蛋白質分子認識過程における水和構造のダイナミクス

• Project/Area Number: 15076210
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Science and Engineering
• Research Institution: Keio University
• Principal Investigator: 中迫 雅由 Keio University, 理工学部, 教授 (30227764)
• Co-Investigator(Kenkyū-buntansha): 岡 俊彦 慶應義塾大学, 理工学部, 助手 (60344389)
• Project Period (FY): 2003 – 2007
• Project Status: Completed (Fiscal Year 2007)
• Budget Amount: ¥77,200,000 (Direct Cost: ¥77,200,000); Fiscal Year 2007: ¥11,500,000 (Direct Cost: ¥11,500,000); Fiscal Year 2006: ¥11,500,000 (Direct Cost: ¥11,500,000); Fiscal Year 2005: ¥11,500,000 (Direct Cost: ¥11,500,000); Fiscal Year 2004: ¥26,600,000 (Direct Cost: ¥26,600,000); Fiscal Year 2003: ¥16,100,000 (Direct Cost: ¥16,100,000)
• Keywords: 生物物理 / ナノマシン / 表面・界面物性 / 放射線・X線・粒子線 / 生体分子 / 蛋白質構造 / 蛋白質水和 / 低温結晶構造解析 / 光受容蛋白質 / X線散乱・回折 / 蛋白質立体構造 / 蛋白質水和構造 / X線回折・散乱 / 結晶構造 / 時間相関 / 阻害剤

Research Abstract

1.グルタミン酸脱水素酵素のドメイン運動と水和構造変化
高分解能X線結晶構造解析により、グルタミン酸脱水素酵素のドメイン運動のスナップショットの立体構造を水和水も含めて明らかにし、液中GDH六量体の分子動力学(MD)計算を実施した。また、原子間力顕微鏡(AFM)によるGDH結晶の表面構造観察して、ドメイン運動の頻度を見積もった。
2.蛋白質のX線結晶構造解析と水和構造解析
ブラストサイジンSデアミナーゼの結晶構造をその反応機構を追跡可能な、様々な反応中間状態で明らかにした。SPring-8のマイクロビームを用いて青色光受容蛋白質Phototoropin1,2 L0V1 ドメインの結晶構造解析を行った。いずれのドメインも結晶学的非対称単位中で二量体を形成しており、その安定化に水分子が大きく寄与していた。アリールマロン酸脱炭酸酵素(AMDase)の立体構造を実験室での重原子多重同型置換解析によって結晶構造を決定した。結晶学的非対称単位中に4分子が含まれ、それぞれが微妙にドメイン運動によって構造変化するとともに、活性ポケットの入り口にあるループがヘリックス-コイル変化をし、水和水や基質の出入りを妨げる可能性が示唆された。
3.データベースを用いた蛋白質水和の研究
水和構造予測プログラムの開発を行った。PDBに登録されている高分解能X線結晶構造解析による立体構造モデル座標から、親水性アミノ酸残基、極性原子周囲の水和構造を抽出し、各原子団周囲での経験的な分布確率密度を得た。これを利用して、任意の水溶性蛋白質の水和構造を予測するプログラムコードを開発した。また、抗体FVフラグメント高次構造決定因子を統計的に解析するためのプログラムコードを開発し、決定因子が重鎖の相補性決定領域3内の特定の領域にあることを見出すことができた。
4.サブゼロ温度領域X線回折実験用結晶冷却装置の開発前年度からの開発を継続して行い、より冷却効率の高い熱交換器を搭載した。シタロン脱水酵素の置換体結晶を用いて、データ収集のための手技を検討中した。

Research Products

• [Journal Article] タンパク質の水和構造を探る (2008)
  • Author(s): 中迫雅由
  • Journal Title: Molecular Science Journal 2 Pages: 22-22
  • NAID: 130002126190
  • Peer Reviewed
• [Journal Article] 非弾性散乱の生物への応用について (2008)
  • Author(s): 城地保昌、中迫雅由、難波啓一
  • Journal Title: 日本結晶学会誌 50 Pages: 78-82
  • NAID: 10021085479
  • Peer Reviewed
• [Journal Article] Crystallization and preliminary X-ray diffraction experiments of arylmalonate decarboxylase from Alcaligenes bronchisepticus (2008)
  • Author(s): Masayoshi Nakasako, Rika Obata, Ryosuke Okubo, Shyuichi Nakayama, Kenji Miyamoto and Hiromichi Ohta
  • Journal Title: Acta Crystallographica Section F
  • Peer Reviewed
• [Journal Article] Structural basis of LOVI-dimerization of phototropin 1and 2 from Arabidopsis (2008)
