2001 Fiscal Year Annual Research Report

生体イオンポンプの反応中間体のX線結晶構造解析

Research Project

Project/Area Number	12480199
Research Institution	Nagoya University
Principal Investigator	神山勉名古屋大学, 大学院・理学研究科, 教授 (30170210)
Co-Investigator(Kenkyū-buntansha)	倭剛久名古屋大学, 大学院・理学研究科, 助教授 (90251587) 垣谷俊昭名古屋大学, 大学院・理学研究科, 教授 (90027350) 村上緑名古屋大学, 大学院・工学研究科, 助手 (20324387) 足立伸一理化学研究所, 播磨研究所, 先任研究員 (60260220) 神谷信夫理化学研究所, 播磨研究所, 室長 (60152865) 井原邦夫名古屋大学, 大学院・理学研究科, 助手 (90223297)
Keywords	X線結晶構造解析 / 膜蛋白質 / バクテリオロドプシン / アーキロドプシン / 光駆動プロトン輸送 / 蛋白質の結晶化 / 生体膜 / 反応中間体
Research Abstract	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。

Research Products
(42 results)

All Other

All Publications (42 results)

[Publications] K.Sakai, et al.: "Monitoring of Freeze-Trapped Reaction Intermediates in Protein Crystals : A Microspectrophotometer for Cryogenic Protein Crystallography"J.Appl.Cryst.. (In press). (2002)
[Publications] H.Sumi, et al.: "Unified theory on rates for electron transfer mediated by midway molecules, bridging between superexchange and sequential processes"J.Phys.Chem. B105. 9603-9622 (2001)
[Publications] T.Kakitani, et al.: "Empirical formula of exciton coherent domain in oligomers and application to LH2","J.Phys.Chem.. (In press). (2002)
[Publications] T.Kakitani, et al.: "Unique mechanisms of excitation energy transfer, electron transfer and photoisomerization in biological systems"J.Biol.Phys.. (In press). (2002)
[Publications] A.Yamada, et al.: "Ab initio MO study on potential energy surfaces for twisting around C7=C8 and C4-C7 bonds of coumaric acid"THEOCHEM. 536. 195-201 (2001)
[Publications] A.Kimura, et al.: "Theory of excitation transfer in the intermediate coupling case"J.Luminescence. 87-89. 815-817 (2000)
[Publications] T.Kawatsu, et al.: "A novel method for determining electron tunneling pathway in protein"Chimica Acta Inorganica. 300-302. 862-868 (2000)
[Publications] T.Kawatsu, et al.: "Worm Model for Electron Tunneling in Proteins : Consolidation of the Pathway Model and the Dutton Plot"J.Phys.Chem.B,. 105. 4424-4435 (2001)
[Publications] S.Yamamoto, et al.: "Ab initio MO study on the potential energy surfaces for twisting around C15=N bond of protonated Schiffbase of retinal"THEOCHEM. 543. 79-87 (2001)
[Publications] T.Kimura, et al.: "Theory of excitation energy transfer in the intermediate coupling case. II.Criterion for intermediate coupling excitation energy transfer mechanism and application to the photosynthetic antenna system"J.Phys.Chem.B. 104. 9276-9287 (2000)
[Publications] A.Yamada, et al.: "Ab initio MO study on potential energy surfaces for twisting around C7=C8 and C4-C7 bonds of coumaric acid"THEOCHEM. 536. 195-201 (2001)
[Publications] A.Kimura, et al.: "Theory of excitation transfer in the intermediate coupling case"J.Luminescence. 87-89. 815-817 (2000)
[Publications] T.Kawatsu, et al.: "A novel method for determining electron tunneling pathway in protein"Chimica Acta Inorganica. 300-302. 862-868 (2000)
[Publications] M.Tanaka, et al.: "Cytochrom aa(3) and its gene duplication in Haloferax volcanii..:"J Bacteriol. 184. 840-845 (2002)
[Publications] A.Oren, et al.: "Halomicrobium mukohataei gen. nov., comb. nov., and emended description of Halomicrobium mukoataei"Int J System Evol Microbiol.. (in press). (2002)
[Publications] R.Yatsunami, et al.: "Gene clusters encoding ATP synthase of Haloarcula japonica strain TR-1. Series No."Nuceic Acids.Symposium. 44. 51-52 (2001)
[Publications] T.Hishiki, et al.: "X-Ray Crystal Structure and Catalytic Properties of Thr252lle Mutant of Cytochrome P450cam : Roles of Thr252 and Water in the Active Center"J.Biochem.. 128. 965-974 (2000)
[Publications] S.-Y.Park, et al.: "Crystallization and Preliminary X-ray Diffraction Analysis of a Cytochrome P450 (CYP119) from Sulfolobus solfataricus"Acta Cryst. D56. 1173-1175 (2000)
