Sensory Rhodopsin

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John L. Spudich - One of the best experts on this subject based on the ideXlab platform.

  • his166 is the schiff base proton acceptor in attractant phototaxis receptor Sensory Rhodopsin i
    Biochemistry, 2014
    Co-Authors: Jun Sasaki, Yuji Furutani, John L. Spudich, Oleg A Sineshchekov, Hazuki Takahashi, Hideki Kandori
    Abstract:

    Photoactivation of attractant phototaxis receptor Sensory Rhodopsin I (SRI) in Halobacterium salinarum entails transfer of a proton from the retinylidene chromophore’s Schiff base (SB) to an unidentified acceptor residue on the cytoplasmic half-channel, in sharp contrast to other microbial Rhodopsins, including the closely related repellent phototaxis receptor SRII and the outward proton pump bacterioRhodopsin, in which the SB proton acceptor is an aspartate residue salt-bridged to the SB in the extracellular (EC) half-channel. His166 on the cytoplasmic side of the SB in SRI has been implicated in the SB proton transfer reaction by mutation studies, and mutants of His166 result in an inverted SB proton release to the EC as well as inversion of the protein’s normally attractant phototaxis signal to repellent. Here we found by difference Fourier transform infrared spectroscopy the appearance of Fermi-resonant X–H stretch modes in light-minus-dark difference spectra; their assignment with 15N labeling and si...

  • opposite displacement of helix f in attractant and repellent signaling by Sensory Rhodopsin htr complexes
    Journal of Biological Chemistry, 2011
    Co-Authors: Jun Sasaki, Ahlim Tsai, John L. Spudich
    Abstract:

    Two forms of the phototaxis receptor Sensory Rhodopsin I distinguished by differences in its photoactive site have been shown to be directly correlated with attractant and repellent signaling by the dual-signaling protein. In prior studies, differences in the photoactive site defined the two forms, namely the direction of light-induced proton transfer from the chromophore and the pKa of an Asp counterion to the protonated chromophore. Here, we show by both in vivo and in vitro measurements that the two forms are distinct protein conformers with structural similarities to two conformers seen in the light-driven proton transport cycle of the related protein bacterioRhodopsin. Measurements of spontaneous cell motility reversal frequencies, an in vivo measure of histidine kinase activity in the phototaxis system, indicate that the two forms are a photointerconvertible pair, with one conformer activating and the other inhibiting the kinase. Protein conformational changes in these photoconversions monitored by site-directed spin labeling show that opposite structural changes in helix F, distant from the photoactive site, correspond to the opposite phototaxis signals. The results provide the first direct evidence that displacements of helix F are directly correlated with signaling and impact our understanding of the Sensory Rhodopsin I signaling mechanism and the evolution of diverse functionality in this protein family.

  • attractant and repellent signaling conformers of Sensory Rhodopsin transducer complexes
    Biochemistry, 2010
    Co-Authors: Oleg A Sineshchekov, Jun Sasaki, Jihong Wang, John L. Spudich
    Abstract:

    Attractant and repellent signaling conformers of the dual-signaling phototaxis receptor Sensory Rhodopsin I and its transducer subunit (SRI−HtrI) have recently been distinguished experimentally by the opposite connection of their retinylidene protonated Schiff bases to the outwardly located periplasmic side and inwardly located cytoplasmic side. Here we show that the pKa of the outwardly located Asp76 counterion in the outwardly connected conformer is lowered by ∼1.5 units from that of the inwardly connected conformer. The pKa difference enables quantitative determination of the relative amounts of the two conformers in wild-type cells and behavioral mutants prior to photoexcitation, comparison of their absorption spectra, and determination of their relative signaling efficiency. We have shown that the one-photon excitation of the SRI−HtrI attractant conformer causes a Schiff base connectivity switch from inwardly connected to outwardly connected states in the attractant signaling photoreaction. Conversel...

  • a schiff base connectivity switch in Sensory Rhodopsin signaling
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Oleg A Sineshchekov, Jun Sasaki, Brian J Phillips, John L. Spudich
    Abstract:

    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 HtrI. Signal-inverting mutations in the SRI–HtrI complex reverse the single-quantum response from attractant to repellent. Fast intramolecular charge movements reported here reveal that the unphotolyzed SRI–HtrI 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 pKa. 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 HtrI 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.

