研究者詳細 - 井原　邦夫

論文 - 井原　邦夫

分割表示 105 件中 61 - 80 件目／全件表示 >>

Crystal structure of the O intermediate of the Leu93->Ala mutant of bacteriorhodopsin.

Zhang J, Yamazaki Y, Hikake M, Murakami M, Ihara K, Kouyama T.

Proteins 80 巻 ( 10 ) 頁： 2384-2396 2012年10月

MarR-type transcriptional regulator ChlR activates expression of tetrapyrrole biosynthesis genes in response to low-oxygen conditions in cyanobacteria.

Aoki R, Takeda T, Omata T, Ihara K, Fujita Y.

Journal of Biological Chemistry 287 巻 ( 16 ) 頁： 13500-13507 2012年4月

Crystal structures of an O-like blue form and an anion-free yellow form of pharaonis halorhodopsin. 査読有り

Kanada S, Takeguchi Y, Murakami M, Ihara K, Kouyama T.

Journal of Molecular Biology 413 巻 ( 1 ) 頁： 162-176 2011年10月

Gene orders in the upstream of 16S rRNA genes divide genera of the family Halobacteriaceae into two groups 査読有り

Minegishi, H. Kamekura, M. Kitajima-Ihara, T. Nakasone, K. Echigo, A. Shimane, Y. Usami, R. Itoh, T. Ihara, K.

Int J Syst Evol Microbiol 2011年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

In many prokaryotic species, 16S rRNA genes are present in multiple copies, and their sequences in general do not differ significantly owing to the concerted evolution. The genus Haloarcula of the family Halobacteriaceae comprises nine validly published species, and all of them possess two to four, highly heterogeneous, 16S rRNA genes. Existence of heterogeneous multiple 16S rRNA genes makes it difficult to reconstruct a biological phylogenetic tree using their sequences data. If the orthologous gene is able to be discriminated from paralogous genes, a tree reconstructed from orthologous genes will reflect a simple biological phylogenetic relationship. At present, however, we have no means to distinguish the orthologous rRNA operon from paralogous one in the members of Halobacteriaceae.In this paper, we found that dihydroorotate oxidase gene, pyrD, was present in the immediate upstream of one 16S rRNA gene, in ten strains of Halobacteriaceae whose genome sequences have been determined, and the direction of pyrD was the opposite of that of the 16S rRNA genes. In two other strains whose genome sequences have been determined, the pyrD was present in far separated positions. We designed PCR primer sets to amplify DNA fragments encompassing from the conserved region of pyrD to a conserved region of tRNA-Ala genes or 23S rRNA genes to determine the 16S rRNA gene sequences preceded by pyrD, and to see if the pyrD is conserved in the immediate upstream of rRNA operon(s) in the type strains of the types species of 28 genera of Halobacteriaceae. Seventeen type strains, including the ten strains mentioned above, gave amplified DNA fragments of approximately 4,000 bp, while eleven type strains including the two strains mentioned above, did not give any PCR products. These eleven strains are members of the Clade I, which was originally defined by Walsh et al. (2004) and expanded by Minegishi et al. (2010). Analysis of contig sequences of three strains belonging to the Clade I also revealed the absence of the pyrD in the immediate upstream of any 16S rRNA genes. It may be scientifically sound to hypothesize that during the evolution of members of Halobacteriaceae, pyrD transposition event happened in one group, and this was followed by subsequent speciation processes in each group, yielding species/genera of the Clade I group and "the rest" of the present family Halobacteriaceae.

DOI： ijs.0.031708-0 [pii] 10.1099/ijs.0.031708-0

A Microbial Rhodopsin with a Unique Retinal Composition Shows Both Sensory Rhodopsin II and Bacteriorhodopsin-like Properties 査読有り

Sudo, Y. Ihara, K. Kobayashi, S. Suzuki, D. Irieda, H. Kikukawa, T. Kandori, H. Homma, M.

Journal of Biological Chemistry 286 巻 ( 8 ) 頁： 5967-5976 2011年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

Rhodopsins possess retinal chromophore surrounded by seven transmembrane alpha-helices, are widespread in prokaryotes and in eukaryotes, and can be utilized as optogenetic tools. Although rhodopsins work as distinctly different photo-receptors 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.