[Publications] C.Sasaki, et al.: "Tracking of Individual Nanocrystals using Diffracted X Rays"Phys.Rev.. E62. 3843-3847 (2000)
[Publications] D.Sun, et al.: "Crystallization and Preliminary X-ray Diffraction Analysis of a Rat Biliverdin Reductase"Acta Crys. D56. 1180-1182 (2000)
[Publications] H.Miyatake, et al.: "Sensory Mechanism of Oxygen Sensor FixL from Rhizobium meliloti : Crystallographic, Mutagenesis and Resonance Raman Spectroscopic Studies"J.Mol.Biol.. 301. 415-431 (2000)
[Publications] H.Shimizu, et al.: "Proton Derivery in NO Reduction by Fungal Nitric Oxide Reductase : Cryogenic-crystallography, Spectroscopy and Kinetics of Ferric-NO Complexes of Wild-Type and Mutant Enzymes"J.Biol.Chem.. 275. 4816-4826 (2000)
[Publications] H.Shimizu, et al.: "X-ray Structure of Nitric Oxide Reductase(Cytochrome P450nor)at Atomic Resolution"ActaCryst.. D58. 81-89 (2002)
[Publications] S.Adachi, et al.: "The RIKEN Structural Biology Beamline II(BL44B2)at the Spring-8"Nucl.Instr.and Meth.. A467-468. 711-714 (2001)
[Publications] C.Sasaki, et al.: "Picometer-Scale Dynamical X-ray Imaging of Single DNA Molecules"Phys.Rev.Lett.. 87. 248102 (2001)
[Publications] Y.Naoi, et al.: "The Functional Similarity and Structural Diversity of Human and Cartilaginous Fish Hemoglobins"J.Mol.Biol.. 307. 259-270 (2001)
[Publications] T.H.Tahirov, et al.: "Crystallization and Preliminary X-ray Diffraction Analysis of the C/EBPb C-terminal Region in Complex with DNA"Acta Cryst.. D57. 854-856 (2001)
[Publications] M.Nakasako, et al.: "Large-Scale Domain Movements and Hydration Structure Changes in the Active-Site Cleft of Unligated Glutamate Dehydrogenase from Thermococcus profundus Studied by Cryogenic X-ray Crystal Structure Analysis and Small-Angle X-ray Scattering"Biochemistry. 40. 3069-3079 (2001)
[Publications] Y.Matsuura, et al.: "Structural Basis for the Higher Ca2+-Activation of the Regulated Actin-activated Myosin ATPase Observed with Dictyosterilum/Tetrahymena Actin Chimeras"J.Mol.Biol.. 296. 579-595 (2000)
[Publications] J.-R.Shen, et al.: "Crystallization and the Crystal Properties of the Oxygen-Evolving Photosystem II from Synechococcus vulcanus"Biochemistry. 39. 14739-14744 (2000)
[Publications] M.Kawamoto, et al.: "The Bio-Crystallography beamline (BL41XU) at Spring-8"Nucl.Instr.and Meth.. A467-468. 1375-1379 (2001)
[Publications] T.H Tahirov, et al.: "Crystallization and preliminary X-ray analysis of C/EBPb C-terminal region in complex with DNA"Acta Cryst.. D57. 854-856 (2001)
[Publications] T.H., Tahirov, et al.: "Crystallization and preliminary X-ray analysis of quaternary, ternary and binary protein-DNA compleces with involvement of AML1/Runx-1/CBFa Runt domain, CBFb and the C/EBPb bZip region□"Acta Cryst.. D57. 850-843 (2001)
[Publications] Y.Toriumi, et al.: "Crystallization and Preliminary X-ray Diffraction Analysis of Light-harvesting Protein Phycocyanin from the Thermophilic Cyanobacterium Synechococcus elongatus"Acta Cryst□. D57. 1326-1328 (2001)
[Publications] 竹田一旗, 神山勉: "膜融合法を利用したバクテリオロドプシンの結晶化法"日本結晶学会誌. 42. 171-175 (2000)
[Publications] 河本正秀, 他: "生体高分子結晶構造解析ビームライン (BL41XU) の到達点とアンジュレータX線の拓く構造生物学"日本結晶学会誌. 42. 87-96 (2000)
[Publications] 足立伸一, 他: "理研ビームライインII(BL44B2)の現状"日本結晶学会誌. 42. 106-113 (2000)
[Publications] 神谷信夫, 他: "構造生物学のための大型実験施設, つくばと播磨の放射光施設"蛋白質核酸酵素. 45. 1279-1288 (2000)
[Publications] 倉光成紀, 他: "構造ゲノム科学"日本結晶学会誌. 43. 45-54 (2001)
[Publications] 竹田一旗, 神山勉: "光をプロトンの流れに - バクテリオロドプシンにおけるエネルギー変換"光が拓く生理科学 - 生命を支える光 (共立出版). 42. 88-101 (2000)
[Publications] 安岡則武, 神谷信夫: "生体超分子システムに迫る"光が拓く生命科学 : 夢の光り-放射光が拓く生命の神秘 (共立出版). 8. 18-31 (2000)
[Publications] 神谷信夫: "理化学研究所におけるタンパク質構造・機構解析の現状と今後の動向"バイオサイエンスとインダストリー. 59. 786-787 (2001)

