Updated on 2021/05/25

写真a

 
MURAKAMI Midori
 
Organization
Graduate School of Science Division of Material Science Biophysics Lecturer
Graduate School
Graduate School of Science
Undergraduate School
School of Science Department of Physics
Title
Lecturer

Degree 1

  1. 博士(理学) ( 2000.4   名古屋大学 ) 

Research Interests 1

  1. ロドプシン

Research Areas 2

  1. Life Science / Biophysics

  2. Life Science / Structural biochemistry

Research History 5

  1. Nagoya University   Graduate School of Science Division of Material Science Biophysics   Lecturer

    2016.4

  2. ETH Zürich   Guest Professor

    2016.4 - 2017.3

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    Country:Switzerland

  3. Paul Scherrer Institut   Guest Scientist

    2016.4 - 2017.3

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    Country:Switzerland

  4. Nagoya University   Graduate School of Science Division of Material Science   Assistant Professor

    2007.4 - 2016.3

  5. Nagoya University   Graduate School of Science Division of Material Science   Assistant

    2000.4 - 2007.3

Professional Memberships 4

  1. 日本生物物理学会

    1993.4

  2. 日本結晶学会

  3. 日本蛋白質科学会

  4. 日本物理学会

Committee Memberships 17

  1. IGER International Symposium on Physics of Life   実行委員長  

    2017.3   

  2. Biophysics and Physicobiology   Associate Editor  

    2015.7   

  3. 16th International Conference on Retinal Proteins   実行委員  

    2014.10   

  4. 日本生物物理学会中部支部   会計幹事  

    2012.4 - 2013.3   

  5. 第50回日本生物物理学会年会実行委員会   実行委員  

    2012   

  6. 日本生物物理学会運営委員/理事   委員/理事  

    2011.1 - 2015.6   

  7. Biophysics and Physicobiology   Editorial Board  

    2011.1 - 2015.6   

  8. 日本生物物理学会   運営委員/理事  

    2011.1 - 2015.6   

  9. 日本生物物理学会会誌編集委員会   委員  

    2007.1 - 2009.12   

  10. 日本生物物理学会   会誌編集委員  

    2007.1 - 2008.12   

  11. 日本比較生理生化学会   会長選挙選挙管理委員  

    2007   

  12. 日本生物物理学会中部支部幹事   会計  

    2005.4 - 2006.3   

  13. 日本生物物理学会中部支部   会計幹事  

    2005.4 - 2006.3   

  14. 第40回日本生物物理学会年会実行委員会   実行委員  

    2001   

  15. 第 40 回日本生物物理学会年会   実行委員  

    2001   

  16. 日本生物物理学会会誌地区編集委員   中部地区委員  

    2000.1 - 2000.12   

  17. 日本生物物理学会   編集中部地区委員  

    2000.1 - 2000.12   

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Papers 33

  1. Crystal Structures of the Putative Isocitrate Dehydrogenase from Sulfolobus tokodaii Strain 7 in the Apo and NADP+-Bound Forms. Reviewed International journal

    Hisanori Kondo, Midori Murakami

    Archaea (Vancouver, B.C.)   Vol. 2018   page: 7571984 - 7571984   2018

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    Language:English   Publishing type:Research paper (scientific journal)  

    Isocitrate dehydrogenase is a catabolic enzyme that acts during the third step of the tricarboxylic acid cycle. The hypothetical protein ST2166 from the archaeon Sulfolobus tokodaii was isolated and crystallized. It shares high primary structure homology with prokaryotic NADP+-dependent IDHs, suggesting that these enzymes share a common enzymatic mechanism. The crystal structure of ST2166 was determined at 2.0 Å resolution in the apo form, and then the structure of the crystal soaked with NADP+ was also determined at 2.4 Å resolution, which contained NADP+ bound at the putative active site. Comparisons between the structures of apo and NADP+-bound forms and NADP-IDHs from other prokaryotes suggest that prokaryotic NADP-IDHs recognize their cofactors using conserved Lys335, Tyr336, and Arg386 in ST2166 at the opening cleft before the domain closure.

    DOI: 10.1155/2018/7571984

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  2. Crystal Structure of the 11-cis Isomer of Pharaonis Halorhodopsin: Structural Constraints on Interconversions among Different Isomeric States. Reviewed International journal

    Siu Kit Chan, Haruki Kawaguchi, Hiroki Kubo, Midori Murakami, Kunio Ihara, Kosuke Maki, Tsutomu Kouyama

    Biochemistry   Vol. 55 ( 29 ) page: 4092 - 4104   2016.7

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    Publishing type:Research paper (scientific journal)   Publisher:AMER CHEMICAL SOC  

    Like other microbial rhodopsins, the light driven chloride pump halorhodopsin from Natronomonas pharaonis (pHR) contains a mixture of all-trans/15-anti and 13-cis/15-syn isomers in the dark adapted state. A recent crystallographic study of the reaction states of pHR has shown that reaction states with 13-cis/15-syn retinal occur in the anion pumping cycle that is initiated by excitation of the all-trans isomer. In this study, we investigated interconversions among different isomeric states of pHR in the absence of chloride ions. The illumination of chloride free pHR with red light caused a large blue shift in the absorption maximum of the retinal visible band. During this "red adaptation", the content of the 11-cis isomer increased significantly, while the molar ratio of the 13-cis isomer to the all-trans isomer remained unchanged. The results suggest that the thermally activated interconversion between the 13-cis and the all-trans isomers is very rapid. Diffraction data from red adapted crystals showed that accommodation of the retinal chromophore with the 11-cis/15-syn configuration was achieved without a large change in the retinal binding pocket. The measurement of absorption kinetics under illumination showed that the 11-cis isomer, with a λmax at 565 nm, was generated upon excitation of a red-shifted species (λmax = 625 nm) that was present as a minor component in the dark adapted state. It is possible that this red-shifted species mimics an O-like reaction state with 13-cis/15-syn retinal, which was hypothesized to occur at a late stage of the anion pumping cycle.

    DOI: 10.1021/acs.biochem.6b00277

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  3. Crystal Structures of Two Isozymes of Citrate Synthase from Sulfolobus tokodaii Strain 7. Reviewed International journal

    Midori Murakami, Tsutomu Kouyama

    Biochemistry research international   Vol. 2016   page: 7560919 - 7560919   2016

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Hindawi Limited  

    Thermoacidophilic archaeon Sulfolobus tokodaii strain 7 has two citrate synthase genes (ST1805-CS and ST0587-CS) in the genome with 45% sequence identity. Because they exhibit similar optimal temperatures of catalytic activity and thermal inactivation profiles, we performed structural comparisons between these isozymes to elucidate adaptation mechanisms to high temperatures in thermophilic CSs. The crystal structures of ST1805-CS and ST0587-CS were determined at 2.0 Å and 2.7 Å resolutions, respectively. Structural comparison reveals that both of them are dimeric enzymes composed of two identical subunits, and these dimeric structures are quite similar to those of citrate synthases from archaea and eubacteria. ST0587-CS has, however, 55 ion pairs within whole dimer structure, while having only 36 in ST1805-CS. Although the number and distributions of ion pairs are distinct from each other, intersubunit ion pairs between two domains of each isozyme are identical especially in interterminal region. Because the location and number of ion pairs are in a trend with other CSs from thermophilic microorganisms, the factors responsible for thermal adaptation of ST-CS isozymes are characterized by ion pairs in interterminal region.

    DOI: 10.1155/2016/7560919

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  4. Crystal structures of the L1, L2, N, and O states of pharaonis halorhodopsin. Reviewed International journal

    Tsutomu Kouyama, Haruki Kawaguchi, Taichi Nakanishi, Hiroki Kubo, Midori Murakami

    Biophysical journal   Vol. 108 ( 11 ) page: 2680 - 2690   2015.6

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:CELL PRESS  

    Halorhodopsin from Natronomonas pharaonis (pHR) functions as a light-driven halide ion pump. In the presence of halide ions, the photochemical reaction of pHR is described by the scheme: K→ L1 → L2 → N → O → pHR' → pHR. Here, we report light-induced structural changes of the pHR-bromide complex observed in the C2 crystal. In the L1-to-L2 transition, the bromide ion that initially exists in the extracellular vicinity of retinal moves across the retinal Schiff base. Upon the formation of the N state with a bromide ion bound to the cytoplasmic vicinity of the retinal Schiff base, the cytoplasmic half of helix F moves outward to create a water channel in the cytoplasmic interhelical space, whereas the extracellular half of helix C moves inward. During the transition from N to an N-like reaction state with retinal assuming the 13-cis/15-syn configuration, the translocated bromide ion is released into the cytoplasmic medium. Subsequently, helix F relaxes into its original conformation, generating the O state. Anion uptake from the extracellular side occurs when helix C relaxes into its original conformation. These structural data provide insight into the structural basis of unidirectional anion transport.