DOI： 10.1074/jbc.M110.190058

Photochemistry of a putative new class of sensory rhodopsin (SRIII) coded by xop2 of Haloarcular marismortui 査読有り

Nakao, Y. Kikukawa, T. Shimono, K. Tamogami, J. Kimitsuki, N. Nara, T. Unno, M. Ihara, K. Kamo, N.

Journal of Photochemistry and Photobiology B-Biology 102 巻 ( 1 ) 頁： 45-54 2011年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

Baliga et al. (2004) [1] reported the existence of a functionally unpredictable opsin gene, named xop2, in Haloarcula marismortui, a holophilic archaeon. Ihara et al. [38] performed molecular phylogenetic analysis and determined that the product of xop2 belonged to a new class of opsins in the sensory rhodopsins. This microbial rhodopsin was therefore named H. marismortui sensory rhodopsin III (HmSRIII). Here, we functionally expressed HmSRIII in Escherichia coli cell membranes to examine the photochemistry. The wavelength of maximum absorption (lambda(max)) for HmSRIII was 506 nm. We observed a very slow photocycle that completed in similar to 50 s. Intermediates were defined as M (lambda(max) similar to 380 nm), N (lambda(max) similar to 460 nm) and O (lambda(max) similar to 530 nm) 0.01 s after the flash excitation. The nomenclature for these intermediates was based on their locations along the absorption maxima of bacteriorhodopsin. Analysis of laser-flash-photolysis data in the presence and absence of azide gave the following results: (1) an equilibrium between N and 0 was attained, (2) the direct product of the M-decay was 0 but not N, and (3) the last photo-intermediate (HmSRIII') had a lambda(max) similar to that of the original, and its decay rate was very slow. Resonance Raman spectroscopy revealed that this N-intermediate had 13-cis retinal conformation. Proton uptake occurred during the course of M-decay, whereas proton release occurred during the course of O-decay (or exactly N-O equilibrium). Very weak proton-pumping activity was observed whose direction is the same as that of bacteriorhodopsin, a typical light-driven proton pump. (C) 2010 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jphotobiol.2010.09.004

Spectroscopic studies of a sensory rhodopsin I homologue from the archaeon Haloarcula vallismortis. 査読有り

Yagasaki J, Suzuki D, Ihara K, Inoue K, Kikukawa T, Sakai M, Fujii M, Homma M, Kandori H, Sudo Y.

Biochemistry 49 巻 ( 6 ) 頁： 1183-1190 2010年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

Sensory rhodopsin I (SRI) functions as a dual receptor regulating both negative and positive phototaxis. It transmits light signals through changes in protein-protein interactions with its transducer protein, HtrI. The phototaxis function of Halobacterium salinarum SRI (HsSRI) has been well characterized using genetic and molecular techniques, whereas that of Salinibacter ruber SRI (SrSRI) has not. SrSRI has the advantage of high protein stability compared with HsSRI and, therefore, provided new information about structural changes and Cl(-) binding of SRI. However, nothing is known about the functional role of SrSRI in phototaxis behavior. In this study, we expressed a SRI homologue from the archaeon Haloarcula vallismortis (HvSRI) as a recombinant protein which uses all-trans-retinal as a chromophore. Functionally important residues of HsSRI are completely conserved in HvSRI (unlike in SrSRI), and HvSRI is extremely stable in buffers without Cl(-). Taking advantage of the high stability, we characterized the photochemical properties of HvSRI under acidic and basic conditions and observed the effects of Cl(-) on the protein under both conditions. Fourier transform infrared results revealed that the structural changes in HvSRI were quite similar to those in HsSRI and SrSRI. Thus, HvSRI can become a useful protein model for improving our understanding of the molecular mechanism of the dual photosensing by SRI.