2001 Fiscal Year Annual Research Report

生体イオンポンプの反応中間体のX線結晶構造解析

Principal Investigator

神山 勉 名古屋大学, 大学院・理学研究科, 教授 (30170210)

Research Products

[Publications] K.Sakai, et al.: "Monitoring of Freeze-Trapped Reaction Intermediates in Protein Crystals : A Microspectrophotometer for Cryogenic Protein Crystallography"J.Appl.Cryst.. (In press). (2002)

[Publications] H.Sumi, et al.: "Unified theory on rates for electron transfer mediated by midway molecules, bridging between superexchange and sequential processes"J.Phys.Chem. B105. 9603-9622 (2001)

[Publications] T.Kakitani, et al.: "Empirical formula of exciton coherent domain in oligomers and application to LH2","J.Phys.Chem.. (In press). (2002)

[Publications] T.Kakitani, et al.: "Unique mechanisms of excitation energy transfer, electron transfer and photoisomerization in biological systems"J.Biol.Phys.. (In press). (2002)

[Publications] A.Yamada, et al.: "Ab initio MO study on potential energy surfaces for twisting around C7=C8 and C4-C7 bonds of coumaric acid"THEOCHEM. 536. 195-201 (2001)

[Publications] A.Kimura, et al.: "Theory of excitation transfer in the intermediate coupling case"J.Luminescence. 87-89. 815-817 (2000)

[Publications] T.Kawatsu, et al.: "A novel method for determining electron tunneling pathway in protein"Chimica Acta Inorganica. 300-302. 862-868 (2000)

