber (SrSRI), the early steps in the photocycle were studied by time-resolved spectroscopic techniques. All of the temporal behaviors of the Sn←S1 absorption, ground-state bleaching, K intermediate absorption, and stimulated emission were observed in the femto- to picosecond time region by absorption spectroscopy. The primary process exhibited four dynamics similar to other microbial Rhodopsins. The first dynamics (τ1 ∼ 54 fs) corresponds to the population branching process from the Franck–Condon region to the reactive (S1r) and nonreactive (S1nr) S1 states. The second dynamics (τ2 = 0.64 ps) is the isomerization process of the S1r state to generate the grou...
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a microbial Rhodopsin with a unique retinal composition shows both sensory Rhodopsin ii and bacterioRhodopsin like properties
Journal of Biological Chemistry, 2011Co-Authors: Yuki Sudo, Takashi Kikukawa, Hideki Kandori, Kunio Ihara, Daisuke Suzuki, Shiori Kobayashi, Hiroki Irieda, Michio HommaAbstract:Rhodopsins possess retinal chromophore surrounded by seven transmembrane α-helices, are widespread in prokaryotes and in eukaryotes, and can be utilized as optogenetic tools. Although Rhodopsins work as distinctly different photoreceptors in various organisms, they can be roughly divided according to their two basic functions, light-energy conversion and light-signal transduction. In microbes, light-driven proton transporters functioning as light-energy converters have been modified by evolution to produce sensory receptors that relay signals to transducer proteins to control motility. In this study, we cloned and characterized two newly identified microbial Rhodopsins from Haloquadratum walsbyi. One of them has photochemical properties and a proton pumping activity similar to the well known proton pump bacterioRhodopsin (BR). The other, named middle Rhodopsin (MR), is evolutionarily transitional between BR and the phototactic sensory Rhodopsin II (SRII), having an SRII-like absorption maximum, a BR-like photocycle, and a unique retinal composition. The wild-type MR does not have a light-induced proton pumping activity. On the other hand, a mutant MR with two key hydrogen-bonding residues located at the interaction surface with the transducer protein HtrII shows robust phototaxis responses similar to SRII, indicating that MR is potentially capable of the signaling. These results demonstrate that color tuning and insertion of the critical threonine residue occurred early in the evolution of sensory Rhodopsins. MR may be a missing link in the evolution from type 1 Rhodopsins (microorganisms) to type 2 Rhodopsins (animals), because it is the first microbial Rhodopsin known to have 11-cis-retinal similar to type 2 Rhodopsins.
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Spectral tuning in sensory Rhodopsin I from Salinibacter ruber
Journal of Biological Chemistry, 2011Co-Authors: Yuki Sudo, Yasufumi Yuasa, Jun Shibata, Daisuke Suzuki, Michio HommaAbstract:Organisms utilize light as energy sources and as signals. Rhodopsins, which have seven transmembrane α-helices with retinal covalently linked to a conserved Lys residue, are found in various organisms as distant in evolution as bacteria, archaea, and eukarya. One of the most notable properties of Rhodopsin molecules is the large variation in their absorption spectrum. Sensory Rhodopsin I (SRI) and sensory Rhodopsin II (SRII) function as photosensors and have similar properties (retinal composition, photocycle, structure, and function) except for their λ(max) (SRI, ∼560 nm; SRII, ∼500 nm). An expression system utilizing Escherichia coli and the high protein stability of a newly found SRI-like protein, SrSRI, enables studies of mutant proteins. To determine the residue contributing to the spectral shift from SRI to SRII, we constructed various SRI mutants, in which individual residues were substituted with the corresponding residues of SRII. Three such mutants of SrSRI showed a large spectral blue-shift (>14 nm) without a large alteration of their retinal composition. Two of them, A136Y and A200T, are newly discovered color tuning residues. In the triple mutant, the λ(max) was 525 nm. The inverse mutation of SRII (F134H/Y139A/T204A) generated a spectral-shifted SRII toward longer wavelengths, although the effect is smaller than in the case of SRI, which is probably due to the lack of anion binding in the SRII mutant. Thus, half of the spectral shift from SRI to SRII could be explained by only those three residues taking into account the effect of Cl(-) binding.
