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

  • direct observation of the structural change of tyr174 in the primary reaction of sensory rhodopsin ii
    Biochemistry, 2011
    Co-Authors: Misao Mizuno, Michio Homma, Yuki Sudo, Yasuhisa Mizutani
    Abstract:

    Sensory rhodopsin II (SRII) is a negative phototaxis receptor containing retinal as its chromophore, which mediates the avoidance of blue light. The signal transduction is initiated by the photoisomerization of the retinal chromophore, resulting in conformational changes of the Protein which are transmitted to a Transducer Protein. To gain insight into the SRII sensing mechanism, we employed time-resolved ultraviolet resonance Raman spectroscopy monitoring changes in the Protein structure in the picosecond time range following photoisomerization. We used a 450 nm pump pulse to initiate the SRII photocycle and two kinds of probe pulses with wavelengths of 225 and 238 nm to detect spectral changes in the tryptophan and tyrosine bands, respectively. The observed spectral changes of the Raman bands are most likely due to tryptophan and tyrosine residues located in the vicinity of the retinal chromophore, i.e., Trp76, Trp171, Tyr51, or Tyr174. The 225 nm UVRR spectra exhibited bleaching of the intensity for al...

  • a microbial rhodopsin with a unique retinal composition shows both sensory rhodopsin ii and bacteriorhodopsin like properties
    Journal of Biological Chemistry, 2011
    Co-Authors: Yuki Sudo, Hideki Kandori, Kunio Ihara, Shiori Kobayashi, Hiroki Irieda, Daisuke Suzuki, Takashi Kikukawa, Michio Homma
    Abstract:

    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.

  • characterization of a signaling complex composed of sensory rhodopsin i and its cognate Transducer Protein from the eubacterium salinibacter ruber
    Biochemistry, 2009
    Co-Authors: Yuki Sudo, Yuji Furutani, Keiichi Inoue, Hideki Kandori, Hiroki Irieda, Masaaki Fujii, Daisuke Suzuki, Akiko Okada, Michio Homma
    Abstract:

    Sensory rhodopsin I (SRI) exists in the cell membranes of microorganisms such as the archaeon Halobacterium salinarum and is a photosensor responsible for positive and negative phototaxis. SRI forms a signaling complex with its cognate Transducer Protein, HtrI, in the membrane. That complex transmits light signals to the flagellar motor through changes in Protein−Protein interactions with the kinase CheA and the adaptor Protein CheW, which controls the direction of the rotation of the flagellar motor. Recently, we cloned and characterized Salinibacter sensory rhodopsin I (SrSRI), which is the first SRI-like Protein identified in eubacteria [Kitajima-Ihara, T., et al. (2008) J. Biol. Chem. 283, 23533−23541]. Here we cloned and expressed SrSRI with its full-length Transducer Protein, SrHtrI, as a fusion construct. We succeeded in producing the complex in Escherichia coli as a recombinant Protein with high quality having all-trans-retinal as a chromophore for SRI, although the expression level was low (0.10 ...

  • characterization of a signaling complex composed of sensory rhodopsin i and its cognate Transducer Protein from the eubacterium salinibacter ruber
    Biochemistry, 2009
    Co-Authors: Yuji Furutani, Yuki Sudo, Keiichi Inoue, Hiroki Irieda, Masaaki Fujii, Daisuke Suzuki, Akiko Okada, Makoto Sakai, Hideki Kandori
    Abstract:

    Sensory rhodopsin I (SRI) exists in the cell membranes of microorganisms such as the archaeon Halobacterium salinarum and is a photosensor responsible for positive and negative phototaxis. SRI forms a signaling complex with its cognate Transducer Protein, HtrI, in the membrane. That complex transmits light signals to the flagellar motor through changes in Protein-Protein interactions with the kinase CheA and the adaptor Protein CheW, which controls the direction of the rotation of the flagellar motor. Recently, we cloned and characterized Salinibacter sensory rhodopsin I (SrSRI), which is the first SRI-like Protein identified in eubacteria [Kitajima-Ihara, T., et al. (2008) J. Biol. Chem. 283, 23533-23541]. Here we cloned and expressed SrSRI with its full-length Transducer Protein, SrHtrI, as a fusion construct. We succeeded in producing the complex in Escherichia coli as a recombinant Protein with high quality having all-trans-retinal as a chromophore for SRI, although the expression level was low (0.10 mg/L of culture). In addition, we report here the photochemical properties of the SrSRI-SrHtrI complex using time-resolved laser flash spectroscopy and other spectroscopic techniques and compare them to SrSRI without SrHtrI.

