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Now showing items 1 - 16 of 14512

  • Proteomonas sulcata ACR1: A Fast Anion Channelrhodopsin

    Govorunova, Elena G.   Sineshchekov, Oleg A.   Spudich, John L.  

    Natural channelrhodopsins with strictly anion selectivity and high unitary conductance have been recently discovered in the cryptophyte alga Guillardia theta. These proteins, called anion channelrhodopsins (ACRs), are of interest for their novel function and also because they were shown to be highly efficient tools to inhibit neuronal action potentials with light. We show that a homologous protein from the cryptophyte alga Proteomonas sulcata (named here PsuACR1) exhibits similar strict anion selectivity as the previously identified G. theta ACRs. Like G. theta ACRs, PsuACR1 lacks a protonatable residue at the position of the proton acceptor Asp-85 in bacteriorhodopsin, which may be a key characteristic of ACR family members shared by haloarchaeal chloride pumps. Of importance for its potential use in optogenetics, despite its 10-fold lower channel activity than the GtACRs, PsuACR1 exhibits an eightfold more rapid channel closing half-time making it uniquely suitable for silencing the subclass of high-frequency firing neurons when high-time resolution is needed. The existence of a rhodopsin with properties similar to G. theta ACRs in a different cryptophyte genus indicates that such proteins may be widespread in the phylum of cryptophyte algae.
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  • Mechanism divergence in microbial rhodopsins

    Spudich, John L.   Sineshchekov, Oleg A.   Govorunova, Elena G.  

    A fundamental design principle of microbial rhodopsins is that they share the same basic light-induced conversion between two conformers. Alternate access of the Schiff base to the outside and to the cytoplasm in the outwardly open "E" conformer and cytoplasmically open "C" conformer, respectively, combined with appropriate timing of pKa changes controlling Schiff base proton release and uptake make the proton path through the pumps vectorial. Phototaxis receptors in prokaryotes, sensory rhodopsins I and II, have evolved new chemical processes not found in their proton pump ancestors, to alter the consequences of the conformational change or modify the change itself. Like proton pumps, sensory rhodopsin II undergoes a photoinduced E -> C transition, with the C conformer a transient intermediate in the photocycle. In contrast, one light-sensor (sensory rhodopsin I bound to its transducer Htrl) exists in the dark as the C conformer and undergoes a light-induced C -> E transition, with the E conformer a transient photocycle intermediate. Current results indicate that algal phototaxis receptors channelrhodopsins undergo redirected Schiff base proton transfers and a modified E -> C transition which, contrary to the proton pumps and other sensory rhodopsins, is not accompanied by the closure of the external half-channel. The article will review our current understanding of how the shared basic structure and chemistry of microbial rhodopsins have been modified during evolution to create diverse molecular functions: light-driven ion transport and photosensory signaling by protein-protein interaction and light-gated ion channel activity. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks. (C) 2013 Elsevier B.V. All rights reserved.
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  • Intramolecular Proton Transfer in Channelrhodopsins

    Sineshchekov, Oleg A.   Govorunova, Elena G.   Wang, Jihong   Li, Hai   Spudich, John L.  

    Channelrhodopsins serve as photoreceptors that control the motility behavior of green flagellate algae and act as light-gated ion channels when heterologously expressed in animal cells. Here, we report direct measurements of proton transfer from the retinylidene Schiff base in several channelrhodopsin variants expressed in HEK293 cells. A fast outward-directed current precedes the passive channel current that has the opposite direction at physiological holding potentials. This rapid charge movement occurs on the timescale of the M intermediate formation in microbial rhodopsins, including that for channelrhodopsin from Chlamydomonas augustae and its mutants, reported in this study. Mutant analysis showed that the glutamate residue corresponding to Asp(85) in bacteriorhodopsin acts as the primary acceptor of the Schiff-base proton in low-efficiency channelrhodopsins. Another photoactive-site residue corresponding to Asp(212) in bacteriorhodopsin serves as an alternative proton acceptor and plays a more important role in channel opening than the primary acceptor. In more efficient channelrhodopsins from Chlamydomonas reinhardtii, Mesostigma viride, and Platymonas (Tetraselmis) subcordiformis, the fast current was apparently absent. The inverse correlation of the outward proton transfer and channel activity is consistent with channel function evolving in channelrhodopsins at the expense of their capacity for active proton transport.
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  • In Vitro Demonstration of Light-Driven Na+/H+ Pumping by a Microbial Rhodopsin

    Li, Hai   Sineshchekov, Oleg A.   da Silva, Giordano F. Z.   Spudich, John L.  