  • Author(s): Masayoshi Nakasako, Kazunori zikihara, Daisuke Matsuoka, Hitomi Katsura and Satoru Tokutomi
  • Journal Title: Journal of Molecular Biology
  • Peer Reviewed
• [Journal Article] Crystal Structures of Blasticidin S Deaminase (BSD):Implications for Dynamic Properties of Catalytic Zinc (2007)
  • Author(s): Takashi Kumasaka, Masaki Yamamoto, Makio Furuichi, Masayoshi Nakasako, Aik-Hong The, Makoto Kimura, Isamu Yamaguchi, Tatzuo Ueki
  • Journal Title: Journal of Biological Chemistry 228 Pages: 37103-37111
  • Peer Reviewed
• [Journal Article] Discovery of MBI-D resistant buster compounds - inquiry of effective compound among derivatives of MBI-D products. (2006)
  • Author(s): Y.Kurahashi, S.Kagabu, N.Yamada, Y.Mitsugi, M.Shimizu, M.Nakasako, I.Yamaguchi
  • Journal Title: J. Pesticide. Sci 31 Pages: 85-94
• [Journal Article] A fluorescent antibiotic resistance marker for rapid production of transgenic rice plants. (2006)
  • Author(s): T.Ochiai-Fukuda, N.Takahashi-Ando, S.Ohsato, T.Igawa, K.Kadokura, H.Hamamoto, M.Nasako, T.Kudo, T.Shibata, I.Yamaguchi, M.Kimura
  • Journal Title: J. Biotech 122 Pages: 521-527
• [Journal Article] High-resolution powder diffraction study of purple membrane with a large Guinier-type camera. (2006)
  • Author(s): T.Oka, K.Miura, K.Inoue, T.Ueki, N.Yagi
  • Journal Title: J. Synch. Rad. 13 Pages: 281-284
• [Journal Article] タンパク質の水和水と機能 (2006)
  • Author(s): 肥後順一, 中迫雅由
  • Journal Title: 生物物理 46 Pages: 220-223
  • NAID: 110004751712
• [Journal Article] A fluorescent antibiotic resistance marker for rapid production of transgenic rice plants. (2006)
  • Author(s): Ochiai-Fukuda, T. et al.
  • Journal Title: J.Biotech. in press(印刷中)
• [Journal Article] Hydrogen-bond patterns in the hydration structure of a protein. (2005)
  • Author(s): Yokomizo, T.Nakasako, M., Yamazaki, T., Shindo, H.Higo, J.
  • Journal Title: Chem.Phys.Lett. 401 Pages: 332-336
• [Journal Article] Light-induced global structural changes of phytochrome A regulating photomorphogenesis in plants. (2005)
  • Author(s): Nakasako, M., Iwata, T., Inoue K., Tokutomi S.
  • Journal Title: FEBS J. 272 Pages: 603-612
• [Journal Article] Structural transition of bacteriorhodopsin is preceded by deprotonation of Schiff base : Microsecond time-resolved X-ray diffraction study of purple membrane. (2005)
  • Author(s): Oka, T., Inoue, K., Kataoka, M., Yagi, N.
  • Journal Title: Biophys.J. 88 Pages: 436-442
• [Journal Article] Quaternary structure of LOV-domain containing polypeptide of Arabidopsis FKF1 protein. (2005)
  • Author(s): Nakasako M., Matsuoka, D., Zikihara.K., Tokutomi, S.