  • characterization of a signaling complex composed of sensory rhodopsin i and its cognate Transducer Protein from the eubacterium salinibacter ruber
    Biochemistry, 2009
    Co-Authors: Yuki Sudo, Yuji Furutani, Keiichi Inoue, Hideki Kandori, Hiroki Irieda, Masaaki Fujii, Daisuke Suzuki, Akiko Okada, Makoto Sakai, Michio Homma
    Abstract:

    Sensory rhodopsin I (SRI) exists in the cell membranes of microorganisms such as the archaeon Halobacterium salinarum and is a photosensor responsible for positive and negative phototaxis. SRI form...

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

  • transient dissociation of the Transducer Protein from anabaena sensory rhodopsin concomitant with formation of the m state produced upon photoactivation
    Journal of the American Chemical Society, 2011
    Co-Authors: Masato Kondoh, John L Spudich, Keiichi Inoue, Jun Sasaki, Masahide Terazima
    Abstract:

    Anabaena sensory rhodopsin (ASR), a microbial rhodopsin in the cyanobacterium sp. PCC7120, has been suggested to regulate cell processes in a light-quality-dependent manner (color-discrimination) through interaction with a water-soluble Transducer Protein (Tr). However, light-dependent ASR-Tr interaction changes have yet to be demonstrated. We applied the transient grating (TG) method to investigate Protein-Protein interaction between ASR with Tr. The molecular diffusion component of the TG signal upon photostimulation of ASR(AT) (ASR with an all-trans retinylidene chromophore) revealed that Tr dissociates from ASR upon formation of the M-intermediate and rebinds to ASR during the decay of M; that is, light induces transient dissociation of ASR and Tr during the photocycle. Further correlating the dissociation of the ASR-Tr pair with the M-intermediate, no transient dissociation was observed after the photoexcitation of the blue-shifted ASR(13C) (ASR with 13-cis, 15-syn chromophore), which does not produce M. This distinction between ASR(AT) and ASR(13C), the two isomeric forms in a color-sensitive equilibrium in ASR, provides a potential mechanism for color-sensitive signaling by ASR.

  • transient dissociation of the Transducer Protein from anabaena sensory rhodopsin concomitant with formation of the m state produced upon photoactivation
    Journal of the American Chemical Society, 2011
    Co-Authors: Masato Kondoh, John L Spudich, Keiichi Inoue, Jun Sasaki, Masahide Terazima
    Abstract:

    Anabaena sensory rhodopsin (ASR), a microbial rhodopsin in the cyanobacterium sp. PCC7120, has been suggested to regulate cell processes in a light-quality-dependent manner (color-discrimination) through interaction with a water-soluble Transducer Protein (Tr). However, light-dependent ASR–Tr interaction changes have yet to be demonstrated. We applied the transient grating (TG) method to investigate Protein–Protein interaction between ASR with Tr. The molecular diffusion component of the TG signal upon photostimulation of ASRAT (ASR with an all-trans retinylidene chromophore) revealed that Tr dissociates from ASR upon formation of the M-intermediate and rebinds to ASR during the decay of M; that is, light induces transient dissociation of ASR and Tr during the photocycle. Further correlating the dissociation of the ASR–Tr pair with the M-intermediate, no transient dissociation was observed after the photoexcitation of the blue-shifted ASR13C (ASR with 13-cis, 15-syn chromophore), which does not produce M....