*Crystal structure of the light-driven chloride pump halorhodopsin from Natronomonas pharaonis 査読有り

Tsutomu Kouyama; Soun Kanada; Yu Takeguchi; Akihiro Narusawa; Midori Murakami; Kunio Ihara

J. Mol. Biology 396 巻 ( 3 ) 頁： 564-579 2010年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

The light-driven chloride pump halorhodopsin from Natronomonas pharaonis (phR) crystallised into the monoclinic space group C2, with a phR trimer per the asymmetric unit. Diffraction data at 2.0-A resolution showed that the carotenoid bacterioruberin binds to crevices between adjacent protein subunits in the trimeric assembly. Besides seven transmembrane helices (A to G) that characterise archaeal rhodopsins, the phR protomer possesses an amphipathic alpha-helix (A') at the N-terminus. This helix, together with a long loop between helices B and C, forms a hydrophobic cap that covers the extracellular surface and prevents a rapid ion exchange between the active centre and the extracellular medium. The retinal bound to Lys256 in helix G takes on an all-trans configuration with the Schiff base being hydrogen-bonded to a water molecule. The Schiff base also interacts with Asp252 and a chloride ion, the latter being fixed by two polar groups (Thr126 and Ser130) in helix C. In the anion uptake pathway, four ionisable residues (Arg123, Glu234, Arg176 and His100) and seven water molecules are aligned to form a long hydrogen-bonding network. Conversely, the cytoplasmic half is filled mostly by hydrophobic residues, forming a large energetic barrier against the transport of anion. The height of this barrier would be lowered substantially if the cytoplasmic half functions as a proton/HCl antiporter. Interestingly, there is a long cavity extending from the main-chain carbonyl of Lys256 to Thr71 in helix B. This cavity, which is commonly seen in halobacterial light-driven proton pumps, is one possible pathway that is utilised for a water-mediated proton transfer from the cytoplasmic medium to the anion, which is relocated to the cytoplasmic channel during the photocycle. Copyright (c) 2009. Elsevier Ltd. All rights reserved.

*Halorubrum chaoviator sp. nov., a haloarchaeon isolated from sea salt in Baja California, Mexico, Western Australia and Naxos, Greece. 査読有り

Mancinelli R. L., Landheim R., Sanchez-Porro C., Dornmayr-Pfaffenhuemer M., Gruber C., Legat A., Ventosa A., Radax C., Ihara K., White M. R. andStan-Lotter H.

Int. J. Syst. Evol. Microbiol. 59 巻頁： 1908-1913 2009年8月

記述言語：英語掲載種別：研究論文（学術雑誌）

Three halophilic isolates, strains Halo-G*T, AUS-1 and Naxos II, were compared. Halo-G* was isolated from an evaporitic salt crystal from Baja California, Mexico, whereas AUS-1 and Naxos II were isolated from salt pools in Western Australia and the Greek island of Naxos, respectively. Halo-G*T had been exposed previously to conditions of outer space and survived 2 weeks on the Biopan facility. Chemotaxonomic and molecular comparisons suggested high similarity between the three strains. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that the strains clustered with Halorubrum species, showing sequence similarities of 99.2-97.1%. The DNA-DNA hybridization values of strain Halo-G*T and strains AUS-1 and Naxos II are 73 and 75%, respectively, indicating that they constitute a single species. The DNA relatedness between strain Halo-G*T and the type strains of 13 closely related species of the genus Halorubrum ranged from 39 to 2%, suggesting that the three isolates constitute a different genospecies. The G+C content of the DNA of the three strains was 65.5-66.5 mol%. All three strains contained C20C20 derivatives of diethers of phosphatidylglycerol, phosphatidylglyceromethylphosphate and phosphatidylglycerolsulfate, together with a sulfated glycolipid. On the basis of these results, a novel species that includes the three strains is proposed, with the name Halorubrum chaoviator sp. nov. The type strain is strain Halo-G*T (=DSM 19316T=NCIMB 14426T=ATCC BAA-1602T).

A photochromic photoreceptor from a eubacterium. 招待有り

Suzuki D, Kitajima-Ihara T, Furutani Y, Ihara K, Kandori H, Homma M, Sudo Y.