  • Journal Title: FEBS Lett. 579 Pages: 1067-1071
• [Journal Article] Patterns and networks of hydrogen-bonds in the hydration structure of human lysozyme. (2005)
  • Author(s): Yokomizo, T., Higo, J., Nakasako, M.
  • Journal Title: Chem.Phys.Lett. 410 Pages: 31-35
• [Journal Article] Conformational dynamics of complementarity determining region H3 of an anti-dansyl Fv fragment in the presence of its hapten. (2005)
  • Author(s): Nakasako, M., Oka, T., Masumo, M., Takahashi, H., Shimada, I.et al.
  • Journal Title: J.Mol.Biol. 351 Pages: 627-640
• [Journal Article] Structural analysis of cell membrane complex of a hair fibre by micro-beam X-ray diffraction. (2005)
  • Author(s): Ohta, N., Oka, T.Inoue, K., Yagi, N., Kato, S., Hatta, I.
  • Journal Title: J.Appl.Cryst. 38 Pages: 274-279
• [Journal Article] Discovery of MBI-D resistant buster compounds-inquiry of effective compound among derivatives of MBI-D products. (2005)
  • Author(s): Kurahashi, Y., Kagabu, S., Yamada, N., Mitsugi, Y., Shimizu, M., Nakasako, M., Yamaguchi, I.
  • Journal Title: J.Pesticide.Sci. 88 Pages: 436-442
• [Journal Article] Tertiary and quaternary structures of phytochrome A. (2005)
  • Author(s): Nakasako M., Tokutomi, S.
  • Journal Title: Light Sensing in Plants(Eds. Wada, M., Shimazaki, K., Iino, M.) Springer Verlag 第一版 Pages: 43-49
• [Journal Article] Water-protein interactions studied by cryogenic X-ray crystallography. (2005)
  • Author(s): Nakasako, M.
  • Journal Title: Hydrogen and Hydration in Proteins and Nucleic Acids(Eds.Niimura, N., Mizuno, H., Helliwell, J., Westhof, E.)(KubaPro) 第一版 Pages: 135-139
• [Journal Article] LOV photoreceptors in Plants ; Molecular structure, photoreaction and signal transduction. (2005)
  • Author(s): Matsuoka, D., Nakasako, M., Zikihara, K., Tokutomi, S.
  • Journal Title: Proceedings of the 15th International Symposium on Flavins and Flavoproteins in press
• [Journal Article] 結晶を冷却する (2005)
  • Author(s): 中迫雅由
  • Journal Title: タンパク質の結晶化(京都大学出版会) 第一版 Pages: 267-281
• [Journal Article] タンパク質の水和構造 (2005)
  • Author(s): 中迫雅由, 肥後順一
  • Journal Title: 現代化学 No.413 Pages: 48-54
• [Journal Article] X線結晶解析 (2005)
  • Author(s): 中迫雅由
  • Journal Title: タンパク質科学 -構造・物性・機能(後藤, 桑島, 谷澤編)(化学同人) 第一版 Pages: 33-43
• [Journal Article] Hydrogen-bond patterns in the hydration structure of a protein. (2005)
  • Author(s): Yokomizo, T., Nakasako, M., Yamazaki, T., Shindo, H., Higo, J.
  • Journal Title: Chemical Physics Letters 401 Pages: 332-336
• [Journal Article] Structural transition of bacteriorhodopsin is preceded by deprotonation of Schiff base : Microsecond time-resolved X-ray diffraction study of purple membrane. (2005)
  • Author(s): Oka, T., Inoue, K., Kataoka, M., Yagi, N.
  • Journal Title: Biophysical Journal 88 Pages: 436-442
• [Journal Article] Light-induced global structural changes of phytochrome A regulating photomorphogenesis in plants. (2005)
  • Author(s): Nakasako, M., Iwata, T., Inoue, K., Tokutomi, S.