  • laser induced transient grating analysis of dynamics of interaction between sensory rhodopsin ii d75n and the htrii Transducer
    Biophysical Journal, 2007
    Co-Authors: Keiichi Inoue, John L Spudich, Jun Sasaki, Masahide Terazima
    Abstract:

    The interaction between sensory rhodopsin II (SRII) and its Transducer HtrII was studied by the time-resolved laser-induced transient grating method using the D75N mutant of SRII, which exhibits minimal visible light absorption changes during its photocycle, but mediates normal phototaxis responses. Flash-induced transient absorption spectra of Transducer-free D75N and D75N joined to 120 amino-acid residues of the N-terminal part of the SRII Transducer Protein HtrII (ΔHtrII) showed only one spectrally distinct K-like intermediate in their photocycles, but the transient grating method resolved four intermediates (K1–K4) distinct in their volumes. D75N bound to HtrII exhibited one additional slower kinetic species, which persists after complete recovery of the initial state as assessed by absorption changes in the UV-visible region. The kinetics indicate a conformationally changed form of the Transducer portion (designated Tr*), which persists after the photoreceptor returns to the unphotolyzed state. The largest conformational change in the ΔHtrII portion was found to cause a ΔHtrII-dependent increase in volume rising in 8 μs in the K4 state and a drastic decrease in the diffusion coefficient (D) of K4 relatively to those of the unphotolyzed state and Tr*. The magnitude of the decrease in D indicates a large structural change, presumably in the solvent-exposed HAMP domain of ΔHtrII, where rearrangement of interacting molecules in the solvent would substantially change friction between the Protein and the solvent.

  • photochromicity of anabaena sensory rhodopsin an atypical microbial receptor with a cis retinal light adapted form
    Journal of Biological Chemistry, 2005
    Co-Authors: Oleg A. Sineshchekov, Vishwa D Trivedi, Jun Sasaki, John L Spudich
    Abstract:

    Abstract We characterize changes in isomeric states of the retinylidene chromophore during light-dark adaptation and photochemical reactions of Anabaena (Nostoc) sp. PCC7120 sensory rhodopsin (ASR). The results show that ASR represents a new type of microbial rhodopsin with a number of unusual characteristics. The three most striking are: (i) a primarily all-trans configuration of retinal in the dark-adapted state and (ii) a primarily 13-cis light-adapted state with a blue-shifted and lower extinction absorption spectrum, opposite of the case of bacteriorhodopsin; and (iii) efficient reversible light-induced interconversion between the 13-cis and all-trans unphotolyzed states of the pigment. The relative amount of ASR with cis and trans chromophore forms depends on the wavelength of illumination, providing a mechanism for single-pigment color sensing analogous to that of phytochrome pigments. In addition ASR exhibits unusually slow formation of L-like and M-like intermediates, with a dominant accumulation of M during the photocycle. Co-expression of ASR with its putative cytoplasmic Transducer Protein shifts the absorption maximum and strongly decreases the rate of dark adaptation of ASR, confirming interaction between the two Proteins. Thus ASR, the first non-haloarchaeal sensory rhodopsin character-ized, demonstrates the diversity of photochemistry of microbial rhodopsins. Its photochromic properties and the position of its two ground state absorption maxima suggest it as a candidate for controlling differential photosynthetic light-harvesting pigment synthesis (chromatic adaptation) or other color-sensitive physiological responses in Anabaena cells.

  • photostimulation of a sensory rhodopsin ii htrii tsr fusion chimera activates chea autophosphorylation and chey phosphotransfer in vitro
    Biochemistry, 2003
    Co-Authors: Vishwa D Trivedi, John L Spudich
    Abstract:

    A chimeric fusion Protein consisting of Natronomonas pharaonis sensory rhodopsin II (SRII), fused by a flexible linker to the two transmembrane helices of its cognate Transducer Protein, HtrII, followed by the HtrII membrane-proximal cytoplasmic fragment joined to the cytoplasmic domains of the Escherichia coli chemotaxis receptor Tsr, was expressed in E. coli. Purified fusion chimera Protein reconstituted in liposomes binds to E. coli CheA kinase in the presence of the coupling Protein CheW, and activates CheA autophosphorylation activity. CheA kinase activity is stimulated by photoexcitation of the SRII domain of the fusion Protein, as shown by the wavelength-dependence of photostimulated phosphotransfer to the E. coli flagellar motor response regulator CheY in the purified in vitro liposomal system. Further confirming the fidelity of the in vitro system, increased and decreased levels of CheA activation in vitro result from overmethylated and undermethylated fusion Protein purified from methylesterase and methyltransferase-deficient E. coli, respectively. Photoexcitation of the undermethylated fusion Protein resulted in a 3-fold increase in phosphotransfer over that of the dark state. The results directly demonstrate the coupling of SRII photoactivated states to histidine kinase activity, previously predicted on the basis of sequence homologies of the haloarchaeal phototaxis system components to those of E. coli chemotaxis. The fusion chimera provides the first tool for in vitro measurement of photosignaling activity of SRII-HtrII molecular complexes.

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

  • a microbial rhodopsin with a unique retinal composition shows both sensory rhodopsin ii and bacteriorhodopsin like properties
    Journal of Biological Chemistry, 2011
    Co-Authors: Yuki Sudo, Hideki Kandori, Kunio Ihara, Shiori Kobayashi, Hiroki Irieda, Daisuke Suzuki, Takashi Kikukawa, Michio Homma
    Abstract:

    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.

  • photoreactions and structural changes of anabaena sensory rhodopsin
    Sensors, 2009
    Co-Authors: Akira Kawanabe, Hideki Kandori
    Abstract:

    Anabaena sensory rhodopsin (ASR) is an archaeal-type rhodopsin found in eubacteria. The gene encoding ASR forms a single operon with ASRT (ASR Transducer) which is a 14 kDa soluble Protein, suggesting that ASR functions as a photochromic sensor by activating the soluble Transducer. This article reviews the detailed photoreaction processes of ASR, which were studied by low-temperature Fourier-transform infrared (FTIR) and UV-visible spectroscopy. The former research reveals that the retinal isomerization is similar to bacteriorhodopsin (BR), but the hydrogen-bonding network around the Schiff base and cytoplasmic region is different. The latter study shows the stable photoproduct of the all-trans form is 100% 13-cis, and that of the 13-cis form is 100% all-trans. These results suggest that the structural changes of ASR in the cytoplasmic domain play important roles in the activation of the Transducer Protein, and photochromic reaction is optimized for its sensor function.

  • characterization of a signaling complex composed of sensory rhodopsin i and its cognate Transducer Protein from the eubacterium salinibacter ruber
    Biochemistry, 2009
    Co-Authors: Yuki Sudo, Yuji Furutani, Keiichi Inoue, Hideki Kandori, Hiroki Irieda, Masaaki Fujii, Daisuke Suzuki, Akiko Okada, Michio Homma
    Abstract:

    Sensory rhodopsin I (SRI) exists in the cell membranes of microorganisms such as the archaeon Halobacterium salinarum and is a photosensor responsible for positive and negative phototaxis. SRI forms a signaling complex with its cognate Transducer Protein, HtrI, in the membrane. That complex transmits light signals to the flagellar motor through changes in Protein−Protein interactions with the kinase CheA and the adaptor Protein CheW, which controls the direction of the rotation of the flagellar motor. Recently, we cloned and characterized Salinibacter sensory rhodopsin I (SrSRI), which is the first SRI-like Protein identified in eubacteria [Kitajima-Ihara, T., et al. (2008) J. Biol. Chem. 283, 23533−23541]. Here we cloned and expressed SrSRI with its full-length Transducer Protein, SrHtrI, as a fusion construct. We succeeded in producing the complex in Escherichia coli as a recombinant Protein with high quality having all-trans-retinal as a chromophore for SRI, although the expression level was low (0.10 ...