  • Signal transfer in haloarchaeal Sensory Rhodopsin- transducer complexes.
    Photochemistry and Photobiology, 2008
    Co-Authors: Jun Sasaki, John L. Spudich
    Abstract:

    Membrane-inserted complexes consisting of two photochemically reactive Sensory Rhodopsin (SR) subunits flanking a homodimer of a transducing protein subunit (Htr) are used by halophilic archaea for sensing light gradients to modulate their swimming behavior (phototaxis). The SR–Htr complexes extend into the cytoplasm where the Htr subunits bind a his-kinase that controls a phosphorylation system that regulates the flagellar motors. This review focuses on current progress primarily on the mechanism of signal relay within the SRII–HtrII complexes from Natronomonas pharaonis and Halobacterium salinarum. The recent elucidation of a photoactive site steric trigger crucial for signal relay, advances in understanding the role of proton transfer from the chromophore to the protein in SRII activation, and the localization of signal relay to the membrane-embedded portion of the SRII–HtrII interface, are beginning to produce a clear picture of the signal transfer process. The SR–Htr complexes offer unprecedented opportunities to resolve first examples of the chemistry of signal relay between membrane proteins at the atomic level, which would provide a major contribution to the general understanding of dynamic interactions between integral membrane proteins.

Martin Engelhard - One of the best experts on this subject based on the ideXlab platform.

  • Minireview The archaeal Sensory Rhodopsin II/transducer complex: a model for transmembrane signal transfer
    2020
    Co-Authors: Johann P. Klare, Valentin I. Gordeliy, Georg Bu, Martin Engelhard
    Abstract:

    Archaebacterial photoreceptors mediate phototaxis by regulating cell motility through two-component signalling cascades. Homologs of this Sensory pathway occur in all three kingdoms of life, most notably in enteric bacteria in which the chemotaxis has been extensively studied. Recent structural and functional studies on the Sensory Rhodopsin II/transducer com- plex mediating the photophobic response of Natronomonas pharaonis have yielded new insights into the mechanisms of signal transfer across the membrane. Electron paramagnetic resonance data and the atomic resolution structure of the recep- tor molecule in complex with the transmembrane segment of its cognate transducer provided a model for signal transfer from the receptor to the cytoplasmic side of the transducer. This mecha- nism might also be relevant for eubacterial chemoreceptor sig-

  • Sensory Rhodopsin i and Sensory Rhodopsin ii form trimers of dimers in complex with their cognate transducers
    Photochemistry and Photobiology, 2017
    Co-Authors: Philipp S Orekhov, Johann P. Klare, Heinzjurgen Steinhoff, Konstantin V. Shaitan, Arne Bothe, Stefan Raunser, Dimitrios Fotiadis, Ramona Schlesinger, Martin Engelhard
    Abstract:

    : Archaeal photoreceptors consist of Sensory Rhodopsins in complex with their cognate transducers. After light excitation, a two-component signaling chain is activated, which is homologous to the chemotactic signaling cascades in enterobacteria. The latter system has been studied in detail. From structural and functional studies, a picture emerges which includes stable signaling complexes, which assemble to receptor arrays displaying hexagonal structural elements. At this higher order structural level, signal amplification and Sensory adaptation occur. Here, we describe electron microscopy data, which show that also the archaeal phototaxis receptors Sensory Rhodopsin I and II in complex with their cognate transducers can form hexagonal lattices even in the presence of a detergent. This result could be confirmed by molecular dynamics calculations, which revealed similar structural elements. Calculations of the global modes of motion displayed one mode, which resembles the "U"-"V" transition of the NpSRII:NpHtrII complex, which was previously argued to represent a functionally relevant global conformational change accompanying the activation process [Ishchenko et al. (2013) J. Photochem. Photobiol. B 123, 55-58]. A model of cooperativity at the transmembrane level is discussed.