    DOI: 10.1016/j.bpj.2015.04.027

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  5. Crystallographic Study of the LUMI Intermediate of Squid Rhodopsin. Reviewed International journal

    Midori Murakami, Tsutomu Kouyama

    PloS one   Vol. 10 ( 5 ) page: e0126970   2015.5

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:PUBLIC LIBRARY SCIENCE  

    Upon absorption of light, the retinal chromophore in rhodopsin isomerizes from the 11-cis to the trans configuration, initiating a photoreaction cycle. The primary photoreaction state, bathorhodopsin (BATHO), relaxes thermally through lumirhodopsin (LUMI) into a photoactive state, metarhodopsin (META), which stimulates the conjugated G-protein. Previous crystallographic studies of squid and bovine rhodopsins have shown that the structural change in the primary photoreaction of squid rhodopsin is considerably different from that observed in bovine rhodopsin. It would be expected that there is a fundamental difference in the subsequent thermal relaxation process between vertebrate and invertebrate rhodopsins. In this work, we performed crystallographic analyses of the LUMI state of squid rhodopsin using the P62 crystal. When the crystal was illuminated at 100 K with blue light, a half fraction of the protein was converted into BATHO. This reaction state relaxed into LUMI when the illuminated crystal was warmed in the dark to 170 K. It was found that, whereas trans retinal is largely twisted in BATHO, it takes on a more planar configuration in LUMI. This relaxation of retinal is accompanied by reorientation of the Schiff base NH bond, the hydrogen-bonding partner of which is switched to Asn185 in LUMI. Unlike bovine rhodopsin, the BATHO-to-LUMI transition in squid rhodopsin was accompanied by no significant change in the position/orientation of the beta-ionone ring of retinal.

    DOI: 10.1371/journal.pone.0126970

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  6. Structural comparison between the open and closed forms of citrate synthase from Thermus thermophilus HB8. Reviewed

    Eiji Kanamori, Shin-Ichi Kawaguchi, Seiki Kuramitsu, Tsutomu Kouyama, Midori Murakami

    Biophysics and physicobiology   Vol. 12   page: 47 - 56   2015

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    The crystal structures of citrate synthase from the thermophilic eubacteria Thermus thermophilus HB8 (TtCS) were determined for an open form at 1.5 Å resolution and for closed form at 2.3 Å resolution, respectively. In the absence of ligands TtCS in the open form was crystalized into a tetragonal form with a single subunit in the asymmetric unit. TtCS was also co-crystallized with citrate and coenzyme-A to form an orthorhombic crystal with two homodimers in the asymmetric unit. Citrate and CoA are found in the active site situated between the large domain and the small domain in all subunit whereas the complex shows two distinct closed conformations, the fully closed form and partially closed form. Structural comparisons are performed to describe conformational changes associated with binding of products of TtCS. Upon binding of citrate, basic residues in the active site move toward citrate and make a hydrogen bond network in the active site, inducing a large-scale rotation of the small domain relative to the large domain. CoA is sandwiched between the small and large domains and then the cysteamine tail is inserted into the active site with a cooperative rotation around mainchain dihedrals in the hinge region connecting helices M and N. According to this rotation these helices are extended to close the active site completely. The considerable flexibility and structural rearrangements in the hinge region are crucial for an ordered bibi reaction in catalysis for microbial CSs.

    DOI: 10.2142/biophysico.12.0_47

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  7. Structure of archaerhodopsin-2 at 1.8 Å resolution. Reviewed International journal

    Tsutomu Kouyama, Ryudo Fujii, Soun Kanada, Taichi Nakanishi, Siu Kit Chan, Midori Murakami

    Acta crystallographica. Section D, Biological crystallography   Vol. 70 ( Pt 10 ) page: 2692 - 2701   2014.10

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

    Archaerhodopsin-2 (aR2), the sole protein found in the claret membrane of Halorubrum sp. Aus-2, functions as a light-driven proton pump. In this study, structural analysis of aR2 was performed using a novel three-dimensional crystal prepared by the successive fusion of claret membranes. The crystal is made up of stacked membranes, in each of which aR2 trimers are arranged on a hexagonal lattice. This lattice structure resembles that found in the purple membrane of H. salinarum, except that lipid molecules trapped within the trimeric structure are not distributed with perfect threefold symmetry. Nonetheless, diffraction data at 1.8 Å resolution provide accurate structural information about functionally important residues. It is shown that two glutamates in the proton-release channel form a paired structure that is maintained by a low-barrier hydrogen bond. Although the structure of the proton-release pathway is highly conserved among proton-pumping archaeal rhodopsins, aR2 possesses the following peculiar structural features: (i) the motional freedom of the tryptophan residue that makes contact with the C13 methyl group of retinal is restricted, affecting the formation/decay kinetics of the L state, and (ii) the N-terminal polypeptide folds into an Ω-loop, which may play a role in organizing the higher-order structure.

    DOI: 10.1107/S1399004714017313

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  8. Crystal structure of Cruxrhodopsin-3 from Haloarcula vallismortis. Reviewed International journal

    Siu Kit Chan, Tomomi Kitajima-Ihara, Ryudoh Fujii, Toshiaki Gotoh, Midori Murakami, Kunio Ihara, Tsutomu Kouyama

    PloS one   Vol. 9 ( 9 ) page: e108362   2014.9

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    Cruxrhodopsin-3 (cR3), a retinylidene protein found in the claret membrane of Haloarcula vallismortis, functions as a light-driven proton pump. In this study, the membrane fusion method was applied to crystallize cR3 into a crystal belonging to space group P321. Diffraction data at 2.1 Å resolution show that cR3 forms a trimeric assembly with bacterioruberin bound to the crevice between neighboring subunits. Although the structure of the proton-release pathway is conserved among proton-pumping archaeal rhodopsins, cR3 possesses the following peculiar structural features: 1) The DE loop is long enough to interact with a neighboring subunit, strengthening the trimeric assembly; 2) Three positive charges are distributed at the cytoplasmic end of helix F, affecting the higher order structure of cR3; 3) The cytoplasmic vicinity of retinal is more rigid in cR3 than in bacteriorhodopsin, affecting the early reaction step in the proton-pumping cycle; 4) the cytoplasmic part of helix E is greatly bent, influencing the proton uptake process. Meanwhile, it was observed that the photobleaching of retinal, which scarcely occurred in the membrane state, became significant when the trimeric assembly of cR3 was dissociated into monomers in the presence of an excess amount of detergent. On the basis of these observations, we discuss structural factors affecting the photostabilities of ion-pumping rhodopsins.

    DOI: 10.1371/journal.pone.0108362

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  9. Crystal structure of deltarhodopsin-3 from Haloterrigena thermotolerans. Reviewed International journal

    Jin Zhang, Katsuhide Mizuno, Yuki Murata, Hideaki Koide, Midori Murakami, Kunio Ihara, Tsutomu Kouyama

    Proteins   Vol. 81 ( 9 ) page: 1585 - 1592   2013.9

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:WILEY-BLACKWELL  

    Deltarhodopsin, a new member of the microbial rhodopsin family, functions as a light-driven proton pump. Here, we report the three-dimensional structure of deltarhodopsin (dR3) from Haloterrigena thermotolerans at 2.7 Å resolution. A crystal belonging to space group R32 (a, b = 111.71 Å, c = 198.25 Å) was obtained by the membrane fusion method. In this crystal, dR3 forms a trimeric structure as observed for bacteriorhodopsin (bR). Structural comparison of dR with bR showed that the inner part (the proton release and uptake pathways) is highly conserved. Meanwhile, residues in the protein-protein contact region are largely altered so that the diameter of the trimeric structure at the cytoplasmic side is noticeably larger in dR3. Unlike bR, dR3 possesses a helical segment at the C-terminal region that fills the space between the AB and EF loops. A significant difference is also seen in the FG loop, which is one residue longer in dR3. Another peculiar property of dR3 is a highly crowded distribution of positively charged residues on the cytoplasmic surface, which may be relevant to a specific interaction with some cytoplasmic component.