  • characterization of a signaling complex composed of sensory rhodopsin i and its cognate Transducer Protein from the eubacterium salinibacter ruber
    Biochemistry, 2009
    Co-Authors: Yuji Furutani, Yuki Sudo, Keiichi Inoue, Hiroki Irieda, Masaaki Fujii, Daisuke Suzuki, Akiko Okada, Makoto Sakai, Hideki Kandori
    Abstract:

    Sensory rhodopsin I (SRI) exists in the cell membranes of microorganisms such as the archaeon Halobacterium salinarum and is a photosensor responsible for positive and negative phototaxis. SRI forms a signaling complex with its cognate Transducer Protein, HtrI, in the membrane. That complex transmits light signals to the flagellar motor through changes in Protein-Protein interactions with the kinase CheA and the adaptor Protein CheW, which controls the direction of the rotation of the flagellar motor. Recently, we cloned and characterized Salinibacter sensory rhodopsin I (SrSRI), which is the first SRI-like Protein identified in eubacteria [Kitajima-Ihara, T., et al. (2008) J. Biol. Chem. 283, 23533-23541]. Here we cloned and expressed SrSRI with its full-length Transducer Protein, SrHtrI, as a fusion construct. We succeeded in producing the complex in Escherichia coli as a recombinant Protein with high quality having all-trans-retinal as a chromophore for SRI, although the expression level was low (0.10 mg/L of culture). In addition, we report here the photochemical properties of the SrSRI-SrHtrI complex using time-resolved laser flash spectroscopy and other spectroscopic techniques and compare them to SrSRI without SrHtrI.

  • characterization of a signaling complex composed of sensory rhodopsin i and its cognate Transducer Protein from the eubacterium salinibacter ruber
    Biochemistry, 2009
    Co-Authors: Yuki Sudo, Yuji Furutani, Keiichi Inoue, Hideki Kandori, Hiroki Irieda, Masaaki Fujii, Daisuke Suzuki, Akiko Okada, Makoto Sakai, Michio Homma
    Abstract:

    Sensory rhodopsin I (SRI) exists in the cell membranes of microorganisms such as the archaeon Halobacterium salinarum and is a photosensor responsible for positive and negative phototaxis. SRI form...

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

  • 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 ...

  • functional expression of the signaling complex sensory rhodopsin ii Transducer ii from halobacterium salinarum in escherichia coli
    Photochemistry and Photobiology, 2009
    Co-Authors: Igor Chizhov, Martin Engelhard
    Abstract:

    : Sensory rhodopsin II, a photoreceptor from Halobacterium salinarum (HsSRII), in complex with its cognate Transducer Protein (HsHtrII) triggers the photophobic response via a cytoplasmic two-component signaling cascade. HsHtrII possess in addition to the HsSRII binding and the cytoplasmic domains an extracellular serine-receptor domain. Here we describe the properties of HsSRII and HsHtrII and those of various shortened Transducer analogs, heterologously expressed in Escherichia coli. HsSRII displays the photocycle typical of archaeal photosensors with prolonged kinetics. Using an isothermal titration calorimetric analysis for this complex a dissociation constant of 1.1 microm was obtained similar to that of the corresponding Transducer/receptor pair from Natronobacterium pharaonis. A shortened Transducer lacking the extracellular and cytoplasmic domain is also sufficient to bind the receptor with a slightly lower affinity. The dissociation constant of serine binding to the extracellular domain was determined to be about 5 microm. This result is in line with the proposal that the extracellular domain indeed is a serine receptor.