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structural changes of salinibacter sensory Rhodopsin i upon formation of the k and m photointermediates
Biochemistry, 2008Co-Authors: Daisuke Suzuki, Yuji Furutani, Michio Homma, Yuki Sudo, Hazuki Takahashi, Hideki KandoriAbstract:Sensory Rhodopsin I (SRI) is one of the most interesting photosensory receptors in nature because of its ability to mediate opposite signals depending on light color by photochromic one-photon and two-photon reactions. Recently, we characterized SRI from eubacterium Salinibacter ruber (SrSRI). This protein allows more detailed information about the structure and structural changes of SRI during its action to be obtained. In this paper, Fourier transform infrared (FTIR) spectroscopy is applied to SrSRI, and the spectral changes upon formation of the K and M intermediates are compared with those of other archaeal Rhodopsins, SRI from Halobacterium salinarum (HsSRI), sensory Rhodopsin II (SRII), bacterioRhodopsin (BR), and haloRhodopsin (HR). Spectral comparison of the hydrogen out-of-plane (HOOP) vibrations of the retinal chromophore in the K intermediates shows that extended choromophore distortion takes place in SrSRI and HsSRI, as well as in SRII, whereas the distortion is localized in the Schiff base re...
Yuji Furutani - One of the best experts on this subject based on the ideXlab platform.
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An inward proton transport using anabaena sensory Rhodopsin
The Journal of Microbiology, 2011Co-Authors: Akira Kawanabe, Yuji Furutani, Kwang-hwan Jung, Hideki KandoriAbstract:ATP is synthesized by an enzyme that utilizes proton motive force and thus nature creates various proton pumps. The best understood proton pump is bacterioRhodopsin (BR), an outward-directed light-driven proton pump in Halobacterium salinarum . Many archaeal and eubacterial Rhodopsins are now known to show similar proton transport activity. Proton pumps must have a specific mechanism to exclude transport in the reverse direction to maintain a proton gradient, and in the case of BR, a highly hydrophobic cytoplasmic domain may constitute such machinery. Although an inward proton pump has neither been created naturally nor artificially, we recently reported that an inward-directed proton transport can be engineered from a bacterial Rhodopsin by a single amino acid replacement Anabaena sensory Rhodopsin (ASR) is a photochromic sensor in freshwater cyanobacteria, possessing little proton transport activity. When we replace Asp217 at the cytoplasmic domain (distance ∼15 Å from the retinal chromophore) to Glu, ASR is converted into an inward proton transport, driven by absorption of a single photon. FTIR spectra clearly show an increased proton affinity for Glu217, which presumably controls the unusual directionality opposite to normal proton pumps.
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structural changes of salinibacter sensory Rhodopsin i upon formation of the k and m photointermediates
Biochemistry, 2008Co-Authors: Daisuke Suzuki, Yuji Furutani, Michio Homma, Yuki Sudo, Hazuki Takahashi, Hideki KandoriAbstract:Sensory Rhodopsin I (SRI) is one of the most interesting photosensory receptors in nature because of its ability to mediate opposite signals depending on light color by photochromic one-photon and two-photon reactions. Recently, we characterized SRI from eubacterium Salinibacter ruber (SrSRI). This protein allows more detailed information about the structure and structural changes of SRI during its action to be obtained. In this paper, Fourier transform infrared (FTIR) spectroscopy is applied to SrSRI, and the spectral changes upon formation of the K and M intermediates are compared with those of other archaeal Rhodopsins, SRI from Halobacterium salinarum (HsSRI), sensory Rhodopsin II (SRII), bacterioRhodopsin (BR), and haloRhodopsin (HR). Spectral comparison of the hydrogen out-of-plane (HOOP) vibrations of the retinal chromophore in the K intermediates shows that extended choromophore distortion takes place in SrSRI and HsSRI, as well as in SRII, whereas the distortion is localized in the Schiff base re...