Commun. Integr. Biol. 1 巻頁： 150-152 2008年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

Sensory rhodopsin I (SRI) is one of the most interesting photo- sensory receptors because of its function in using the photochromic reaction to mediate opposing signals which depend on the color of light. It was initially thought that SRI exists only in the archaea, but we recently reported for the first time a newly functional SRI from a eubacterium, Salinibacter ruber (SrSRI). The amino acid sequence of SrSRI shows 43% identity with the well-known SRI (HsSRI) and contains most of the amino acid residues identified as necessary for SRI function. The photochemical properties of SrSRI are similar to those of HsSRI. In addition, SrSRI is a highly stable protein, even in dilute salt conditions. Thus, SrSRI could be a key protein for characterizing its association with the SrSRI transducer protein, SrHtrI, and for elucidating structural changes of SRI and HtrI that occur during their function. Recently, new approaches to manipulate cellular functions with rhodopsins have been established. SRI can activate and deactivate a kinase, CheA, by the photochromic reaction. Kinases are key molecules for signal transduction in various organisms, and SRI could potentially manipulate their cellular functions.

Salinibacter sensory rhodopsin: sensory rhodopsin I-like protein from a eubacterium. 査読有り

Kitajima-Ihara T, Furutani Y, Suzuki D, Ihara K, Kandori H, Homma M, Sudo Y.

Journal of Biological Chemistry 283 巻頁： 23533-23541 2008年8月

記述言語：英語掲載種別：研究論文（学術雑誌）

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 ( approximately 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.

*A halorhodopsin-overproducing mutant isolated from an extremely haloalkaliphilic archaeon Natronomonas pharaonis. 査読有り

Ihara K, Narusawa A, Maruyama K, Takeguchi M, Kouyama T.

FEBS Letter 582 巻頁： 2931-2936 2008年8月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）

The halorhodopsin (hR)-overproducing mutant strain KM-1 was isolated from the extremely haloalkaliphilic archaeon Natronomonas pharaonis type strain DSM2160(T). hR-enriched membranes were easily obtained by washing the cells with distilled water. The membranes were claret colored owing to two pigments: hR and bacterioruberin. The hR component in the absorption spectra changed from blue to purple upon the addition of Cl(-) and had a K(m) value of 1.7 mM. Overexpression of hR in strain KM-1 might be caused by the point mutation Asp324-->Asn in the bacteriorhodopsin activator homologues of N. pharaonis. The mutation changed the hR-expression pattern from inducible to constitutive in the late exponential phase.

Halide binding by the D212N mutant of Bacteriorhodopsin affects hydrogen bonding of water in the active site. 査読有り

Shibata M, Yoshitsugu M, Mizuide N, Ihara K, Kandori H.

Biochemistry 46 巻 ( 25 ) 頁： 7525-7535 2007年6月

Hydrogen-bonding interaction of the protonated schiff base with halides in a chloride-pumping bacteriorhodopsin mutant. 査読有り

Shibata M,Ihara K,Kandori H.

Biochemistry 45 巻 ( 35 ) 頁： 10633-10640 2006年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

Bacteriorhodopsin (BR) and halorhodopsin (HR) are light-driven proton and chloride ion pumps, respectively, in Halobacterium salinarum. The amino acid identity of these proteins is about 25%, suggesting that each has been optimized for their own functions during evolution. However, it is known that the BR mutants, D85T and D85S, can pump chloride ions. This fact implies that the Schiff base region is important in determining ionic selectivity. The X-ray crystallographic structure of D85S(Br(-)) showed the presence of a bromide ion in the Schiff base region (Facciotti, M. T., Cheung, V. S., Nguyen, D., Rouhani, S., and Glaeser, R. M. (2003) Biophys. J. 85, 451-458). In this article, we report on the study of hydrogen bonds of the Schiff base and water molecules in D85S in the absence and presence of various halides, assigning their N-D and O-D stretching vibrations in D(2)O, respectively, in low-temperature Fourier-transform infrared (FTIR) spectroscopy. We found that the hydrogen bond of the Schiff base in D85S(Cl(-)) is much stronger than that in HR, being as strong as that in wild-type BR. Similar halide dependence in D85S and in solution implies that the Schiff base forms a direct hydrogen bond with a halide, consistent with the X-ray structure. Photoisomerization causes a weakened hydrogen bond of the Schiff base, and halide dependence on the stretching frequency is lost. These spectral features are similar to those in the photocycle of proton-pumping BR, though the weakened hydrogen bond is more significant for BR. However, the spectral features of water bands in D85S are closer to chloride-pumping HR because O-D stretching vibrations of water are observed only at >2500 cm(-)(1). Unlike in BR, we did not observe strongly hydrogen-bonded water molecules for halide-pumping D85S mutants. This observation agrees with our recent hypothesis that strongly hydrogen-bonded water molecules are required for the proton-pumping activity of archaeal rhodopsins. Hydrogen-bond