    DOI: 10.1002/prot.24316

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  10. Large deformation of helix F during the photoreaction cycle of Pharaonis halorhodopsin in complex with azide. Reviewed International journal

    Taichi Nakanishi, Soun Kanada, Midori Murakami, Kunio Ihara, Tsutomu Kouyama

    Biophysical journal   Vol. 104 ( 2 ) page: 377 - 385   2013.1

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:CELL PRESS  

    Halorhodopsin from Natronomonas pharaonis (pHR), a retinylidene protein that functions as a light-driven chloride ion pump, is converted into a proton pump in the presence of azide ion. To clarify this conversion, we investigated light-induced structural changes in pHR using a C2 crystal that was prepared in the presence of Cl(-) and subsequently soaked in a solution containing azide ion. When the pHR-azide complex was illuminated at pH 9, a profound outward movement (∼4 Å) of the cytoplasmic half of helix F was observed in a subunit with the EF loop facing an open space. This movement created a long water channel between the retinal Schiff base and the cytoplasmic surface, along which a proton could be transported. Meanwhile, the middle moiety of helix C moved inward, leading to shrinkage of the primary anion-binding site (site I), and the azide molecule in site I was expelled out to the extracellular medium. The results suggest that the cytoplasmic half of helix F and the middle moiety of helix C act as different types of valves for active proton transport.

    DOI: 10.1016/j.bpj.2012.12.018

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  11. Crystal structure of the O intermediate of the Leu93→Ala mutant of bacteriorhodopsin. Reviewed International journal

    Jin Zhang, Yoshikazu Yamazaki, Masanori Hikake, Midori Murakami, Kunio Ihara, Tsutomu Kouyama

    Proteins   Vol. 80 ( 10 ) page: 2384 - 2396   2012.10

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    The lifetime of the O intermediate of bacteriorhodopsin (BR) is extended by a factor of ∼250 in the Leu93-to-Ala mutant (BR_L93A). To clarify the structural changes occurring in the last stage of the proton pumping cycle of BR, we crystallized BR_L93A into a hexagonal P622 crystal. Diffraction data from the unphotolyzed state showed that the deletion of three carbon atoms from Leu93 is compensated by the insertion of four water molecules in the cytoplasmic vicinity of retinal. This insertion of water is suggested to be responsible for the blue-shifted λ(max) (540 nm) of the mutant. A long-lived substate of O with a red-shifted λ(max) (~565 nm) was trapped when the crystal of BR_L93A was flash-cooled after illumination with green light. This substate (O(slow)) bears considerable similarity to the M intermediate of native BR; that is, it commonly shows deformation of helix C and the FG loop, downward orientation of the side chain of Arg82, and disruption of the Glu194/Glu204 pair. In O(slow), however, the main chain of Lys216 is less distorted and retinal takes on the 13-cis/15-syn configuration. Another significant difference is seen in the pH dependence of the structure of the proton release group, the pK(a) value of which is suggested to be much lower in O(slow) than in M.

    DOI: 10.1002/prot.24124

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  12. Crystallographic analysis of the primary photochemical reaction of squid rhodopsin. Reviewed International journal

    Midori Murakami, Tsutomu Kouyama

    Journal of molecular biology   Vol. 413 ( 3 ) page: 615 - 627   2011.10

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

    Visual signal transduction is initiated by the photoisomerization of 11-cis retinal upon rhodopsin ligation. Unlike vertebrate rhodopsin, which interacts with Gt-type G-protein to stimulate the cyclic GMP signaling pathway, invertebrate rhodopsin interacts with Gq-type G-protein to stimulate a signaling pathway that is based on inositol 1,4,5-triphosphate. Since the inositol 1,4,5-triphosphate signaling pathway is utilized by mammalian nonvisual pigments and a large number of G-protein-coupled receptors, it is important to elucidate how the activation mechanism of invertebrate rhodopsin differs from that of vertebrate rhodopsin. Previous crystallographic studies of squid and bovine rhodopsins have shown that there is a profound difference in the structures of the retinal-binding pockets of these photoreceptors. Here, we report the crystal structures of all-trans bathorhodopsin (Batho; the first photoreaction intermediate) and the artificial 9-cis isorhodopsin (Iso) of squid rhodopsin. Upon the formation of Batho, the central moiety of the retinal was observed to move largely towards the cytoplasmic side, while the Schiff base and the ionone ring underwent limited movements (i.e., the all-trans retinal in Batho took on a right-handed screwed configuration). Conversely, the 9-cis retinal in Iso took on a planar configuration. Our results suggest that the light energy absorbed by squid rhodopsin is mostly converted into the distortion energy of the retinal polyene chain and surrounding residues.

    DOI: 10.1016/j.jmb.2011.08.044

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  13. Crystal structures of an O-like blue form and an anion-free yellow form of pharaonis halorhodopsin. Reviewed International journal

    Soun Kanada, Yuu Takeguchi, Midori Murakami, Kunio Ihara, Tsutomu Kouyama

    Journal of molecular biology   Vol. 413 ( 1 ) page: 162 - 176   2011.10

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

    Halorhodopsin from Natronomonas pharaonis (pHR) was previously crystallized into a monoclinic space group C2, and the structure of the chloride-bound purple form was determined. Here, we report the crystal structures of two chloride-free forms of pHR, that is, an O-like blue form and an M-like yellow form. When the C2 crystal was soaked in a chloride-free alkaline solution, the protein packing was largely altered and the yellow form containing all-trans retinal was generated. Upon neutralization, this yellow form was converted into the blue form. From structural comparison of the different forms of pHR, it was shown that the removal of a chloride ion from the primary binding site (site I), which is located between the retinal Schiff base and Thr126, is accompanied by such a deformation of helix C that the side chain of Thr126 moves toward helix G, leading to a significant shrinkage of site I. A large structural change is also induced in the chloride uptake pathway, where a flip motion of the side chain of Glu234 is accompanied by large movements of the surrounding aromatic residues. Irrespective of different charge distributions at the active site, there was no large difference in the structures of the yellow form and the blue form. It is shown that the yellow-to-purple transition is initiated by the entrance of one water and one HCl to the active site, where the proton and the chloride ion in HCl are transferred to the Schiff base and site I, respectively.

    DOI: 10.1016/j.jmb.2011.08.021

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  14. 無脊椎動物のロドプシン研究

    村上緑

    生物物理   Vol. 50   page: 274-275   2010.12

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    Language:Japanese  

  15. Crystal structure of the light-driven chloride pump halorhodopsin from Natronomonas pharaonis. Reviewed International journal

    Tsutomu Kouyama, Soun Kanada, Yuu Takeguchi, Akihiro Narusawa, Midori Murakami, Kunio Ihara

    Journal of molecular biology   Vol. 396 ( 3 ) page: 564 - 579   2010.2

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    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.

    DOI: 10.1016/j.jmb.2009.11.061

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  16. Structural divergence and functional versatility of the rhodopsin superfamily. Invited Reviewed International journal

    Tsutomu Kouyama, Midori Murakami

    Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology   Vol. 9 ( 11 ) page: 1458 - 1465   2010

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ROYAL SOC CHEMISTRY  

    Seven-transmembrane-helix retinylidene proteins, which constitute the rhodopsin superfamily, have been discovered in diverse species, including Archaea, Eubacteria, fungi, algae and animals. Some members of this super-family were specialized to function as light-driven proton pumps, light-driven chloride pumps, photoisomerases, or light-gated ion channels, where the photochemical reactions are self-completed without interactions with other proteins. Other members evolved to acquire the ability to modulate soluble cytoplasmic or membrane-embedded signal transducers. During the last decade, high-resolution crystal structures were reported for ten members of the rhodopsin superfamily; viz., four proton pumps, two chloride pumps, two microbial photosensors and two visual pigments. Comparison of these structures provides us with a hint to elucidate the common structural motif that is utilized to stabilize their tertiary structures as well as unique architectures that are relevant to specific functions.