[Publications] T.Kawatsu, et al.: "Worm Model for Electron Tunneling in Proteins : Consolidation of the Pathway Model and the Dutton Plot"J.Phys.Chem.B,. 105. 4424-4435 (2001)

[Publications] S.Yamamoto, et al.: "Ab initio MO study on the potential energy surfaces for twisting around C15=N bond of protonated Schiffbase of retinal"THEOCHEM. 543. 79-87 (2001)

[Publications] T.Kimura, et al.: "Theory of excitation energy transfer in the intermediate coupling case. II.Criterion for intermediate coupling excitation energy transfer mechanism and application to the photosynthetic antenna system"J.Phys.Chem.B. 104. 9276-9287 (2000)

[Publications] A.Yamada, et al.: "Ab initio MO study on potential energy surfaces for twisting around C7=C8 and C4-C7 bonds of coumaric acid"THEOCHEM. 536. 195-201 (2001)

[Publications] A.Kimura, et al.: "Theory of excitation transfer in the intermediate coupling case"J.Luminescence. 87-89. 815-817 (2000)

[Publications] T.Kawatsu, et al.: "A novel method for determining electron tunneling pathway in protein"Chimica Acta Inorganica. 300-302. 862-868 (2000)

[Publications] M.Tanaka, et al.: "Cytochrom aa(3) and its gene duplication in Haloferax volcanii..:"J Bacteriol. 184. 840-845 (2002)

[Publications] A.Oren, et al.: "Halomicrobium mukohataei gen. nov., comb. nov., and emended description of Halomicrobium mukoataei"Int J System Evol Microbiol.. (in press). (2002)

[Publications] R.Yatsunami, et al.: "Gene clusters encoding ATP synthase of Haloarcula japonica strain TR-1. Series No."Nuceic Acids.Symposium. 44. 51-52 (2001)

[Publications] T.Hishiki, et al.: "X-Ray Crystal Structure and Catalytic Properties of Thr252lle Mutant of Cytochrome P450cam : Roles of Thr252 and Water in the Active Center"J.Biochem.. 128. 965-974 (2000)

[Publications] S.-Y.Park, et al.: "Crystallization and Preliminary X-ray Diffraction Analysis of a Cytochrome P450 (CYP119) from Sulfolobus solfataricus"Acta Cryst. D56. 1173-1175 (2000)

[Publications] C.Sasaki, et al.: "Tracking of Individual Nanocrystals using Diffracted X Rays"Phys.Rev.. E62. 3843-3847 (2000)

[Publications] D.Sun, et al.: "Crystallization and Preliminary X-ray Diffraction Analysis of a Rat Biliverdin Reductase"Acta Crys. D56. 1180-1182 (2000)

[Publications] H.Miyatake, et al.: "Sensory Mechanism of Oxygen Sensor FixL from Rhizobium meliloti : Crystallographic, Mutagenesis and Resonance Raman Spectroscopic Studies"J.Mol.Biol.. 301. 415-431 (2000)

[Publications] H.Shimizu, et al.: "Proton Derivery in NO Reduction by Fungal Nitric Oxide Reductase : Cryogenic-crystallography, Spectroscopy and Kinetics of Ferric-NO Complexes of Wild-Type and Mutant Enzymes"J.Biol.Chem.. 275. 4816-4826 (2000)

[Publications] H.Shimizu, et al.: "X-ray Structure of Nitric Oxide Reductase(Cytochrome P450nor)at Atomic Resolution"ActaCryst.. D58. 81-89 (2002)

[Publications] S.Adachi, et al.: "The RIKEN Structural Biology Beamline II(BL44B2)at the Spring-8"Nucl.Instr.and Meth.. A467-468. 711-714 (2001)

[Publications] C.Sasaki, et al.: "Picometer-Scale Dynamical X-ray Imaging of Single DNA Molecules"Phys.Rev.Lett.. 87. 248102 (2001)