Comparative study of archaeal light driven proton pumps 招待有り

Ihara K

12th International Conference on Reinal Protein satellite meeting ; Structure, Fuction, and Evolution of Rhodopsins -Mechanism of proton transfer and color tuning 頁： 10-11 2006年6月

Comparative study of archaeal light driven proton pumps for functional expression in Escherichia coli. 招待有り

Ihara K

12th International Conference on Reinal Protein 頁： p092b 2006年6月

Crystal structure of archaerhodopsin-1 and -2: Common structural motif in archaeal light-driven proton pumps 査読有り

Enami N, Yoshimura K, Murakami M., Okunmura H., Ihara K. and Kouyama T.

J.Mol. Biol. 358 巻 ( 3 ) 頁： 675-685 2006年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

Archaerhodopsin-1 and -2 (aR-1 and aR-2) are light-driven proton pumps found in Halorubrum sp. aus-1 and -2, which share 55-58% sequence identity with bacteriorhodopsin (bR), a proton pump found in Halobacterium salinarum. In this study, aR-1 and aR-2 were crystallized into 3D crystals belonging to P4(3)2(1)2 (a = b = 128.1 A, c = 117.6 A) and C222(1) (a = 122.9 A, b = 139.5 A, c = 108.1 A), respectively. In both the crystals, the asymmetric unit contains two protein molecules with slightly different conformations. Each subunit is composed of seven helical segments as seen in bR but, unlike bR, aR-1 as well as aR-2 has a unique omega loop near the N terminus. It is found that the proton pathway in the extracellular half (i.e. the proton release channel) is more opened in aR-2 than in aR-1 or bR. This structural difference accounts for a large variation in the pKa of the acid purple-to-blue transition among the three proton pumps. All the aromatic residues surrounding the retinal polyene chain are conserved among the three proton pumps, confirming a previous argument that these residues are required for the stereo-specificity of the retinal isomerization. In the cytoplasmic half, the region surrounded by helices B, C and G is highly conserved, while the structural conservation is very low for residues extruded from helices E and F. Structural conservation of the hydrophobic residues located on the proton uptake pathway suggests that their precise arrangement is necessary to prevent a backward flow of proton in the presence of a large pH gradient and membrane potential. An empty cavity is commonly seen in the vicinity of Leu93 contacting the retinal C13 methyl. Existence of such a cavity is required to allow a large rotation of the side-chain of Leu93 at the early stage of the photocycle, which has been shown to accompany water translocation across the Schiff base.

*A light-driven proton pump from Haloterrigene turkmenica: functional expression in Escherichia coli membrane and coupling with a H+ co-transporter. 査読有り

Kamo N., Hashiba T., Kikukawa T., Araiso T., Ihara K. and Nara T.

Biochem. Biophys. Res. Commun. 341 巻頁： 285-290 2006年1月

*Internal water molecules of light-driven chloride pump proteins. 査読有り

Shibata M., Muneda N., Ihara K., Sasaki T., Demura M. , and Kandori H.

Chem. Phys. Lett. 392 巻頁： 330-333 2004年

Search for the retinal-type photosynthetic microorganisms from the japanease sea. 招待有り

K.Ihara, K. Sugiura, and S. Ito

Ann. Report Interdiscipl. Res. Inst. Environ. Sci 22 巻頁： 51-60 2003年

　前のページ - 次のページ　

<井原 邦夫>

論文 - 井原 邦夫

<井原　邦夫>

論文 - 井原　邦夫