    DOI: 10.1039/c0pp00236d

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  17. Corrigendum to "Crystal Structures of Different Substates of Bacteriorhodopsin's M Intermediate at Various pH Levels" [J. Mol. Biol. (2009) 393, 559-573] (DOI:10.1016/j.jmb.2009.08.047) Reviewed

    Masataka Yamamoto, Naoki Hayakawa, Midori Murakami, Tsutomu Kouyama

    Journal of Molecular Biology   Vol. 394 ( 4 ) page: 804 - 804   2009.12

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    DOI: 10.1016/j.jmb.2009.10.037

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  18. Crystal structures of different substrates of bacteriorhodopsin's M intermediate at various pH levels. Reviewed International journal

    Masataka Yamamoto, Naoki Hayakawa, Midori Murakami, Tsutomu Kouyama

    Journal of molecular biology   Vol. 393 ( 3 ) page: 559 - 573   2009.10

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    The hexagonal P622 crystal of bacteriorhodopsin, which is made up of stacked membranes, is stable provided that the precipitant concentration in the soaking solution is higher than a critical value (i.e., 1.5 M ammonium sulfate). Diffraction data showed that the crystal lattice shrank linearly with increasing precipitant concentration, due primarily to narrowing of intermembrane spaces. Although the crystal shrinkage did not affect the rate of formation of the photoreaction M intermediate, its lifetime increased exponentially with the precipitant concentration. It was suggested that the energetic barrier of the M-to-N transition becomes higher when the motional freedom of the EF loop is reduced by crystal lattice force. As a result of this property, the M state accumulated predominantly when the crystal that was soaked at a high precipitant concentration was illuminated at room temperature. Structural data obtained at various pH levels showed that the overall structure of M is not strongly dependent on pH, except that Glu194 and Glu204 in the proton release complex are more separated at pH 7 than at pH 4.4. This result suggests that light-induced disruption of the paired structure of Glu194 and Glu204 is incomplete when external pH is lower than the pK(a) value of the proton release group in the M state.

    DOI: 10.1016/j.jmb.2009.08.047

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  19. Functionally important structural elements of the cyanobacterial clock-related protein Pex. Reviewed International journal

    Shunsuke Kurosawa, Reiko Murakami, Kiyoshi Onai, Megumi Morishita, Daisuke Hasegawa, Ryo Iwase, Tatsuya Uzumaki, Fumio Hayashi, Tomomi Kitajima-Ihara, Shuhei Sakata, Midori Murakami, Tsutomu Kouyama, Masahiro Ishiura

    Genes to cells : devoted to molecular & cellular mechanisms   Vol. 14 ( 1 ) page: 1 - 16   2009.1

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    Pex, a clock-related protein involved in the input pathway of the cyanobacterial circadian clock system, suppresses the expression of clock gene kaiA and lengthens the circadian period. Here, we determined the crystal structure of Anabaena Pex (AnaPex; Anabaena sp. strain PCC 7120) and Synechococcus Pex (SynPex; Synechococcus sp. strain PCC 7942). Pex is a homodimer that forms a winged-helix structure. Using the DNase I protection and electrophoresis mobility shift assays on a Synechococcus kaiA upstream region, we identified a minimal 25-bp sequence that contained an imperfectly inverted repeat sequence as the Pex-binding sequence. Based on crystal structure, we predicted the amino acid residues essential for Pex's DNA-binding activity and examined the effects of various Ala-substitutions in the alpha3 helix and wing region of Pex on in vitro DNA-binding activity and in vivo rhythm functions. Mutant AnaPex proteins carrying a substitution in the wing region displayed no specific DNA-binding activity, whereas those carrying a substitution in the alpha3 helix did display specific binding activity. But the latter were less thermostable than wild-type AnaPex and their in vitro functions were defective. We concluded that Pex binds a kaiA upstream DNA sequence via its wing region and that its alpha3 helix is probably important to its stability.

    DOI: 10.1111/j.1365-2443.2008.01245.x

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  20. イカロドプシンの結晶構造と無脊椎動物の視覚 Invited Reviewed

    村上緑

    生物物理   Vol. 49   page: 15-16   2009

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  21. Functionally important structural elements of the cyanobacterial clock-related protein Pex Reviewed

    Kurosawa S, Murakami R, Onai K, Morishita M, Hasegawa D, Iwase R, Uzumaki T, Hayashi F, Kitajima-Ihara T, Sakata S, Murakami M, Kouyama T, Ishiura M.

    Genes Cells   Vol. 14   page: 1-16   2009

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  22. Structure of squid rhodopsin Invited Reviewed

    Murakami M, Kouyama T

    Research Frontiers 2008     page: 30-31   2009

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  23. Effect of xenon binding to a hydrophobic cavity on the proton pumping cycle in bacteriorhodopsin. International journal

    Naoki Hayakawa, Takashi Kasahara, Daisuke Hasegawa, Keiko Yoshimura, Midori Murakami, Tsutomu Kouyama

    Journal of molecular biology   Vol. 384 ( 4 ) page: 812 - 823   2008.12

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    To understand the functional role of apolar cavities in bacteriorhodopsin, a light-driven proton pump found in Halobacterium salinarum, we investigated the crystal structure in pressurized xenon or krypton. Diffraction data from the P622 crystal showed that one Xe or Kr atom binds to a preexisting hydrophobic cavity buried between helices C and D, located at the same depth from the membrane surface as Asp96, a key residue in the proton uptake pathway. The occupation fraction of Xe or Kr was calculated as approximately 0.32 at a pressure of 1 MPa. In the unphotolyzed state, the binding of Xe or Kr caused no large deformation of the cavity. However, the proton pumping cycle was greatly perturbed when an aqueous suspension of purple membrane was pressurized with xenon gas; that is, the decay of the M state was accelerated significantly (~5 times at full occupancy), while the decay of an equilibrium state of N and O was slightly decelerated. A similar but much smaller perturbation in the reaction kinetics was observed upon pressurization with krypton gas. In a glycerol/water mixture, xenon-induced acceleration of M decay became less significant in proportion to the water activity. Together with the structure of the xenon-bound protein, these observations suggest that xenon binding helps water molecules permeate into apolar cavities in the proton uptake pathway, thereby accelerating the water-mediated proton transfer from Asp96 to the Schiff base.

    DOI: 10.1016/j.jmb.2008.09.075

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  24. Crystal structure of squid rhodopsin

    Murakami Midori, Kouyama Tsutomu

    NATURE   Vol. 453 ( 7193 ) page: 363 - U33   2008.5

  25. スルメイカロドプシンのX線結晶構造解析 Invited

    村上緑、神山勉

    実験医学   Vol. 26   page: 2237-2240   2008

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  26. Crystallization of visual pigments and archaeal rhodopsins

    Kouyama Tsutomu, Murakami Midori, Yoshimura Keiko, Yamamoto Masataka

    ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES   Vol. 64   page: C133 - C133   2008

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  27. Crystallization and crystal properties of squid rhodopsin. Reviewed International journal

    Midori Murakami, Rei Kitahara, Toshiaki Gotoh, Tsutomu Kouyama

    Acta crystallographica. Section F, Structural biology and crystallization communications   Vol. 63 ( Pt 6 ) page: 475 - 479   2007.6

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    Rhodopsin, a photoreceptor membrane protein in the retina, is a prototypical member of the G-protein-coupled receptor family. In this study, rhodopsin from the retina of the squid Todarodes pacificus was treated with V8 protease to remove the C-terminal extension. Truncated rhodopsin was selectively extracted from the microvillar membranes using alkyl glucoside in the presence of zinc ions and was then crystallized by the sitting-drop vapour-diffusion method. Of the various crystals obtained, hexagonal crystals grown in the presence of octylglucoside and ammonium sulfate diffracted to 2.8 A resolution. The diffraction data suggested that the crystal belongs to space group P6(2), with unit-cell parameters a = b = 122.1, c = 158.6 A. Preliminary crystallographic analysis, together with linear dichroism results, suggested that the rhodopsin dimers are packed in such a manner that their transmembrane helices are aligned nearly parallel to the c axis.

    DOI: 10.1107/S1744309107017423

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  28. Crystal structures of archaerhodopsin-1 and -2: Common structural motif in archaeal light-driven proton pumps. Reviewed International journal

    Nobuo Enami, Keiko Yoshimura, Midori Murakami, Hideo Okumura, Kunio Ihara, Tsutomu Kouyama

    Journal of molecular biology   Vol. 358 ( 3 ) page: 675 - 685   2006.5

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    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.

    DOI: 10.1016/j.jmb.2006.02.032

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  29. Crystal structure of the 13-cis isomer of bacteriorhodopsin in the dark-adapted state. Reviewed International journal

    Taichi Nishikawa, Midori Murakami, Tsutomu Kouyama

    Journal of molecular biology   Vol. 352 ( 2 ) page: 319 - 328   2005.9

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    The atomic structure of the trans isomer of bacteriorhodopsin was determined previously by using a 3D crystal belonging to the space group P622. Here, a structure is reported for another isomer with the 13-cis, 15-syn retinal in a dark-adapted crystal. Structural comparison of the two isomers indicates that retinal isomerization around the C13[double bond]C14 and the C15[double bond]N bonds is accompanied by noticeable displacements of a few residues in the vicinity of the retinal Schiff base and small re-arrangement of the hydrogen-bonding network in the proton release channel. On the other hand, aromatic residues surrounding the retinal polyene chain were found to scarcely move during the dark/light adaptation. This result suggests that variation in the structural rigidity within the retinal-binding pocket is one of the important factors ensuring the stereospecific isomerization of retinal.