  • expression of the halobacterial Transducer Protein htrii from natronomonas pharaonis in escherichia coli
    FEBS Letters, 2007
    Co-Authors: Nadine Mennes, Johann P. Klare, Ramona Schlesinger, Ralf Seidel, Igor Chizhov, Martin Engelhard
    Abstract:

    Abstract Archaeal phototaxis is mediated by sensory rhodopsins which form complexes with their cognate Transducers. Whereas the receptors sensory rhodopsin I and sensory rhodopsin II (SRII) have been expressed in Escherichia coli (E. coli) only shortened fragments of HtrII from Natronomonas pharaonis (NpHtrII) are available. Here we describe the heterologous expression of full length NpHtrII which was achieved in yields of up to 0.9 mg per litre cell culture. Gel filtration analysis reveals the tendency of the Transducer to form dimers and higher-order oligomers which was also observed when complexed to NpSRII. A circular dichroism (CD) spectrum of NpHtrII is comparable to those obtained for the E. coli chemoreceptors indicating a similar folding with predominantly α-helical structure. NpHtrII dissociates from the NpSRII/HtrII complex with an apparent KD of about 0.6 μM. Photocycle kinetics of the complex is comparable to that obtained for NpSRII in complex with a truncated Transducer with slight differences in the M-decay. The data indicate that the heterologously expressed NpHtrII adopt a native like structure, providing the means for elucidating transmembrane signal transduction and activation of microbial signalling cascades.

  • development of the signal in sensory rhodopsin and its transfer to the cognate Transducer
    Nature, 2006
    Co-Authors: Rouslan Moukhametzianov, Johann P. Klare, Martin Engelhard, Rouslan G Efremov, Christian Baeken, Annika Goppner, Jorg Labahn, Georg Buldt, V Gordeliy
    Abstract:

    The microbial phototaxis receptor sensory rhodopsin II (NpSRII, also named phoborhodopsin) mediates the photophobic response of the haloarchaeon Natronomonas pharaonis1,2 by modulating the swimming behaviour of the bacterium3. After excitation by blue-green light NpSRII triggers, by means of a tightly bound Transducer Protein (NpHtrII), a signal transduction chain homologous with the two-component system of eubacterial chemotaxis4. Two molecules of NpSRII and two molecules of NpHtrII form a 2:2 complex in membranes as shown by electron paramagnetic resonance5 and X-ray structure analysis6. Here we present X-ray structures of the photocycle intermediates K and late M (M2) explaining the evolution of the signal in the receptor after retinal isomerization and the transfer of the signal to the Transducer in the complex. The formation of late M has been correlated with the formation of the signalling state2,7. The observed structural rearrangements allow us to propose the following mechanism for the light-induced activation of the signalling complex. On excitation by light, retinal isomerization leads in the K state to a rearrangement of a water cluster that partly disconnects two helices of the receptor. In the transition to late M the changes in the hydrogen bond network proceed further. Thus, in late M state an altered tertiary structure establishes the signalling state of the receptor. The Transducer responds to the activation of the receptor by a clockwise rotation of about 15° of helix TM2 and a displacement of this helix by 0.9 A at the cytoplasmic surface.

Michio Homma - One of the best experts on this subject based on the ideXlab platform.

  • direct observation of the structural change of tyr174 in the primary reaction of sensory rhodopsin ii
    Biochemistry, 2011
    Co-Authors: Misao Mizuno, Michio Homma, Yuki Sudo, Yasuhisa Mizutani
    Abstract:

    Sensory rhodopsin II (SRII) is a negative phototaxis receptor containing retinal as its chromophore, which mediates the avoidance of blue light. The signal transduction is initiated by the photoisomerization of the retinal chromophore, resulting in conformational changes of the Protein which are transmitted to a Transducer Protein. To gain insight into the SRII sensing mechanism, we employed time-resolved ultraviolet resonance Raman spectroscopy monitoring changes in the Protein structure in the picosecond time range following photoisomerization. We used a 450 nm pump pulse to initiate the SRII photocycle and two kinds of probe pulses with wavelengths of 225 and 238 nm to detect spectral changes in the tryptophan and tyrosine bands, respectively. The observed spectral changes of the Raman bands are most likely due to tryptophan and tyrosine residues located in the vicinity of the retinal chromophore, i.e., Trp76, Trp171, Tyr51, or Tyr174. The 225 nm UVRR spectra exhibited bleaching of the intensity for al...