  • Transient Conformational Changes of Sensory Rhodopsin II Investigated by Vibrational Stark Effect Probes
    Journal of Physical Chemistry B, 2016
    Co-Authors: Hendrik Mohrmann, Martin Engelhard, Ines Kube, Víctor A. Lórenz-fonfría, Joachim Heberle
    Abstract:

    Sensory Rhodopsin II (SRII) is the primary light sensor in the photophobic reaction of the halobacterium Natronomonas pharaonis. Photoactivation of SRII results in a movement of helices F and G of this seven-helical transmembrane protein. This conformational change is conveyed to the transducer protein (HtrII). Global changes in the protein backbone have been monitored by IR difference spectroscopy by recording frequency shifts in the amide bands. Here we investigate local structural changes by judiciously inserting thiocyanides at different locations of SRII. These vibrational Stark probes absorb in a frequency range devoid of any protein vibrations and respond to local changes in the dielectric, electrostatics, and hydrogen bonding. As a proof of principle, we demonstrate the use of Stark probes to test the conformational changes occurring in SRII 12 ms after photoexcitation and later. Thus, a methodology is provided to trace local conformational changes in membrane proteins by a minimal invasive probe ...

  • Ground state structure of D75N mutant of Sensory Rhodopsin II in complex with its cognate transducer.
    Journal of Photochemistry and Photobiology B-biology, 2013
    Co-Authors: Andrii Ishchenko, Johann P. Klare, Martin Engelhard, Ekaterina Round, Valentin Borshchevskiy, Sergei Grudinin, Ivan Gushchin, Taras Balandin, Alina Remeeva, Georg Buldt
    Abstract:

    The complex of Sensory Rhodopsin II (NpSRII) with its cognate transducer (NpHtrII) mediates negative phototaxis in halobacteria Natronomonas pharaonis. Upon light activation NpSRII triggers, by means of NpHtrII, a signal transduction chain homologous to the two component system in eubacterial chemotaxis. Here we report on the crystal structure of the ground state of the mutant NpSRII-D75N/NpHtrII complex in the space group I212121. Mutations of this aspartic acid in light-driven proton pumps dramatically modify or/and inhibit protein functions. However, in vivo studies show that the similar D75N mutation retains functionality of the NpSRII/NpHtrII complex. The structure provides the molecular basis for the explanation of the unexpected observation that the wild and the mutant complexes display identical physiological response on light excitation.

  • Active State of Sensory Rhodopsin II: Structural Determinants for Signal Transfer and Proton Pumping
    Journal of Molecular Biology, 2011
    Co-Authors: Ivan Gushchin, Georg Buldt, Martin Engelhard, Andrii Ishchenko, Ekaterina Round, Valentin Borshchevskiy, Sergei Grudinin, Anastasia Reshetnyak, Valentin I. Gordeliy
    Abstract:

    The molecular mechanism of transmembrane signal transduction is still a pertinent question in cellular biology. Generally, a receptor can transfer an external signal via its cytoplasmic surface as found for GPCRs like Rhodopsin or via the membrane domain like it is utilized by Sensory Rhodopsin II (SRII) in complex with its transducer HtrII. In the absence of HtrII SRII functions as a proton pump. Here, we report on the crystal structure of the active state of SRII from Natronomonas pharaonis (NpSRII). The problem of a dramatic loss of the diffraction quality upon loading of the active state was overcome by growing better crystals and reducing the occupancy of the state. The determined conformational changes at the region comprising helices F and G are similar to those observed for the NpSRII-transducer complex but they are much more pronounced. Meaning of these differences for proton pumping ability and understanding of the signal transduction by NpSRII is discussed.

Yuki Sudo - One of the best experts on this subject based on the ideXlab platform.

  • with PhoboRhodopsin (Sensory Rhodopsin II)
    2020
    Co-Authors: Satoru Yamaguchi, Yuki Sudo, Naoki Kamo, Kazumi Shimono, Satoru Tuzi, Akira Naito, Hazime Saitô
    Abstract:

    We have recorded 13 C NMR spectra of the (3- 13 C)Ala, (1- 13 C)Val-labeled pharaonis transducer pHtrII(1-159) in the presence and absence of phoboRhodopsin (ppR or Sensory Rhodopsin II) in egg phosphatidylcholine or dimyristoylphos- phatidylcholine bilayers by means of site-directed (amino acid specific) solid-state NMR. Two kinds of 13 C NMR signals of (3- 13 C)Ala-pHtrII complexed with ppR were clearly seen with dipolar decoupled magic angle spinning (DD-MAS) NMR. One of these resonances was at the peak position of the low-field a-helical peaks (aII-helix) and is identified with cytoplasmic a-helices protruding from the bilayers; the other was the high-field a-helical peak (aI-helix) and is identified with the transmembrane a-helices. The first peaks, however, were almost completely suppressed by cross-polarization magic angle spinning (CP-MAS) regardless of the presence or absence of ppR or by DD-MAS NMR in the absence of ppR. This is caused by an increased fluctuation frequency of the cytoplasmic a-helix from 10 5 Hz in the uncomplexed states to .10 6 Hz in the complexed states, leading to the appearance of peaks that were suppressed because of the interference of the fluctuation frequency with the frequency of proton decoupling (10 5 Hz), as viewed from the 13 C NMR spectra of (3- 13 C)Ala-labeled pHtrII. Consistent with this view, the 13 C DD-MAS NMR signals of the cytoplasmic a-helices of the complexed (3- 13 C)Ala-pHtrII in the dimyristoylphos- phatidylcholine (DMPC) bilayer were partially suppressed at 0C due to a decreased fluctuation frequency at the low temperature. In contrast, examination of the 13 C CP-MAS spectra of (1- 13 C)Val-labeled complexed pHtrII showed that the 13 C NMR signals of the transmembrane a-helix were substantially suppressed. These spectral changes are again interpreted in terms of the increased fluctuation frequency of the transmembrane a-helices from 10 3 Hz of the uncomplexed states to 10 4 Hz of the complexed states. These findings substantiate the view that the transducers alone are in an aggregated or clustered state but the ppR-pHtrII complex is not aggregated. We show that 13 C NMR is a very useful tool for achieving a better understanding of membrane proteins which will serve to clarify the molecular mechanism of signal transduction in this system.

  • color discriminating retinal configurations of Sensory Rhodopsin i by photo irradiation solid state nmr spectroscopy
    Angewandte Chemie, 2014
    Co-Authors: Hiroki Yomoda, Yuki Sudo, Akimori Wada, Takashi Okitsu, Yoshiteru Makino, Yuya Tomonaga, Tetsurou Hidaka, Izuru Kawamura, Akira Naito
    Abstract:

    : SRI (Sensory Rhodopsin I) can discriminate multiple colors for the attractant and repellent phototaxis. Studies aimed at revealing the color-dependent mechanism show that SRI is a challenging system not only in photobiology but also in photochemistry. During the photoreaction of SRI, an M-intermediate (attractant) transforms into a P-intermediate (repellent) by absorbing blue light. Consequently, SRI then cycles back to the G-state. The photoreactions were monitored with the (13)C NMR signals of [20-(13)C]retnal-SrSRI using in situ photo-irradiation solid-state NMR spectroscopy. The M-intermediate was trapped at -40 °C by illumination at 520 nm. It was transformed into the P-intermediate by subsequent illumination at 365 nm. These results reveal that the G-state could be directly transformed to the P-intermediate by illumination at 365 nm. Thus, the stationary trapped M- and P-intermediates are responsible for positive and negative phototaxis, respectively.

  • the early steps in the photocycle of a photosensor protein Sensory Rhodopsin i from salinibacter ruber
    Journal of Physical Chemistry B, 2014
    Co-Authors: Yuki Sudo, Misao Mizuno, Satoshi Takeuchi, Tahei Tahara, Yasuhisa Mizutani
    Abstract:

    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...

  • photo induced regulation of the chromatic adaptive gene expression by anabaena Sensory Rhodopsin
    Journal of Biological Chemistry, 2012
    Co-Authors: Hiroki Irieda, Yuki Sudo, Michio Homma, Teppei Morita, Kimika Maki, Hiroji Aiba
    Abstract:

    Rhodopsin molecules are photochemically reactive membrane-embedded proteins, with seven transmembrane α-helices, which bind the chromophore retinal (vitamin A aldehyde). They are roughly divided into two groups according to their basic functions: (i) ion transporters such as proton pumps, chloride pumps, and cation channels; and (ii) photo-sensors such as Sensory Rhodopsin from microbes and visual pigments from animals. Anabaena Sensory Rhodopsin (ASR), found in 2003 in the cyanobacterium Anabaena PCC7120, is categorized as a microbial Sensory Rhodopsin. To investigate the function of ASR in vivo, ASR and the promoter sequence of the pigment protein phycocyanin were co-introduced into Escherichia coli cells with the reporter gene crp. The result clearly showed that ASR functions as a repressor of the CRP protein expression and that this is fully inhibited by the light activation of ASR, suggesting that ASR would directly regulate the transcription of crp. The repression is also clearly inhibited by the truncation of the C-terminal region of ASR, or mutations on the C-terminal Arg residues, indicating the functional importance of the C-terminal region. Thus, our results demonstrate a novel function of Rhodopsin molecules and raise the possibility that the membrane-spanning protein ASR could work as a transcriptional factor. In the future, the ASR activity could be utilized as a tool for arbitrary protein expression in vivo regulated by visible light.