    DOI: 10.1016/j.jmb.2005.07.021

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  30. Crystal structures of acid blue and alkaline purple forms of bacteriorhodopsin. Reviewed International journal

    Hideo Okumura, Midori Murakami, Tsutomu Kouyama

    Journal of molecular biology   Vol. 351 ( 3 ) page: 481 - 495   2005.8

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    Bacteriorhodopsin, a light-driven proton pump found in the purple membrane of Halobacterium salinarum, exhibits purple at neutral pH but its color is sensitive to pH. Here, structures are reported for an acid blue form and an alkaline purple form of wild-type bacteriorhodopsin. When the P622 crystal prepared at pH 5.2 was acidified with sulfuric acid, its color turned to blue with a pKa of 3.5 and a Hill coefficient of 2. Diffraction data at pH 2-5 indicated that the purple-to-blue transition accompanies a large structural change in the proton release channel; i.e. the extracellular half of helix C moves towards helix G, narrowing the proton release channel and expelling a water molecule from a micro-cavity in the vicinity of the retinal Schiff base. In this respect, the acid-induced structural change resembles the structural change observed upon formation of the M intermediate. But, the acid blue form contains a sulfate ion in a site(s) near Arg82 that is created by re-orientations of the carboxyl groups of Glu194 and Glu204, residues comprising the proton release complex. This result suggests that proton uptake by the proton release complex evokes the anion binding, which in turn induces protonation of Asp85, a key residue regulating the absorption spectrum of the chromophore. Interestingly, a pronounced structural change in the proton release complex was also observed at high pH; i.e. re-orientation of Glu194 towards Tyr83 was found to take place at around pH 10. This alkaline transition is suggested to be accompanied by proton release from the proton release complex and responsible for rapid formation of the M intermediate at high pH.

    DOI: 10.1016/j.jmb.2005.06.026

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  31. Specific damage induced by X-ray radiation and structural changes in the primary photoreaction of bacteriorhodopsin Invited Reviewed

    Yasuhiro Matsui, Keisuke Sakai, Midori Murakami, Yoshitsugu Shiro, Shin ichi Adachi, Hideo Okumura, Tsutomu Kouyama

    JOURNAL OF MOLECULAR BIOLOGY   Vol. 324 ( 3 ) page: 469 - 481   2002.11

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  32. Transduction ion channels directly gated by sugars on the insect taste cell Reviewed

    M Murakami, H Kijima

    JOURNAL OF GENERAL PHYSIOLOGY   Vol. 115 ( 4 ) page: 455 - 466   2000.4

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    Insects detect sugars and amino acids by a specialized taste cell, the sugar receptor cell, in the taste hairs located on their labela and tarsi. We patch-clamped sensory processes of taste cells regenerated from the cut end of the taste hairs on the labelum of the flashfly isolated from the pupa similar to 20 h before emergence. We recorded both single channel and ensemble currents of novel ion channels located on the distal membrane of the sensory process of the sugar receptor cell. In the stable outside-out patch membrane excised from the sensory processes, we could repeatedly record sucrose-induced currents for tens of minutes without appreciable decrease. An inhibitor of G-protein activation, GDP-beta-S, did not significantly decrease the sucrose response. These results strongly suggested that the channel is an ionotropic receptor (a receptor/channel complex), activated directly by sucrose without mediation by second messengers or G protein. The channel was shown to be a nonselective cation channel. Analyses of single channel currents showed that the sucrose-gated channel has a single channel conductance of similar to 30 pS and has a very short mean open time of similar to 0.23 ms. It is inhibited by external Ca2+ and the dose-current amplitude relation could be described by a Michaelis-Menten curve with an apparent dissociation constant of similar to 270 mM. We also report transduction ion channels of the receptor/channel complex type directly gated by fructose and those gated by L-valine located on the sensory process.

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  33. 4-aminopyridine and l-cis-diltiazem block the cGMP-activated K+ channels closed by light in the molluscan extra-ocular photoreceptors Reviewed

    Gotow T, Nishi T, Murakami M.

    Brain Res.   Vol. 745   page: 303-308   1997

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Books 1

  1. 「日本の結晶学(Ⅱ)―輝かしき発展―」・多様化するロドプシンの構造と機能,その応用

    日本結晶学会, 日本の結晶学, I, 出版編集委員会( Role: Joint author)

    日本結晶学会  2014.7 

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    Responsible for pages:336   Language:Japanese

MISC 13

  1. イカロドプシンの光異性化‐熱的緩和反応の結晶構造解析

    村上緑, 神山勉

    日本物理学会講演概要集(CD-ROM)   Vol. 70 ( 1 ) page: ROMBUNNO.22PPSA-52   2015.3

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    Language:Japanese  

    J-GLOBAL

  2. 発色団を導入したイカロドプシン結晶の吸光特性

    村上緑, 神山勉

    日本物理学会講演概要集   Vol. 69 ( 1 ) page: 422   2014.3

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    J-GLOBAL

  3. X線結晶構造から観るイカロドプシンの光反応初期過程

    村上緑, 神山勉

    日本物理学会講演概要集   Vol. 68 ( 1 ) page: 447   2013.3

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    J-GLOBAL

  4. イカロドプシンルミ中間体の結晶構造解析

    村上緑, 神山勉

    日本結晶学会年会講演要旨集   Vol. 2011   page: 94   2011

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    J-GLOBAL

  5. 無脊椎動物のロドプシン研究―津田基之先生に応えて―

    村上緑

    生物物理   Vol. 50   page: 274-275   2010.12

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  6. イカロドプシン光反応初期中間体のX線結晶構造解析

    村上緑, 神山勉

    日本薬学会年会要旨集   Vol. 130th ( 1 ) page: 341   2010.3

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    J-GLOBAL

  7. イカロドプシンの結晶構造と無脊椎動物の視覚 Invited Reviewed

    村上緑

    生物物理   Vol. 49   page: 15-16 - 16   2009

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    Language:Japanese   Publishing type:Article, review, commentary, editorial, etc. (scientific journal)  

  8. スルメイカロドプシンのX線結晶構造解析 Invited

    村上緑, 神山勉

    実験医学   Vol. 26   page: 2237-2240   2008

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  9. バクテリオロドプシンの光誘起構造変化に及ぼす結晶格子力の影響

    山本昌孝, 早川直紀, 村上緑, 神山勉

    日本結晶学会年会講演要旨集   Vol. 2007   page: 85   2007

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    J-GLOBAL

  10. バクテリオロドプシンの光反応サイクルに及ぼすXeガスの影響

    早川直紀, 笠原隆史, 長谷川大祐, 村上緑, 神山勉

    日本結晶学会年会講演要旨集   Vol. 2007   page: 84   2007

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    J-GLOBAL

  11. イカロドプシンの結晶構造

    村上緑, 神山勉

    日本結晶学会年会講演要旨集   Vol. 2007   page: 48   2007

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    J-GLOBAL

  12. 低温顕微分光法によるバクテリオロドプシン反応中間体の構造解析

    徳久武, 村上緑, 神山勉

    日本生物物理学会年会講演予稿集   Vol. 40th   page: S170   2002.11

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    J-GLOBAL

  13. 高度好熱菌Thermus thermophilus HB8由来クエン酸シンターゼリガンド複合体のX線結晶構造解析

    村上緑, 金森英司, 河口真一, 倉光成紀, 横山茂之, 神山勉

    日本生物物理学会年会講演予稿集   Vol. 39th   page: S37   2001.10

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    J-GLOBAL

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Presentations 43

  1. Structural basis of bistable photoreaction in invertebrate rhodopsin International conference

    Midori Murakami

    16th Conference of the Asian Crystallographic Association  2019.12.19 

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    Event date: 2019.12

    Language:English   Presentation type:Oral presentation (general)  

    Venue:National University of Singapore   Country:Singapore  

  2. Structure of the photo-activated acid-meta state of squid rhodopsin International conference

    Murakami M

    Asian Biophysics Association Symposium in conjunction with the Australian Society for Biophysics Meeting 

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    Event date: 2018.12

    Language:English   Presentation type:Oral presentation (general)  