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ftir study of the l intermediate of anabaena sensory Rhodopsin structural changes in the cytoplasmic region
Biochemistry, 2008Co-Authors: Akira Kawanabe, Yuji Furutani, Kwang-hwan Jung, Sa Ryong Yoon, Hideki KandoriAbstract:Anabaena sensory Rhodopsin (ASR) is an archaeal-type Rhodopsin found in eubacteria. The gene encoding ASR forms a single operon with ASRT (ASR transducer) that is a 14 kDa soluble protein, suggesting that ASR functions as a photochromic sensor by activating the soluble transducer. One of the characteristics of ASR is that the formation of the M intermediate accompanies a proton transfer from the Schiff base to Asp217 in the cytoplasmic side [Shi, L., Yoon, S. R., Bezerra, A. G., Jr., Jung, K. H., and Brown, L. S. (2006) J. Mol. Biol. 358, 686−700], in remarkable contrast to other archaeal-type Rhodopsins such as a light-driven proton-pump, bacterioRhodopsin (BR). In this study, we applied low-temperature Fourier transform infrared (FTIR) spectroscopy to the all-trans form of ASR at 170 K, and compared the structural changes in the L intermediate with those of BR. The ASRL minus ASR difference spectra were essentially similar to those for BR, suggesting common structures for the L state in ASR and BR. On t...
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photochromism of anabaena sensory Rhodopsin
Journal of the American Chemical Society, 2007Co-Authors: Akira Kawanabe, Yuji Furutani, Kwang-hwan Jung, Hideki KandoriAbstract:Protein-controlled photochemical reactions often mediate biological light-signal and light-energy conversions. Microbial Rhodopsins possess all-trans or 13-cis retinal as the chromophore in the dark, and in the light-driven proton pump, bacterioRhodopsin (BR), the stable photoproduct at the end of the functional cycle of the all-trans form is 100% all-trans. In contrast, a microbial Rhodopsin discovered in Anabaena PCC7120 is believed to function as a photochromic sensor. For Anabaena sensory Rhodopsin (ASR), the photoreaction is expected to be not cyclic, but photochromic. The present low-temperature UV−visible spectroscopy of ASR indeed revealed that the stable photoproduct of the all-trans form in ASR is 100% 13-cis, and that of the 13-cis form is 100% all-trans. The complete photocycle for the proton pump in BR and the complete photochromism for the chromatic sensor of ASR are highly advantageous for their functions. Thus, the microbial Rhodopsins have acquired unique photoreactions, in spite of their...
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ftir study of the photoisomerization processes in the 13 cis and all trans forms of anabaena sensory Rhodopsin at 77 k
Biochemistry, 2006Co-Authors: Akira Kawanabe, Yuji Furutani, Kwang-hwan Jung, Hideki KandoriAbstract:Archaeal-type Rhodopsins can accommodate either all-trans- or 13-cis,15-syn-retinal in their chromophore binding site in the dark, but only the former isomer is functionally important. In contrast, Anabaena sensory Rhodopsin (ASR), an archaeal-type Rhodopsin found in eubacteria, exhibits a photochromic interconversion of both forms, suggesting that ASR functions as a photosensor which interacts with its 14 kDa soluble transducer differently in the all-trans and 13-cis,15-syn forms. In this study, we applied low-temperature Fourier transform infrared (FTIR) spectroscopy to the 13-cis,15-syn form of ASR (13C-ASR) at 77 K and compared the local structure around the chromophore and its structural changes upon retinal photoisomerization with those of the all-trans form (AT-ASR) [Furutani, Y., Kawanabe, A., Jung, K. H., and Kandori, H. (2005) Biochemistry 44, 12287−12296]. By use of [ζ-15N]lysine-labeled ASR, we identified the N−D stretching vibrations of the Schiff base (in D2O) at 2165 cm-1 for 13C-ASR and at...