  • a microbial rhodopsin with a unique retinal composition shows both sensory rhodopsin ii and bacteriorhodopsin like properties
    Journal of Biological Chemistry, 2011
    Co-Authors: Yuki Sudo, Hideki Kandori, Kunio Ihara, Shiori Kobayashi, Hiroki Irieda, Daisuke Suzuki, Takashi Kikukawa, Michio Homma
    Abstract:

    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.

  • characterization of a signaling complex composed of sensory rhodopsin i and its cognate Transducer Protein from the eubacterium salinibacter ruber
    Biochemistry, 2009
    Co-Authors: Yuki Sudo, Yuji Furutani, Keiichi Inoue, Hideki Kandori, Hiroki Irieda, Masaaki Fujii, Daisuke Suzuki, Akiko Okada, Michio Homma
    Abstract:

    Sensory rhodopsin I (SRI) exists in the cell membranes of microorganisms such as the archaeon Halobacterium salinarum and is a photosensor responsible for positive and negative phototaxis. SRI forms a signaling complex with its cognate Transducer Protein, HtrI, in the membrane. That complex transmits light signals to the flagellar motor through changes in Protein−Protein interactions with the kinase CheA and the adaptor Protein CheW, which controls the direction of the rotation of the flagellar motor. Recently, we cloned and characterized Salinibacter sensory rhodopsin I (SrSRI), which is the first SRI-like Protein identified in eubacteria [Kitajima-Ihara, T., et al. (2008) J. Biol. Chem. 283, 23533−23541]. Here we cloned and expressed SrSRI with its full-length Transducer Protein, SrHtrI, as a fusion construct. We succeeded in producing the complex in Escherichia coli as a recombinant Protein with high quality having all-trans-retinal as a chromophore for SRI, although the expression level was low (0.10 ...

  • characterization of a signaling complex composed of sensory rhodopsin i and its cognate Transducer Protein from the eubacterium salinibacter ruber
    Biochemistry, 2009
    Co-Authors: Yuki Sudo, Yuji Furutani, Keiichi Inoue, Hideki Kandori, Hiroki Irieda, Masaaki Fujii, Daisuke Suzuki, Akiko Okada, Makoto Sakai, Michio Homma
    Abstract:

    Sensory rhodopsin I (SRI) exists in the cell membranes of microorganisms such as the archaeon Halobacterium salinarum and is a photosensor responsible for positive and negative phototaxis. SRI form...

  • salinibacter sensory rhodopsin sensory rhodopsin i like Protein from a eubacterium
    Journal of Biological Chemistry, 2008
    Co-Authors: Tomomi Kitajimaihara, Yuji Furutani, Michio Homma, Hideki Kandori, Kunio Ihara, Daisuke Suzuki, Yuki Sudo
    Abstract:

    Abstract Halobacterium salinarum sensory rhodopsin I (HsSRI), a dual receptor regulating both negative and positive phototaxis in haloarchaea, transmits light signals through changes in Protein-Protein interactions with its Transducer, halobacterial Transducer Protein I (HtrI). Haloarchaea also have another sensor pigment, sensory rhodopsin II (SRII), which functions as a receptor regulating negative phototaxis. Compared with HsSRI, the signal relay mechanism of SRII is well characterized because SRII from Natronomonus pharaonis (NpSRII) is much more stable than HsSRI and HsSRII, especially in dilute salt solutions and is much more resistant to detergents. Two genes encoding SRI homologs were identified from the genome sequence of the eubacterium Salinibacter ruber. Those sequences are distantly related to HsSRI (∼40% identity) and contain most of the amino acid residues identified as necessary for its function. To determine whether those genes encode functional Protein(s), we cloned and expressed them in Escherichia coli. One of them (SrSRI) was expressed well as a recombinant Protein having all-trans retinal as a chromophore. UV-Vis, low-temperature UV-Vis, pH-titration, and flash photolysis experiments revealed that the photochemical properties of SrSRI are similar to those of HsSRI. In addition to the expression system, the high stability of SrSRI makes it possible to prepare large amounts of Protein and enables studies of mutant Proteins that will allow new approaches to investigate the photosignaling process of SRI-HtrI.