1997 Fiscal Year Annual Research Report

生物分子モーターの1分子計測と1分子操作

Research Project

Project/Area Number	09279104
Research Category	Grant-in-Aid for Scientific Research on Priority Areas
Research Institution	Keio University
Principal Investigator	木下一彦慶應義塾大学, 理工学部, 教授 (30124366)
Co-Investigator(Kenkyū-buntansha)	嶋本伸雄国立遺伝学研究所, 構造遺伝学研究センター, 教授 (20127658) 上田太郎工業技術院, 産業技術融合領域研究所, 主任研究官上村慎治東京大学, 大学院・総合文化研究科, 助教授 (90177585) 大岩和弘郵政省通信総合研究所, 関西支所, 主任研究官柳田敏雄大阪大学, 基礎工学部・医学部, 教授 (30089883)
Keywords	ミオシン / アクチン / ATP合成酵素 / ダイニン / RNAポリメラーゼ / 光学顕微鏡 / 蛍光 / 光ピンセット
Research Abstract	蛍光偏光イメージングにより蛍光色素1分子の向きを水溶液中で連続的に観察する手法を開発した。応用として、ミオシン上を滑走するアクチン線維の線維軸周りの回転の定量的測定に成功し、ミオシンはアクチンと間歇的にしか相互作用しないことを示した。一方蛋白質分子よりはるかに大きな目印を用いて構造変化を捉える手段として、アクチン線維を目印に用いることを提案した。応用として、ATP合成酵素の1部であるF_1-ATPaseがたった1分子でそのまま回転モーターであることを示した。直径わずか10nmの分子中の回転を、回転子部分にアクチン線維を結合させることによりより可視化した。(木下) 全反射顕微鏡法と光ピンセット法を組み合わせ、モーター1分子の化学-力学反応を1分子計測する技術を確立した。1分子のミオシンにおいてATPの加水分解反応と滑り運動の間に数十〜数百ミリ秒の遅れが生じることを発見したミオシンがエネルギーを分子内部に蓄積し、仕事が必要なタイミングを計っているという可能性を示唆する。(柳田) 蛍光性ATPアナログを合成し、ミオシン結合に伴う蛍光変化が異性体により異なることを示した。また、ダイニン1分子が微少管の滑り運動を起こすことを示した。(大岩) 精密位置計測装置とUVフラッシュを組み合わせ、CagedATP投与でトリガーされたウニ精子鞭毛運動を解析した。ATP投与後15ミリ秒で運動が開始され、この遅れはダイニン分子のATP結合から滑り力発生中間体形成までの時間と考えられる。(上村) 単頭ミオシンは運動活性・酵素活性が双頭ミオシンより低く、2つのモーター領域間には機能的共同性があることが示唆された。(上田) RNAポリメラーゼの転写開始期に短鎖RNAのみを合成する転写中間体を見いだし、この生成が転写開始因子のRNA保持ドメインの構造に依存すること、ポリメラーゼ同士の衝突で捉進されることを発見した。(嶋本)

Research Products
(32 results)

All Other

All Publications (32 results)