    Country:Australia  

  3. Towards the structural study of large conformational changes of squid metarhodopsin Invited International conference

    Murakami M

    18th International Conference on Retinal Proteins 

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    Event date: 2018.9

    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Hockley Valley Resort   Country:Canada  

  4. Crystallography of squid rhodopsin towards characterizing structural dynamics and bi-stability Invited

    Murakami M

    IGER International Symposium on Physics of Life 

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    Event date: 2017.3

    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Nagoya   Country:Japan  

  5. Another world of Rhodopsin; Structure and Function of squid rhodopsin

    Murakami M

    jLBR Seminar 

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    Event date: 2017.3

    Language:English   Presentation type:Public lecture, seminar, tutorial, course, or other speech  

    Venue:Villigen   Country:Switzerland  

  6. Crystal structure of the LM intermediate of squid rhodopsin Invited International conference

    Murakami M

    17th International Conference on Retinal Proteins 

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    Event date: 2016.10

    Presentation type:Oral presentation (invited, special)  

    Venue:Potsdam   Country:Germany  

  7. Crystallographic studies of squid rhodopsin International conference

    Murakami M

    Membrane Protein Meeting 

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    Event date: 2016.9

    Language:English   Presentation type:Public lecture, seminar, tutorial, course, or other speech  

    Venue:Villigen   Country:Switzerland  

  8. Structural changes of squid rhodopsin during the thermal relaxation process in the P62 crystal Invited International conference

    Murakami M, Kouyama T

    The 2015 International Chemical Congress of Pacific Basin Societies (PACIFICHEM 2015) 

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    Event date: 2015.12

    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Hawaii   Country:United States  

  9. Crystal structure of the lumi intermediate of squid rhodopsin Invited International conference

    Murakami M, Kouyama T

    15th International Conference on Retinal Proteins 

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    Event date: 2012.9 - 2012.10

    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Ascona   Country:Switzerland  

  10. Photo-activation mechanism of squid rhodopsin Invited

    Murakami M

    Photo-activation mechanism of squid rhodopsin 

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    Event date: 2012.9

    Language:English   Presentation type:Oral presentation (invited, special)  

    Country:Japan  

  11. X線結晶構造から観る無脊椎動物ロドプシンの光反応過程 Invited

    村上緑

    第45回生命情報科学研究セミナー 

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    Event date: 2012.3

    Language:Japanese   Presentation type:Public lecture, seminar, tutorial, course, or other speech  

    Venue:東京   Country:Japan  

  12. イカロドプシンルミ中間体の結晶構造解析 Invited

    村上緑

    蛋白研セミナー「結晶学でみるタンパク質の化学と物理」 

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    Event date: 2012.3

    Language:Japanese   Presentation type:Public lecture, seminar, tutorial, course, or other speech  

    Venue:大阪   Country:Japan  

  13. Crystal structure of squid rhodopsin and its photoreaction Invited

    Murakami M

    Physiology and Biophysics Department Seminar Series 

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    Event date: 2010.7

    Language:English   Presentation type:Public lecture, seminar, tutorial, course, or other speech  

    Venue:Irvine   Country:Japan  

  14. イカロドプシンの結晶構造解析-X線でレチナールの光異性化反応を見る- Invited

    村上緑

    第57回生命情報科学研究セミナー 

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    Event date: 2010.2 - 2012.2

    Language:Japanese   Presentation type:Public lecture, seminar, tutorial, course, or other speech  

    Venue:東京   Country:Japan  

  15. イカロドプシンのX線結晶構造解析 Invited

    村上緑

    日本生物物理学会第46回年会シンポジウム「GPCR型受容体を介した生物機能の物理的理解」 

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    Event date: 2008.12

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Country:Japan  

  16. X-ray structure of squid rhodopsin Invited

    Murakami M

    International symposium on membrane proteins and high resolution X-ray structural analysis 

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    Event date: 2008.9

    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Hyogo   Country:Japan  

  17. スルメイカロドプシンのX線結晶構造解析 Invited

    村上緑, 神山勉

    第8回日本蛋白質科学会年会ワークショップ「GPCR解析研究の新展開」 

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    Event date: 2008.6

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Country:Japan  

  18. Crystal Structure of Squid Rhodopsin Invited International conference

    Murakami M

    Gordon Research Conference on "LIGAND RECOGNITION & MOLECULAR GATING" 

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    Event date: 2008.3

    Language:English   Presentation type:Oral presentation (invited, special)  

    Country:United States  

  19. Crystal structure of squid rhodopsin Invited

    International Symposium on RETINAL PROTEINS: EXPERIMENTS AND THEORY 

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    Event date: 2007.9

    Language:English   Presentation type:Oral presentation (invited, special)  

    Country:Germany  

  20. 膜タンパク質の立体構造解析 Invited

    村上緑

    第10回VBLシンポジウム「ナノマテリアルの創生、解析、機能 ―有機、無機、生体」 

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    Event date: 2006.10

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Venue:名古屋   Country:Japan  

  21. X-ray crystallography of squid rhodopsin International conference

    12thICRP Satellite Meeting Nagoya 

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    Event date: 2006.6

    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Nagoya   Country:Japan  

  22. Sugar-gated transduction ion channels localized in sensory processes of the insect taste cells Invited International conference

    Murakami M, Kijima H

    5th International Congress of Comparative Physiology and Biochemistry 

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    Event date: 1999.8

    Language:English   Presentation type:Oral presentation (invited, special)  

    Country:Japan  

  23. Structural basis of bistable photoreaction in invertebrate rhodopsin Invited

    MURAKAMI, Midori

    16th Conference of the Asian Crystallographic Association  2019.12.19 

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    Language:English  

  24. Structure of the photo-activated acid-meta state of squid rhodopsin

    Murakami M

    Asian Biophysics Association Symposium in conjunction with the Australian Society for Biophysics Meeting  2018.12.2 

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    Language:English   Presentation type:Oral presentation (general)  

  25. Towards the structural study of large conformational changes of squid metarhodopsin Invited

    MURAKAMI, Midori

    18th International Conference on Retinal Proteins  2018.9.24 

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    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Hockley Valley Resort  

  26. Crystallography of squid rhodopsin towards characterizing structural dynamics and bi-stability Invited International conference

    MURAKAMI, Midori

    IGER International Symposium on Physics of Life  2017.3.25 

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    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Nagoya  

  27. Another world of Rhodopsin; Structure and Function of squid rhodopsin International conference

    Murakami M

    jLBR Seminar  2017.3.22 

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    Language:English   Presentation type:Public lecture, seminar, tutorial, course, or other speech  

    Venue:Villigen  

  28. Crystal structure of the LM intermediate of squid rhodopsin Invited

    Murakami M

    17th International Conference on Retinal Proteins  2016.10.2 

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    Presentation type:Oral presentation (invited, special)  

    Venue:Potsdam  

  29. Crystallographic studies of squid rhodopsin

    Murakami M

    Membrane Protein Meeting  2016.9.26 

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    Venue:Villigen  

  30. Structural changes of squid rhodopsin during the thermal relaxation process in the P62 crystal Invited

    Murakami M, Kouyama T

    The 2015 International Chemical Congress of Pacific Basin Societies (PACIFICHEM 2015)  2015.12.15 

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    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Hawaii  

  31. Crystal structure of the lumi intermediate of squid rhodopsin Invited

    Murakami M, Kouyama T

    15th International Conference on Retinal Proteins  2012.9.30 

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    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Ascona  

  32. Photo-activation mechanism of squid rhodopsin Invited International conference

    Murakami M

    Photo-activation mechanism of squid rhodopsin  2012.9 

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  33. X線結晶構造から観る無脊椎動物ロドプシンの光反応過程 Invited International conference

    村上緑

    第45回生命情報科学研究セミナー  2012.3.22 

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    Language:Japanese   Presentation type:Public lecture, seminar, tutorial, course, or other speech  

    Venue:東京  

  34. イカロドプシンルミ中間体の結晶構造解析 Invited International conference

    村上緑

    蛋白研セミナー「結晶学でみるタンパク質の化学と物理」  2012.3.6 

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    Language:Japanese   Presentation type:Public lecture, seminar, tutorial, course, or other speech  

    Venue:大阪  

  35. Crystal structure of squid rhodopsin and its photoreaction Invited International conference

    Murakami M

    Physiology and Biophysics Department Seminar Series  2010.7.30 

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    Venue:Irvine  

  36. イカロドプシンの結晶構造解析-X線でレチナールの光異性化反応を見る- Invited International conference

    村上緑

    第57回生命情報科学研究セミナー  2010.2.19 

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    Language:Japanese   Presentation type:Public lecture, seminar, tutorial, course, or other speech  