  • structural characteristics around the β ionone ring of the retinal chromophore in salinibacter Sensory Rhodopsin i
    Biochemistry, 2011
    Co-Authors: Hiroki Irieda, Akira Kawanabe, Hideki Kandori, Michio Homma, Louisa Reissig, Yuki Sudo
    Abstract:

    Organisms sense and respond to environmental stimuli through membrane-embedded receptors and transducers. Sensory Rhodopsin I (SRI) and Sensory Rhodopsin II (SRII) are the photoreceptors for the positive and negative phototaxis in microorganisms, respectively. They form signaling complexes in the membrane with their cognate transducer proteins, HtrI and HtrII, and these SRI–HtrI and SRII–HtrII complexes transmit a light signal through their cytoplasmic Sensory signaling system, inducing opposite effects (i.e., the inactivation or activation of the kinase CheA). Here we found, by using Fourier transformed infrared spectroscopy, that a conserved residue, Asp102 in Salinibacter SRI (SrSRI), which is located close to the β-ionone ring of the retinal chromophore, is deprotonated upon formation of the active M-intermediate. Furthermore, the D102E mutant of SrSRI affects the structure and/or structural changes of Cys130. This mutant shows a large spectral shift and is comparably unstable, especially in the absen...

Johann P. Klare - One of the best experts on this subject based on the ideXlab platform.

  • Minireview The archaeal Sensory Rhodopsin II/transducer complex: a model for transmembrane signal transfer
    2020
    Co-Authors: Johann P. Klare, Valentin I. Gordeliy, Georg Bu, Martin Engelhard
    Abstract:

    Archaebacterial photoreceptors mediate phototaxis by regulating cell motility through two-component signalling cascades. Homologs of this Sensory pathway occur in all three kingdoms of life, most notably in enteric bacteria in which the chemotaxis has been extensively studied. Recent structural and functional studies on the Sensory Rhodopsin II/transducer com- plex mediating the photophobic response of Natronomonas pharaonis have yielded new insights into the mechanisms of signal transfer across the membrane. Electron paramagnetic resonance data and the atomic resolution structure of the recep- tor molecule in complex with the transmembrane segment of its cognate transducer provided a model for signal transfer from the receptor to the cytoplasmic side of the transducer. This mecha- nism might also be relevant for eubacterial chemoreceptor sig-

  • Sensory Rhodopsin i and Sensory Rhodopsin ii form trimers of dimers in complex with their cognate transducers
    Photochemistry and Photobiology, 2017
    Co-Authors: Philipp S Orekhov, Johann P. Klare, Heinzjurgen Steinhoff, Konstantin V. Shaitan, Arne Bothe, Stefan Raunser, Dimitrios Fotiadis, Ramona Schlesinger, Martin Engelhard
    Abstract:

    : Archaeal photoreceptors consist of Sensory Rhodopsins in complex with their cognate transducers. After light excitation, a two-component signaling chain is activated, which is homologous to the chemotactic signaling cascades in enterobacteria. The latter system has been studied in detail. From structural and functional studies, a picture emerges which includes stable signaling complexes, which assemble to receptor arrays displaying hexagonal structural elements. At this higher order structural level, signal amplification and Sensory adaptation occur. Here, we describe electron microscopy data, which show that also the archaeal phototaxis receptors Sensory Rhodopsin I and II in complex with their cognate transducers can form hexagonal lattices even in the presence of a detergent. This result could be confirmed by molecular dynamics calculations, which revealed similar structural elements. Calculations of the global modes of motion displayed one mode, which resembles the "U"-"V" transition of the NpSRII:NpHtrII complex, which was previously argued to represent a functionally relevant global conformational change accompanying the activation process [Ishchenko et al. (2013) J. Photochem. Photobiol. B 123, 55-58]. A model of cooperativity at the transmembrane level is discussed.