[Publications] Y.Naoi, et al.: "The Functional Similarity and Structural Diversity of Human and Cartilaginous Fish Hemoglobins"J.Mol.Biol.. 307. 259-270 (2001)

[Publications] T.H.Tahirov, et al.: "Crystallization and Preliminary X-ray Diffraction Analysis of the C/EBPb C-terminal Region in Complex with DNA"Acta Cryst.. D57. 854-856 (2001)

[Publications] Y.Matsuura, et al.: "Structural Basis for the Higher Ca2+-Activation of the Regulated Actin-activated Myosin ATPase Observed with Dictyosterilum/Tetrahymena Actin Chimeras"J.Mol.Biol.. 296. 579-595 (2000)

[Publications] J.-R.Shen, et al.: "Crystallization and the Crystal Properties of the Oxygen-Evolving Photosystem II from Synechococcus vulcanus"Biochemistry. 39. 14739-14744 (2000)

[Publications] M.Kawamoto, et al.: "The Bio-Crystallography beamline (BL41XU) at Spring-8"Nucl.Instr.and Meth.. A467-468. 1375-1379 (2001)

[Publications] T.H Tahirov, et al.: "Crystallization and preliminary X-ray analysis of C/EBPb C-terminal region in complex with DNA"Acta Cryst.. D57. 854-856 (2001)

[Publications] T.H., Tahirov, et al.: "Crystallization and preliminary X-ray analysis of quaternary, ternary and binary protein-DNA compleces with involvement of AML1/Runx-1/CBFa Runt domain, CBFb and the C/EBPb bZip region□"Acta Cryst.. D57. 850-843 (2001)

[Publications] Y.Toriumi, et al.: "Crystallization and Preliminary X-ray Diffraction Analysis of Light-harvesting Protein Phycocyanin from the Thermophilic Cyanobacterium Synechococcus elongatus"Acta Cryst□. D57. 1326-1328 (2001)

[Publications] 竹田一旗, 神山 勉: "膜融合法を利用したバクテリオロドプシンの結晶化法"日本結晶学会誌. 42. 171-175 (2000)

[Publications] 河本正秀, 他: "生体高分子結晶構造解析ビームライン (BL41XU) の到達点とアンジュレータX線の拓く構造生物学"日本結晶学会誌. 42. 87-96 (2000)

[Publications] 足立伸一, 他: "理研ビームライインII(BL44B2)の現状"日本結晶学会誌. 42. 106-113 (2000)

[Publications] 神谷信夫, 他: "構造生物学のための大型実験施設, つくばと播磨の放射光施設"蛋白質核酸酵素. 45. 1279-1288 (2000)

[Publications] 倉光成紀, 他: "構造ゲノム科学"日本結晶学会誌. 43. 45-54 (2001)

[Publications] 竹田一旗, 神山 勉: "光をプロトンの流れに - バクテリオロドプシンにおけるエネルギー変換"光が拓く生理科学 - 生命を支える光 (共立出版). 42. 88-101 (2000)

[Publications] 安岡則武, 神谷信夫: "生体超分子システムに迫る"光が拓く生命科学 : 夢の光り-放射光が拓く生命の神秘 (共立出版). 8. 18-31 (2000)

[Publications] 神谷信夫: "理化学研究所におけるタンパク質構造・機構解析の現状と今後の動向"バイオサイエンスとインダストリー. 59. 786-787 (2001)

神山勉名古屋大学, 大学院・理学研究科, 教授 (30170210)

[Publications] 竹田一旗, 神山勉: "膜融合法を利用したバクテリオロドプシンの結晶化法"日本結晶学会誌. 42. 171-175 (2000)

[Publications] 竹田一旗, 神山勉: "光をプロトンの流れに - バクテリオロドプシンにおけるエネルギー変換"光が拓く生理科学 - 生命を支える光 (共立出版). 42. 88-101 (2000)