    Venue:東京  

  37. イカロドプシンのX線結晶構造解析 Invited International conference

    村上緑

    日本生物物理学会第46回年会シンポジウム「GPCR型受容体を介した生物機能の物理的理解」  2008.12.3 

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  38. X-ray structure of squid rhodopsin Invited International conference

    Murakami M

    International symposium on membrane proteins and high resolution X-ray structural analysis  2008.9.1 

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    Venue:Hyogo  

  39. スルメイカロドプシンのX線結晶構造解析 Invited International conference

    村上緑, 神山勉

    第8回日本蛋白質科学会年会ワークショップ「GPCR解析研究の新展開」  2008.6 

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  40. Crystal Structure of Squid Rhodopsin Invited

    Murakami M

    Gordon Research Conference on "LIGAND RECOGNITION & MOLECULAR GATING"  2008.3 

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  41. Crystal structure of squid rhodopsin Invited International conference

    International Symposium on RETINAL PROTEINS: EXPERIMENTS AND THEORY  2007.9 

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  42. 膜タンパク質の立体構造解析 Invited International conference

    村上緑

    第10回VBLシンポジウム「ナノマテリアルの創生、解析、機能 ―有機、無機、生体」  2006.10 

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    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Venue:名古屋  

  43. X-ray crystallography of squid rhodopsin Invited

    12thICRP Satellite Meeting Nagoya  2006.6  "Structure, Function and Evolution of Rhodopsins-Mechanisms of Proton Transfer and Color Tuning-"

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    Venue:Nagoya  

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KAKENHI (Grants-in-Aid for Scientific Research) 12

  1. Structual study of photo-activated meta state of squid rhodopsin

    Grant number:16K07317  2016.4 - 2020.3

    Murakami Midori

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    Grant amount:\5070000 ( Direct Cost: \3900000 、 Indirect Cost:\1170000 )

    The helix architecture of squid photo-activated metarhodopsin(Meta) is similar to that of the dark adapted form. The retinal chromophore is in an all-trans configuration with nearly same axial length to that of bovine Meta. The beta-ionone ring rotates ~ 60 degree to direct nearly parallel to the polyene chain plane. This rotation pushes surrounding aromatic rings of nearby residues to enlarge the binding pocket. These structural changes within the active site weaken the interhelical interactions at the cytoplasmic side. The salt bridge of the DRY motif between helices 3 and 6 is unlocked, resulting in being in partially open form in squid Meta.
    The protonated Schiff base of retinal hydrogen bonds to the OH group of Tyr111 in helix 3, and Asn87 and Asn185 are located within 4A from the Schiff base. The counterion Glu180 is kept ~5A from the Schiff base by hydrogen bonds with Ser187, Tyr190, Tyr277 and Asn185. These residues form a hydrogen bonding network within the active site.

  2. Molecular mechanisms in a visual transduction system of invertebrate rhodopsin

    Grant number:24370043  2012.4 - 2016.3

    Murakami Midori

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    Grant amount:\15860000 ( Direct Cost: \12200000 、 Indirect Cost:\3660000 )

    Blue light illumination at 100 K induced a red-shift of the absorption maximum, indicating that an equilibrium state of three isomeric states, Rhodopsin, Batho and 9-cis isorhodopsin was established. When this crystal was warmed to 240K, the absorption maximum was blue-shifted. Accurate estimation of isomer composition under this condition suggested that Batho was converted to LM with no change in the contents of the other states. We collected diffraction data set and calculate the difference electron density maps. It indicates that the conformational changes are located at the vicinity of the active site upon formation the LM intermediate. The larger movements of helices that characterize meta formation are not initiated at the early stage of photocycle in squid rhodopsin, but occur during the transition from LM to Meta. We try to crystallize the Acid Meta state of squid rhodopsin and succeed to obtain some new crystals which can diffract up to high resolution.

  3. Higher order structure and function of retinal proteins

    Grant number:24370066  2012.4 - 2015.3

    KOUYAMA tsutomu

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    ① Archaerhodopsin-2 was crystallized and its structure was determined at 1.8 Åresolution. ② Cruxrhodopsin-3 was crystallized and its structure was determined at 2.1 Å resolution. ③ Deltarhodopsin-3 was crystallized and its trimeric structure was determined at 2.7 Å resolution. ④ The light-driven chloride ion pump halorhodopsin (pHR) from Natronomonas pharonis was crystallized, and the major photoreaction states (L1, L2, N, N’and O states) of pHR-bromide ion complex were determined at 2.2 -2.7 Å resolutions. ⑤ The crystal structure of an M intermediate of pHR-azide complex was determined at 2.3 Å resolution. ⑥ Various mutants of phoborhodopsin from Halobacterium salinarum were prepared and their photoreaction kinetics were investigated. ⑦ The crystal structure of the Lumi state of squid rhodopsin was determined at 2.7 Å resolution. ⑧ Octopus rhodopsin was crystallized by the membrane fusion method.

  4. 無脊椎動物ロドプシンの光活性化機構の解明

    Grant number:23121513  2011.4 - 2013.3

    新学術領域研究(研究領域提案型)

    村上 緑

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    Authorship:Principal investigator 

    Grant amount:\4680000 ( Direct Cost: \3600000 、 Indirect Cost:\1080000 )

    超高速、超高感度の光センサとして独特の進化を遂げた脊椎動物ロドプシンとは対照的に無脊椎動物のロドプシンは一般GPCRと共通の分子特性を多くもつ。ロドプシン活性化の分子機構を調べる目的で、無脊椎動物ロドプシンの代表としてイカロドプシンを研究対象にX線結晶構造解析に取り組んできた。本課題では光反応中間体の構造解析を進め、レチナール光異性化後の最初の熱産物であるルミロドプシンの構造解析を行った。最終年度では、結晶中でのロドプシンの熱的振る舞い、ルミ中間体へのX線照射の影響を顕微分光測定により調べ、立体構造を2.8Aで決定することができた。また、ルミ中間体に続いて230Kへの熱緩和で現れるLM中間体についても熱的振る舞いに関する知見を得る事ができた。全長ロドプシンについては、試料に含まれる脂質の量を界面活性剤の添加量を調整することで結晶化試料を最適化し、天然状態での三次元結晶を再現良く得ることができた。
    25年度が最終年度であるため、記入しない。
    25年度が最終年度であるため、記入しない。

  5. Structural analysis of photo-intermediates of squid rhodopsin

    Grant number:22770146  2010 - 2011

    MURAKAMI Midori

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    Authorship:Principal investigator 

    Grant amount:\4290000 ( Direct Cost: \3300000 、 Indirect Cost:\990000 )

    Photochemical properties of invertebrate rhodopsin are different largely from vertebrate rhodopsin. Recent crystallographic studies of squid and bovine rhodopsins suggest profound differences in the structure of these photoreceptors. Here, we perform X-ray crystallographic studies of squid bathorhodopsin and an artificial 9-cis isomer isorhodopsin. Analyses of diffraction data of frozen crystals that were illuminated at these wavelengths provided the structural information of each isomeric state. The result showed that the formation of bathorhodopsin is accompanied by a large movement of the central moiety of the retinal polyene chain towards the cytoplasmic side with small movements of the s-ionone ring and the Schiff base ; i. e., the retinal takes on a right-handed screwed all-trans configuration. Conversely, the retinal in isorhodopsin takes on a planar 9-cis configuration ; i. e., as compared to the 11-cis retinal in the unphotolysed state, a limited configurational change is observed around the C9=C10 bond.