  • new insights on signal propagation by Sensory Rhodopsin ii transducer complex
    Scientific Reports, 2017
    Co-Authors: Johann P. Klare, Andrii Ishchenko, Ekaterina Round, Valentin Borshchevskiy, Sergei Grudinin, Ivan Gushchin
    Abstract:

    The complex of two membrane proteins, Sensory Rhodopsin II (NpSRII) with its cognate transducer (NpHtrII), mediates negative phototaxis in halobacteria N. pharaonis. Upon light activation NpSRII triggers a signal transduction chain homologous to the two-component system in eubacterial chemotaxis. Here we report on crystal structures of the ground and active M-state of the complex in the space group I212121. We demonstrate that the relative orientation of symmetrical parts of the dimer is parallel (“U”-shaped) contrary to the gusset-like (“V”-shaped) form of the previously reported structures of the NpSRII/NpHtrII complex in the space group P21212, although the structures of the monomers taken individually are nearly the same. Computer modeling of the HAMP domain in the obtained “V”- and “U”-shaped structures revealed that only the “U”-shaped conformation allows for tight interactions of the receptor with the HAMP domain. This is in line with existing data and supports biological relevance of the “U” shape in the ground state. We suggest that the “V”-shaped structure may correspond to the active state of the complex and transition from the “U” to the “V”-shape of the receptor-transducer complex can be involved in signal transduction from the receptor to the signaling domain of NpHtrII.

  • Ground state structure of D75N mutant of Sensory Rhodopsin II in complex with its cognate transducer.
    Journal of Photochemistry and Photobiology B-biology, 2013
    Co-Authors: Andrii Ishchenko, Johann P. Klare, Martin Engelhard, Ekaterina Round, Valentin Borshchevskiy, Sergei Grudinin, Ivan Gushchin, Taras Balandin, Alina Remeeva, Georg Buldt
    Abstract:

    The complex of Sensory Rhodopsin II (NpSRII) with its cognate transducer (NpHtrII) mediates negative phototaxis in halobacteria Natronomonas pharaonis. Upon light activation NpSRII triggers, by means of NpHtrII, a signal transduction chain homologous to the two component system in eubacterial chemotaxis. Here we report on the crystal structure of the ground state of the mutant NpSRII-D75N/NpHtrII complex in the space group I212121. Mutations of this aspartic acid in light-driven proton pumps dramatically modify or/and inhibit protein functions. However, in vivo studies show that the similar D75N mutation retains functionality of the NpSRII/NpHtrII complex. The structure provides the molecular basis for the explanation of the unexpected observation that the wild and the mutant complexes display identical physiological response on light excitation.

  • primary reaction of Sensory Rhodopsin ii mutant d75n and the influence of azide
    Biochemistry, 2009
    Co-Authors: Mirka-kristin Verhoefen, Johann P. Klare, Martin Engelhard, Sergiu Amarie, Martin O. Lenz, Jorg Tittor, Dieter Oesterhelt, Josef Wachtveitl
    Abstract:

    The early steps in the photocycle of Sensory Rhodopsin II mutant D75N are investigated in a comprehensive study using femtosecond visible pump/probe spectroscopy. An overall slower response dynamics after photoexcitation is observed compared to wild-type Sensory Rhodopsin II, which is assigned to changed electrostatics and an altered hydrogen-bonding network within the retinal binding pocket. Furthermore, the influence of azide on the primary reaction is analyzed. The addition of azide accelerates the sub-10 ps dynamics of the D75N mutant nearly to reaction rates found in wild-type. Moreover, a further reaction pathway becomes observable in the investigated time range, which is assigned to a previously described K1 to K2 transition. The specific acceleration of the early steps seems to be a unique feature of the D75N mutant as similar azide effects do not emerge in analogous azide measurements of wild-type Sensory Rhodopsin II, bacterioRhodopsin, and the bacterioRhodopsin mutant D85N.

Hideki Kandori - One of the best experts on this subject based on the ideXlab platform.

  • his166 is the schiff base proton acceptor in attractant phototaxis receptor Sensory Rhodopsin i
    Biochemistry, 2014
    Co-Authors: Jun Sasaki, Yuji Furutani, John L. Spudich, Oleg A Sineshchekov, Hazuki Takahashi, Hideki Kandori
    Abstract:

    Photoactivation of attractant phototaxis receptor Sensory Rhodopsin I (SRI) in Halobacterium salinarum entails transfer of a proton from the retinylidene chromophore’s Schiff base (SB) to an unidentified acceptor residue on the cytoplasmic half-channel, in sharp contrast to other microbial Rhodopsins, including the closely related repellent phototaxis receptor SRII and the outward proton pump bacterioRhodopsin, in which the SB proton acceptor is an aspartate residue salt-bridged to the SB in the extracellular (EC) half-channel. His166 on the cytoplasmic side of the SB in SRI has been implicated in the SB proton transfer reaction by mutation studies, and mutants of His166 result in an inverted SB proton release to the EC as well as inversion of the protein’s normally attractant phototaxis signal to repellent. Here we found by difference Fourier transform infrared spectroscopy the appearance of Fermi-resonant X–H stretch modes in light-minus-dark difference spectra; their assignment with 15N labeling and si...