  • Journal Title: The FEBS Journal 272 Pages: 603-612
• [Journal Article] Quaternary structure of LOV-domain containing polypeptides of Arabidopsis FKF1 protein. (2005)
  • Author(s): Nakasako, M., Matsuoka, D., Zikihara, K., Tokutomi, S.
  • Journal Title: FEBS Letters 579 Pages: 1067-1071
• [Journal Article] Enzymatic characterization of scytalone dehydratase Val75Met variant found in melanin biosynthesis inhibitor (MBI) resistant strains of the rice blast fungus. (2004)
  • Author(s): Yamada, N., Motoyama, T., Nakasako, M., Kagabu, S., Kudo, S., Yamaguchi, I.
  • Journal Title: Bioscience Biotechnology and Biochemistry 68 Pages: 615-621
• [Journal Article] Water-protein interactions from high-resolution protein crystallography. (2004)
  • Author(s): Nakasako, M.
  • Journal Title: Philosophical Transaction B Royal Society London 359 Pages: 1191-1206
• [Journal Article] Light-induced structural changes of LOV-domain containing polypeptides from Arabidopsis phototropin 1 and 2 studied by small-angle X-ray scattering. (2004)
  • Author(s): Nakasako, M., Iwata, T., Matsuoka, D., Tokutomi, S.
  • Journal Title: Biochemistry 43 Pages: 14881-14890
• [Journal Article] X-ray imaging microscopy using Fresnel zone plate objective and quasi-monochromatic undulator radiation. (2004)
  • Author(s): Suzuki, Y., Takeuchi, A., Takano, H., Uesugi, K., Oka, T., Inoue, K.
  • Journal Title: Review of Scientific Instruments 75 Pages: 1155-1157
• [Journal Article] Picometer-scale dynamical observations of individual membrane proteins : the case of bacteriorhodopsin. (2004)
  • Author(s): Okumura, Y., Oka, T., Kataoka, M., Taniguchi, Y., Sasaki, Y.C.
  • Journal Title: Physical Review E 70
• [Journal Article] Microsecond Time-resolved Diffraction and Scattering Measurements System Using Semi-monochromatic X-ray Pulse at SPring-8 BL40XU. (2004)
  • Author(s): Oka, T., Inoue, K., Yagi, N.
  • Journal Title: Proceedings of the 8-th International Conference on Synchrotron Radiation Instrumentation Pages: 1197-1200
• [Journal Article] 30年後の放射光生命科学実験に期待すること-生きた細胞を原子分解能でリアルタイムに観察する (2004)
  • Author(s): 中迫雅由
  • Journal Title: 日本放射光学会誌"放射光" 17 Pages: 268-268
  • NAID: 10013364215
• [Presentation] Surface structure of glutamate dehydrogenase crystal observed by atomic forcemicroscopy (2008)
  • Author(s): Sato Tsunero and Nakasako Masayoshi
  • Organizer: The 5-th open workshop "Chemistry of Biological Processes Created by Water and Biomolecules"
  • Place of Presentation: 奈良
  • Year and Date: 2008-01-24
• [Presentation] Search for determinants regulating the quaternary structures of Fvfiragments of antibodies through a database analysis (2008)
  • Author(s): Masashi Miyawaza and Masayoshi Nakasako
  • Organizer: The 5-th open workshop "Chemistry of BiologicalProcesses Created by Water and Biomolecules"
  • Place of Presentation: 奈良
  • Year and Date: 2008-01-24
• [Presentation] Crystal structure of arylmalonate decarboxylase (2008)
  • Author(s): Masayoshi Nakasako, Rika Obata, Kenji Miyamoto and Hiromichi Ohta
  • Organizer: The 5-th open workshop "Chemistry of Biological Processes Created by Water and Biomolecules"
  • Place of Presentation: 奈良
  • Year and Date: 2008-01-24
• [Presentation] Crystal structures of LOV1 domains of phototropin 1 and 2from Arabidopsis thaliana (2008)