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  • Ultrasensitive Measurements of Microbial Rhodopsin Photocycles Using Photochromic FRET

    Bayraktar, Halil   Fields, Alexander P.   Kralj, Joel M.   Spudich, John L.   Rothschild, Kenneth J.  

    Microbial rhodopsins are an important class of light-activated transmembrane proteins whose function is typically studied on bulk samples. Herein, we apply photochromic fluorescence resonance energy transfer to investigate the dynamics of these proteins with sensitivity approaching the single-molecule limit. The brightness of a covalently linked organic fluorophore is modulated by changes in the absorption spectrum of the endogenous retinal chromophore that occur as the molecule undergoes a light-activated photocycle. We studied the photocycles of blue-absorbing proteorhodopsin and sensory rhodopsin II (SRII). Clusters of 23 molecules of SRII clearly showed a light-induced photocycle. Single molecules of SRII showed a photocycle upon signal averaging over several illumination cycles.
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  • Mechanisms,Diversity and Optogenetic Applications of Channelrhodopsins from Cryptophyte Algae

    Govorunova, Elena G.   Spudich, John L.  

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  • A Schiff base connectivity switch in sensory rhodopsin signaling

    Sineshchekov, Oleg A.   Sasaki, Jun   Phillips, Brian J.   Spudich, John L.  

    Sensory rhodopsin I (SRI) in Halobacterium salinarum acts as a receptor for single-quantum attractant and two-quantum repellent phototaxis, transmitting light stimuli via its bound transducer Htrl. Signal-inverting mutations in the SRI-Htrl complex reverse the single-quantum response from attractant to repellent. Fast intramolecular charge movements reported here reveal that the unphotolyzed SRI-Htrl complex exists in two conformational states, which differ by their connection of the retinylidene Schiff base in the SRI photoactive site to inner or outer half-channels. In single-quantum photochemical reactions, the conformer with the Schiff base connected to the cytoplasmic (CP) half-channel generates an attractant signal, whereas the conformer with the Schiff base connected to the extracellular (EC) half-channel generates a repellent signal. In the wild-type complex the conformer equilibrium is poised strongly in favor of that with CP-accessible Schiff base. Signal-inverting mutations shift the equilibrium in favor of the EC-accessible Schiff base form, and suppressor mutations shift the equilibrium back toward the CP-accessible Schiff base form, restoring the wild-type phenotype. Our data show that the sign of the behavioral response directly correlates with the state of the connectivity switch, not with the direction of proton movements or changes in acceptor pK(a). These findings identify a shared fundamental process in the mechanisms of transport and signaling by the rhodopsin family. Furthermore, the effects of mutations in the Htrl subunit of the complex on SRI Schiff base connectivity indicate that the two proteins are tightly coupled to form a single unit that undergoes a concerted conformational transition.
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  • Single-molecule tour de force: Teasing apart a signaling complex

    Hoff, Wouter D.   Spudich, John L.  

    The phototaxis receptor sensory rhodopsin 11 communicates with its transducer in a membrane-embedded complex. The first application of single-molecule force spectroscopy to receptor-transducer interaction by Cisneros et al. (2008) reveals new structural features in the signaling complex.
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  • Animal Models in Eye Research || The Simplest Eyes

    Spudich, John L.  