  6. Structural and functional divergence of rhodopsin super-family

    Grant number:21370070  2009 - 2011

    KOUYAMA Tsutomu

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    Authorship:Coinvestigator(s) 

    (1) Crystallization of retinal proteins by the membrane fusion method:
    (1) The chloride ion pump from Natoronomonas pharaonis, pharaonis halorhodopsin(pHR), was crystallized, and its structure was determined at 2. 0 Aresolution.
    (2) Deltarhodopsin(dR3), a light-driven proton pomp found in the cell membrane of Haloterrigena thermotolerans, was crystallized, and its structure was determined at 2. 7 A resolution.
    (2) Structural analyses of the photoreaction intermediates of bacteriorhodopsin:
    (1) The structure of the M intermediate was determined at various pH levels. It was shown that the formation of M intermediate at neutral pH is accompanied by a complete destruction of the paired structure of two glutamates in the proton release channel, while this destruction is incomplete at a weakly acidic pH.
    (2) Using the Leu93-to-Ala mutant(BR_L93A) of bacteriorhodopsin, the structure of the O intermediate was determined at 2. 3 A resolution
    (3) Structural analyses of the light-driven chloride ion pump(pHR):
    (1) The crystal structures of the O-like blue form and the yellow anion-depleted form of pHR were determined at 1. 8 ? 2. 0 A resolutions. The result allowed us to elucidate the mechanism by which the chloride ion is removed from the active site near the protonated Schiff base of retinal.
    (2) The crystal structure of an M-like intermediate of the pHR-azide complex was determined at 2. 3 A resolution. It is shown that the cytoplasmic half of helix F undergoes a large structural change during the proton pumping cycle
    (3) A mutant of pHR(pHR_ΔBC) in which a long loop between transmembrane helices B and C was partially deleted was expressed in E. coli. It was shown that the photocycle of pHR_ΔBC is not influenced by the membrane potential, while the uptake process of chloride ion is much faster than observed in the wild-type pHR.
    (4) Structural analyses of squid rhodopsin
    (1) The primary photochemical reaction intermediate(Baso) of squid rhodopsin was determined at 2. 7 A resolution.
    (2) Light-induced structural changes in octopus rhodopsin(oRh) were investigated by small-angle X-ray scattering method.

  7. Gq型G蛋白質共役光受容体のX線構造解析

    Grant number:20054008  2008 - 2009

    村上 緑

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    Authorship:Principal investigator 

    Grant amount:\4000000 ( Direct Cost: \4000000 )

    ロドプシンが担う光情報変換機能の初期過程は、蛋白質内に含む発色団レチナールの11-cis型から全トランス型への光異性化反応である。バソ中間体において吸収した光エネルギーが活性部位レチナールに微小な歪みとして蓄えられ、ルミ中間体を経てメタ中間体で分子全体に広がる構造変化を惹起し、その結果、細胞質側表面に共役したG蛋白質に光情報が伝えられる。本研究課題では、バソ中間体およびその光異性体である9-シス型イソロドプシンの構造解析を行った。
    イカロドプシン結晶に対し液体窒素温度下でさまざまな波長の光を照射すると、11-シス型基底状態、9-シス型イソロドプシン、全トランス型バソ中間体の3状態間で平衡状態を形成した。この平衡状態において、さまざまな光照射条件下での光異性体の形成率を見積もり、バソ中間体およびイソロドプシンの測定に最適な条件を見出した。さらに、3状態間遷移の速度定数を決定することができた。X線回折実験においては、ビームラインに設置されたオンライン顕微分光装置を用いて照射したX線量に依存する結晶の吸収変化を測定し、少量のX線量によって壊れていくタンパク質を定量的に解析し、X線照射下でのバソ中間体の残存率を正確に見積もった。これらの結果に基づき、バソ中間体とイソロドプシンのモデルを精度よく構築することができた。

  8. Analyses of common structural motif of rhodopsin family

    Grant number:19370065  2007 - 2008

    TSUTOMU Kouyama

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  9. X-ray crystallographic study of squid rhodopsin

    Grant number:19770129  2007 - 2008

    MURAKAMI Midori

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    Authorship:Principal investigator 

    Grant amount:\3530000 ( Direct Cost: \3200000 、 Indirect Cost:\330000 )

  10. Four Dimensional Structural Analysis of Biological Ion Pumps

    Grant number:17370056  2005 - 2006

    KOUYAMA Tsutomu

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    Bacteriorhodopsin, a light-driven proton pump found in the cell membrane of halobacteria, is one of well characterized biological ion pumps. Based on the results of four-dimensional X-ray crystallographic analyses, the principal investigator and colleague have proposed a new hypothesis that bacteriorhodopsin actually functions as an antiporter of water and proton. To get supports for this hypothesis, we investigated time-resolved spectroscopy as well as X-ray crystallographic analysis of bacteriorhodopin exposed to pressurized noble gases. It was then found that when Xe or Kr atom binds specificically to a hydrophobic cavity in the cytoplasmic side of bacteriorhodopsin, the decay of the M intermediate is accelerated significantly. We also performed X-ray crystallographic analysis of archaearhodopsin-2, a light-driven proton pump found in Halorubrum, and determined its three-dimensional structure at 2.1 angstrom resolution. It was shown that the crystal of archaerhodopsin-2 is made up of stacked membranes, in each of which the trimeric complexes are arranged on a honeycomb lattice and that the second chromophore, bacterioruberin, mediates interactions between neighboring proteins within the trimer. We succeeded in getting a three-dimensional crystal of proteorhodopsin, a light-driven proton pump from a proteobacterioum. We have established to prepare a large amount of halorhodopsin, a light-driven chloride pump, after a repeated cycle of UV mutagenesis. We obtained a well-ordered three-dimensional crystal of squid rhodopsin and determined its structure at 2.8 angstrom resolution.

  11. X-ray Crystallographic Analyses of Retinal Proteins

    Grant number:15370066  2003 - 2004

    KOUYAMA Tsutomu

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    Bacteriorhodopsin, a transmembrane protein found in halobacterium halobium, functions as a light-driven proton pump. It is composed of 7 transmembrane helices and contains one molecule of retinal. This protein has been extensively studied by various biophysical techniques and its structure and function has been elucidated in detail. Structural analysis of the reaction intermediates is indispensable for better understanding of the proton pumping mechanism and determination of their structures at high resolution has been desired. Our group has developed a novel crystallization technique, so called the membrane fusion method, by which a three-dimensional crystal of bacteriorhodopsin containing native lipids can be prepared. By reducing the possible effect of X-radiation damages, we have succeeded in increasing the resolution of structural determination to such a level that water molecules within the protein can be visualized. In this project, we carried out a crystallographic analysis of the L intermediate of bacteriorhodopsin. It is found that formation of this intermediate accompanies the following structural changes : 1)amino-acid residues (Leu93 and Trp182) contacting the C13 methyl of retinal undergo significant structural changes (rotation and/or displacement of their side chains) ; 2)a water molecule that initially interacts with the retinal Schiff base moves to a micro-cavity that is created by rotation of the side chain of Leu93. Furthermore, we carried out X-ray crystallographic analysis of archaerhodopsin, a protein analogous to bacteriorhodopsin. As compared to bacteriorhodopsin, the proton release channel is more opened in archaerhodopsin. In addition, we crystallized squid rhodopsin and collected a low-resolution diffraction data.

  12. 生体イオンポンプの反応中間体のX線結晶構造解析

    Grant number:12480199  2000 - 2001

    神山 勉

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    光駆動プロトンポンプとして知られるバクテリオロドプシンの反応中間体の立体構造を求めることを目指して研究を行った。これまでに、膜融合を利用して膜蛋白質を結晶化することを試み、脂質分子を含んだバクテリオロドプシンの三次元結晶を作成する技術の開発を行ってきたが、本研究においては、この結晶化法を改良し、構造解析の分解能を2.3オングストロームまで向上させた。これにより、蛋白質に結合した脂質の構造および活性部位の水分子の位置を求めることができた。また、バクテリオロドプシンの反応中間体の構造解析を行うため、単結晶のX線回折と吸収スペクトルの同時測定を行うための装置も作成し、低温下での光照射により結晶中に蓄積した中間体の量を高精度で求める方法を確立した。このような開発研究を積み重ねる中で、蛋白質結晶のX線損傷が従来考えられてきたより遥かに少量のX線照射量により引き起こされることを見出した。この結果を踏まえ、X線損傷の影響を軽減できる低温X線回折測定法を開発し、反応サイクルの初期に現れるK中間体についての信頼度の高い回折データを得た。構造解析の結果、反応サイクル初期に引き起こさせる構造変化は発色段レチナールのシッフ塩基近傍に限定されることが示された。また、反応サイクルの後半に現れるM中間体についても構造解析の精度を高め、蛋白質を構成する7本のヘリックスのうち一本(Gヘリックス)が膜面に垂直方向にスライドすることを確認するとともに、活性部位における水分子の動きも明らかにした。さらに、K中間体が熱緩和して生じるL中間体についても構造解析を行い、構造変化の認められる領域が細胞外側方向に広がることを明らかにした。また、バクテリオロドプシンと類似の蛋白質であるアーキロドプシンの結晶化を行い、4オングストロームの分解能の回折データを収集した。まだ解析途上であるが、アーキロドプシン4量体が集積して結晶ができていることが示唆されている。

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Teaching Experience (On-campus) 2

  1. 物理学実験Ⅱ

    2020

  2. 物理学実験Ⅰ

    2020