  • steady state emission of the fluorescent intermediate of anabaena Sensory Rhodopsin as a function of light adaptation conditions
    Chemical Physics Letters, 2013
    Co-Authors: Alexandre Cheminal, Hideki Kandori, Kwang-hwan Jung, Jeremie Leonard, S Haacke
    Abstract:

    Abstract Steady-state fluorescence measurements of the first excited state of the anabaena Sensory Rhodopsin (ASR), and BacterioRhodopsin are reported for different light stabilization conditions, including the dark-adapted state. We determine the fluorescence spectra of both all-trans (AT), and 13- cis (13C) protonated Schiff base of retinal, and compare the effect of the proteins. Referenced against the fluorescence quantum yield of AT-bR (2.5 × 10 −4 ) we find for AT-ASR, 13C-ASR, and 13C-bR the values of 3.3 × 10 −4 , 0.8 × 10 −4 , and 1.7 × 10 −4 , respectively. Using reported excited state lifetimes, the radiative rates are deduced, and their differences discussed on the basis of a configuration-dependent oscillator strength.

  • structural characteristics around the β ionone ring of the retinal chromophore in salinibacter Sensory Rhodopsin i
    Biochemistry, 2011
    Co-Authors: Hiroki Irieda, Akira Kawanabe, Hideki Kandori, Michio Homma, Louisa Reissig, Yuki Sudo
    Abstract:

    Organisms sense and respond to environmental stimuli through membrane-embedded receptors and transducers. Sensory Rhodopsin I (SRI) and Sensory Rhodopsin II (SRII) are the photoreceptors for the positive and negative phototaxis in microorganisms, respectively. They form signaling complexes in the membrane with their cognate transducer proteins, HtrI and HtrII, and these SRI–HtrI and SRII–HtrII complexes transmit a light signal through their cytoplasmic Sensory signaling system, inducing opposite effects (i.e., the inactivation or activation of the kinase CheA). Here we found, by using Fourier transformed infrared spectroscopy, that a conserved residue, Asp102 in Salinibacter SRI (SrSRI), which is located close to the β-ionone ring of the retinal chromophore, is deprotonated upon formation of the active M-intermediate. Furthermore, the D102E mutant of SrSRI affects the structure and/or structural changes of Cys130. This mutant shows a large spectral shift and is comparably unstable, especially in the absen...

  • spectrally silent intermediates during the photochemical reactions of salinibacter Sensory Rhodopsin i
    Journal of Physical Chemistry B, 2011
    Co-Authors: Keiichi Inoue, Michio Homma, Yuki Sudo, Hideki Kandori
    Abstract:

    Salinibacter Sensory Rhodopsin I (SrSRI) is a microbial Rhodopsin discovered from the eubacterium Salinibacter ruber. It is thought to be a photoreceptor engaging the signal transductions for both positive and negative phototaxis. To elucidate the photoreactions of SrSRI in the presence and absence of chloride ions, we measured the refractive index change after the photoexcitation by the transient grating method. As a result, two spectrally silent processes were identified after the formation of M intermediate, and we named the spectrally identical intermediates M1, M2, and M3. The enthalpy changes (ΔH) were estimated as ΔH = 136, 99, and 63 kJ/mol for K, M1, and M2 intermediates, respectively. The ΔH values were significantly decreased (36−55 kJ/mol) by the removal of chloride ions, suggesting their importance for structural changes of SrSRI. Volume expansions of SrSRI were observed on the spectrally silent steps (44 and 11 mL/mol). They may be related to the signaling process because blue-shifted interm...

  • An inward proton transport using anabaena Sensory Rhodopsin
    The Journal of Microbiology, 2011
    Co-Authors: Akira Kawanabe, Yuji Furutani, Kwang-hwan Jung, Hideki Kandori
    Abstract:

    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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