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  • Cross-protomer interaction with the photoactive site in oligomeric proteorhodopsin complexes

    Ran, Tingting   Ozorowski, Gabriel   Gao, Yanyan   Sineshchekov, Oleg A.   Wang, Weiwu   Spudich, John L.   Luecke, Hartmut  

    Proteorhodopsins (PRs), members of the microbial rhodopsin superfamily of seven-transmembrane-helix proteins that use retinal chromophores, comprise the largest subfamily of rhodopsins, yet very little structural information is available. PRs are ubiquitous throughout the biosphere and their genes have been sequenced in numerous species of bacteria. They have been shown to exhibit ion-pumping activity like their archaeal homolog bacteriorhodopsin (BR). Here, the first crystal structure of a proteorhodopsin, that of a blue-light-absorbing proteorhodopsin (BPR) isolated from the Mediterranean Sea at a depth of 12 m (Med12BPR), is reported. Six molecules of Med12BPR form a doughnut-shaped C-6 hexameric ring, unlike BR, which forms a trimer. Furthermore, the structures of two mutants of a related BPR isolated from the Pacific Ocean near Hawaii at a depth of 75 m (HOT75BPR), which show a C-5 pentameric arrangement, are reported. In all three structures the retinal polyene chain is shifted towards helix C when compared with other microbial rhodopsins, and the putative proton-release group in BPR differs significantly from those of BR and xanthorhodopsin (XR). The most striking feature of proteorhodopsin is the position of the conserved active-site histidine (His75, also found in XR), which forms a hydrogen bond to the proton acceptor from the same molecule (Asp97) and also to Trp34 of a neighboring protomer. Trp34 may function by stabilizing His75 in a conformation that favors a deprotonated Asp97 in the dark state, and suggests cooperative behavior between protomers when the protein is in an oligomeric form. Mutation-induced alterations in proton transfers in the BPR photocycle in Escherichia coli cells provide evidence for a similar cross-protomer interaction of BPR in living cells and a functional role of the inter-protomer Trp34-His75 interaction in ion transport. Finally, Wat402, a key molecule responsible for proton translocation between the Schiff base and the proton acceptor in BR, appears to be absent in PR, suggesting that the ion-transfer mechanism may differ between PR and BR.
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  • Crystal structure of the Anabaena sensory rhodopsin transducer

    Vogeley, Lutz   Trivedi, Vishwa D.   Sineshchekov, Oleg A.   Spudich, Elena N.   Spudich, John L.   Luecke, Hartmut  

    We present crystal structures of the Anabaena sensory rhodopsin transducer (ASRT), a soluble cytoplasmic protein that interacts with the first structurally characterized eubacterial retinylidene photoreceptor Anabaena sensory rhodopsin (ASR). Four crystal structures of ASRT from three different spacegroups were obtained, in all of which ASRT is present as a planar (C4) tetramer, consistent with our characterization of ASRT as a of tetramer in solution. The ASRT tetramer is tightly packed, with large interfaces where the well-structured P-sandwich portion of the monomers provides the bulk of the tetramer-forming interactions, and forms a flat, stable surface on one side of the tetramer (the beta-face). Only one of our four different ASRT crystals reveals a C-terminal a-helix in the otherwise all-beta protein, together with a large loop from each monomer on the opposite face of the tetramer (the a-face), which is flexible and largely disordered in the other three crystal forms. Gel-filtration chromatography demonstrated that ASRT forms stable tetramers in solution and isothermal microcalorimetry showed that the ASRT tetramer binds to ASR with a stoichiometry of one ASRT tetramer per one ASR photoreceptor with a K-d of 8 mu M in the highest affinity measurements. Possible mechanisms for the interaction of this transducer tetramer with the ASR photoreceptor via its flexible a-face to mediate transduction of the light signal are discussed. (c) 2007 Elsevier Ltd. All rights reserved.
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  • Bacteriorhodopsin-like channelrhodopsins:Alternative mechanism for control of cation conductance

    Sineshchekov, Oleg A.   Govorunova, Elena G.   Li, Hai   Spudich, John L.  