  • Author(s): Masayoshi Nakasako, Daisuke, Matsuoka, Kazunori Zikiharaand Satoru Tokutomi
  • Organizer: The 5th open workshop "Chemistry of Biological Processes Created by Water and Biomolecules"
  • Place of Presentation: 奈良
  • Year and Date: 2008-01-24
• [Presentation] Simulation on the X-ray speckle patterns of a hydrated protein moleculewith conformational variations (2007)
  • Author(s): Sakamoto Keita, Hceguchi Mitsunori, Nakasako Masayoshi
  • Organizer: 日本生物物理学会第45回年会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 2007-12-23
• [Presentation] Search for determinants regulating the quaternary structures of Fv fragmentsof antibodies through a database analysis (2007)
  • Author(s): Miyazawa Masashi and Nakasako Masayoshi
  • Organizer: 日本生物物理学会第45回年会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 2007-12-23
• [Presentation] Prediction of Hydration Structures around polar protein atoms througha data-base analysis (2007)
  • Author(s): Matsuoka Daisuke and Nakasako Masavoshi
  • Organizer: 日本生物物理学会第45回年会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 2007-12-23
• [Presentation] Crystal structures of LOV1 domains in Arabidopsis phototropin 1 and 2 (2007)
  • Author(s): Masayoshi Nakasako, Daisuke, Matsuoka, Kazimori Zikihara, Hitomi Katsura and Satoru Tokutomi
  • Organizer: 日本生物物理学会第45回年会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 2007-12-23
• [Presentation] Observation of the surface of glutamate dehydrogenase crystals by atomicforce microscopy (2007)
  • Author(s): Sato Tsunero and Nakasako Masayoshi
  • Organizer: 日本生物物理学会第45回年会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 2007-12-23
• [Presentation] X線溶液散乱で何が分かるのか?なぜ放射光なのか? (2007)
  • Author(s): 中迫 雅由
  • Organizer: 第2回X線溶液散乱研究会-SPring-8における放射光X線溶液散乱-
  • Place of Presentation: 理化学研究所 播磨研究所
  • Year and Date: 2007-10-11
• [Presentation] 蛋白質表面に吸着した水分子と蛋白質運動の相関について (2007)
  • Author(s): 中迫 雅由
  • Organizer: 日本物理学会第62回年次大会
  • Place of Presentation: 北海道大学
  • Year and Date: 2007-09-23
• [Book] タンパク質の結晶化(坂部ら編)第9章 結晶を冷却する (2005)
  • Author(s): 中迫雅由
  • Publisher: 京都大学出版会(印刷中)
• [Book] "Light Sensing in Plants" Tertiary and Quaternary Structures of Phytochrome A. (2005)
  • Author(s): Nakasako, M., Tokutomi, S.
  • Publisher: Springer Verlag(印刷中)
• [Publications] Yamada, N., Motoyama, T., Nakasako, M., Kagabu, S., Kudo, T., Yamaguchi, I: "Enzymatic characterization of scytalone dehydratase Val75Met variant found in melanin biosynthesis inhibitor (MBI-D) resistant strains of the rice blast fungus."Biosci.Biotech.Biochem. (in press). (2004)
• [Publications] Nakasako, M: "Water-protein interactions from high-resolution protein crystallography."Phil.Mag.Trans.B. (in press). (2004)
• [Publications] Oka, T., Inoue, K., Yagi, N.: "Microsecond Time-resolved Diffraction and Scattering Measurements System Using Semi-monochromatic X-ray Pulse at SPring-8 BL40XU."Proceedings of the Eighth International Conference on Synchrotron Radiation Instrumentation. (in press). (2004)
• [Publications] Suzuki, Y., Takeuchi, A., Takano, H., Uesugi, K., Oka, T., Inoue, K: "X-ray imaging microscopy using Fresnel zone plate objective and quasi-monochromatic undulator radiation."Rev.Sci.Instrum.. (in press). (2004)