    The recently discovered cation-conducting channelrhodopsins in cryptophyte algae are far more homologous to haloarchaeal rhodopsins, in particular the proton pump bacteriorhodopsin (BR), than to earlier known channelrhodopsins. They uniquely retain the two carboxylate residues that define the vectorial proton path in BR in which Asp-85 and Asp-96 serve as acceptor and donor, respectively, of the photoactive site Schiff base (SB) proton. Here we analyze laser flash-induced photocurrents and photochemical conversions in Guillardia theta cation channelrhodopsin 2 (GtCCR2) and its mutants. Our results reveal a model in which the GtCCR2 retinylidene SB chromophore rapidly deprotonates to the Asp-85 homolog, as in BR. Opening of the cytoplasmic channel to cations in GtCCR2 requires the Asp-96 homolog to be unprotonated, as has been proposed for the BR cytoplasmic channel for protons. However, reprotonation of the GtCCR2 SB occurs not from the Asp-96 homolog, but by proton return from the earlier protonated acceptor, preventing vectorial proton translocation across the membrane. In GtCCR2, deprotonation of the Asp-96 homolog is required for cation channel opening and occurs > 10-fold faster than reprotonation of the SB, which temporally correlates with channel closing. Hence in GtCCR2, cation channel gating is tightly coupled to intramolecular proton transfers involving the same residues that define the vectorial proton path in BR.
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  • Microbial rhodopsins: functional versatility and genetic mobility

    Sharma, Adrian K.   Spudich, John L.   Doolittle, W. Ford  

    The type 1 (microbial) rhodopsins are a diverse group of photochemically reactive proteins that span the three domains of life. Their broad phylogenetic distribution has motivated conjecture that rhodopsin-like functionality was present in the last common ancestor of all life. Here, we discuss the evolution of the type 1 microbial rhodopsins and document five cases of lateral gene transfer (LGT) between domains. We suggest that, thanks to the functional versatility of these retinylidene proteins and the relative ease with which they can complement the existing energy-generating or photosensory repertoires of many organisms, LGT is in fact the principal force that determines their broad but patchy distribution.
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  • Structural Changes in an Anion Channelrhodopsin:Formation of the K and L Intermediates at 80 K

    Yi, Adrian   Li, Hai   Mamaeva, Natalia   De Cordoba, Roberto E. Fernandez   Lugtenburg, Johan   DeGrip, Willem J.   Spudich, John L.   Rothschild, Kenneth J.  

    A recently discovered natural family of light-gated anion channelrhodopsins (ACRs) from cryptophyte algae provides an effective means of optogenetically silencing neurons. The most extensively studied ACR, is from Guillardia theta (GtACR1). Earlier studies of GtACR1 have established a correlation between formation of a blue-shifted L-like intermediate and the anion channel "open" state. To study structural changes of GtACR1 in the K and L intermediates of the photocycle, a combination of low-temperature Fourier transform infrared (FTIR) and ultraviolet visible absorption difference spectroscopy was used along with stable-isotope retinal labeling and site-directed mutagenesis. In contrast to bacteriorhodopsin (BR) and other microbial rhodopsins, which form only a stable red-shifted K intermediate at 80 K, GtACR1 forms both stable K and L-like intermediates. Evidence includes the appearance of positive ethylenic and fingerprint vibrational hands characteristic of the L intermediate as well as a positive visible absorption band near 485 nm. FTIR difference bands in the carboxylic acid C=3DO stretching region indicate: hat several Asp/Glu residues undergo hydrogen bonding changes at 80 K. The Glu68 -> Gln and, Ser97 -> Glu substitutions, residues located close to the retinylidene Schiff base; altered the K:L ratio and several of the FTIR bands in the carboxylic acid region. In the case of the Ser97 -> Glu substitution, a significant red-shift of the absorption wavelength of the K and L intermediates occurs. Sequence comparisons suggest that L formation in GtACR1 at 80 K is due in part to the substitution of the-highly conserved Leu or Ile at position 93 in helix 3 (BR sequence) with the homologous Met105 in GtACR1.
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  • Sensory rhodopsin II: functional insights from structure

    Spudich, John L.   Luecke, Hartmut  

    The authors review the recent rapid progress in atomic-resolution structural analyses of the first sensory member of the widespread microbial rhodopsin family: the haloarchaeal phototaxis receptor sensory rhodopsin II.
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  • 2SA51 Microbial Rhodopsins : Structure and Mechanism in Sensory Signaling and Transport

    Spudich, John L.  

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