[Publications] K,Akashi,et al.: "Formation of Giant Liposomes Promoted by Divalent Alkali Cations : Critical Role of Electrostatic Repulsion." Biophys. J.(in press).
[Publications] Y.Suzuki,et al.: "Modulation of Actin Filament Sliding by Mutations of the SH2 Cysteine in Dictyostelium Myosin II." Biochem.Biophys.Res.Commun.234. 701-706 (1997)
[Publications] I.Sase,et al.: "Axial Rotation of Sliding Actin Filaments Revealed by Single-Fluorophore Imaging." Proc.Natl.Acad.Sci.USA. 94. 5646-5650 (1997)
[Publications] H.Noji,et al.: "Ditect Observation of the Rotation of Fl-ATPase." Nature. 386. 299-302 (1997)
[Publications] H.Miyata,et al.: "Cooperative Association of Actin Protomers and Crosslinked Actin Oligomers in Filaments at Low Ionic Strength." J.Biochem.121. 527-533 (1997)
[Publications] A.Ishijima,et al.: "Simultaneous Observation of Individual ATPase and Mechanical Events by a Single Myosin Molecule during Interaction with Actin." Cell. 92. 161-171 (1998)
[Publications] T.Aoki,et al.: "Non-Contact Scanning probe Microscopy with Sub-Piconewton Force Sensitivitiy." Ultramicroscopy. 70. 45-55 (1997)
[Publications] K.Saito,et al.: "Dual-Colour Microscopy of Single Fluorophores Bound to Myosin Interacting with Fluorescently Labelled Actin Using Anti-Stoke Fluorescence." J.Microscopy. 183. 255 (1997)
[Publications] T.Funatsu,et al.: "Imaging and Nano-Manipulation of Single Bio-Molecules." Biophysical Chemistry,. (in press).
[Publications] Y.Harada,et al.: "Single Molecule Imaging and Nano-Manipulation of Biomolecules." Methods in Cell Biology. 55. 117-128 (1997)
[Publications] H,Kojima,et al.: "Mechanics of Single Kinesin Molecules Measured by Optical Trapping Nanometry." Biophys. J.73. 2012-2022 (1997)
[Publications] M.Tokunaga,et al.: "Subpiconewton Intermolecular Force Microscopy." Biochem.and Biophys.Res.Commun.231. 565-569 (1997)
[Publications] A.H.Iwane,et al.: "Myosin Subfragment-1 is Fully Equipped with Factors Essential for Motor Function." Biochem.and Biophys.Res.Commun.230. 76-80 (1997)
[Publications] A.H.Iwane,et al.: "Single Molecular Assay of the Individual ATP Turnovers by a Myosin GFP Fusion Protein Expressed in vitro." FEBS Letters. 407. 235-258 (1997)
[Publications] Y.Inoue,et al.: "Movements of Truncated Kinesin Fragments with a Short or an Artificial Flexible Neck." Proc.Natl.Acad.Sci.U.S.A.94. 7275-7280 (1997)
[Publications] H.Higuchi,et al.: "Kinetics of Force Generation by Single Kinesin Molecules Activated by Laser Photolysis of Caged ATP." Proc.Natl.Acad.Sci.U.S.A.94. 4395-4400 (1997)
[Publications] M.Anson,and K.Oiwa: "Singnal-to-noise ratio,spatial and time resolution of measurements with single fluorescent molecules." Biophysical Journal. 74. A185 (1998)
[Publications] K.Oiwa,et al.: "Isolation and characterization of 2'-0- and 3'-0-isomers of Cy3-EDA-ATP:kinetics and single molecule studies." Biophysical Journal.74. A260 (1998)
[Publications] K.Oiwa,et al.: "Direct observation of a central bare zone in a native thick filament isolated from the anterior byssus retractor muscle of Mytilus edulis using fluorescent ATP analogue" Journal of Biochemistry,. (in press).
[Publications] A.Yamada,et al.: "Unidirectional movement of fluorescent microtubules on rows" Journal of Cell Science. 111. 93-98 (1998)
[Publications] H.Suzuki,et al.: "Control of actin moving trajectory by patterned poly(methylmethacrylate)tracks." Biophysical Journal. 72. 1997-2001 (1997)
[Publications] T.Tani,and S.Kamimura: "Reactivation of sea-urchin sperm flagella induced rapid Photolysis of caged ATP." J.Exp.Biol.,. (in press).
[Publications] S.Yumura,et al.: "Transport of myosin II to the equatorial region without its own motor activity in mitotic Dictyostelium cells." Molecular Biology of the Cell. 8. 2089-2099 (1997)
[Publications] H.Nagai,and N.Shimamoto: "Most conserved regions of the E.coli primary sigma factor are involved in interaction with RNA polymerase core enzyme." Gene.Cells 2,. (in press).
[Publications] T.Kinebuchi,et al.: "Functional domain of Esherichia coli Single-stranded DNA binding protein as assessed by analysis of the deletion mutants." Biochemistry. 36. 6732-6738 (1997)
[Publications] T.Kubori,et al.: "Physical interference between Esherichia cole RNA polymerase molecules transcribing in tandem enhances abortive synthesis and misincorporation." Nuc.Acids Res.25. 2640-2647 (1997)
[Publications] R.Sen,et al.: "Reduction in abortive transcription from the lambdaPR promoter by mutations in region 3 of the sigma-70 subunit of E.cole RNA Polymerase." J.Biol.Chem.273(in press). (1998)
[Publications] 加畑博幸、嶋本伸雄: "DNA・蛋白質相互作用の可視化とスライディング運動" 蛋白質・核酸・酵素増刊. 42. 1181-1186 (1997)
[Publications] 久堀智子、嶋本伸雄: "転写開始とその調節の新モデル" 生物物理. 214. 249-253 (1997)
[Publications] 柳田俊雄、石渡信一: "見る"生物物理から見た生命像3" 吉岡書店, (1997)
[Publications] T.Q.P.Uyeda,et al.: "In Dictyostelium-A model system for cell and development biology." Universal Academy Press,Tokyo, 12 (1997)
[Publications] 嶋本伸雄、郷通子: "遺伝子の構造生物学Vol,2 196" 共立出版、東京, (1998)

1997 Fiscal Year Annual Research Report

生物分子モーターの1分子計測と1分子操作

Principal Investigator

木下 一彦 慶應義塾大学, 理工学部, 教授 (30124366)

Research Products

[Publications] K,Akashi,et al.: "Formation of Giant Liposomes Promoted by Divalent Alkali Cations : Critical Role of Electrostatic Repulsion." Biophys. J.(in press).

[Publications] Y.Suzuki,et al.: "Modulation of Actin Filament Sliding by Mutations of the SH2 Cysteine in Dictyostelium Myosin II." Biochem.Biophys.Res.Commun.234. 701-706 (1997)

[Publications] I.Sase,et al.: "Axial Rotation of Sliding Actin Filaments Revealed by Single-Fluorophore Imaging." Proc.Natl.Acad.Sci.USA. 94. 5646-5650 (1997)

[Publications] H.Noji,et al.: "Ditect Observation of the Rotation of Fl-ATPase." Nature. 386. 299-302 (1997)

[Publications] H.Miyata,et al.: "Cooperative Association of Actin Protomers and Crosslinked Actin Oligomers in Filaments at Low Ionic Strength." J.Biochem.121. 527-533 (1997)

[Publications] A.Ishijima,et al.: "Simultaneous Observation of Individual ATPase and Mechanical Events by a Single Myosin Molecule during Interaction with Actin." Cell. 92. 161-171 (1998)

[Publications] T.Aoki,et al.: "Non-Contact Scanning probe Microscopy with Sub-Piconewton Force Sensitivitiy." Ultramicroscopy. 70. 45-55 (1997)

[Publications] K.Saito,et al.: "Dual-Colour Microscopy of Single Fluorophores Bound to Myosin Interacting with Fluorescently Labelled Actin Using Anti-Stoke Fluorescence." J.Microscopy. 183. 255 (1997)

[Publications] T.Funatsu,et al.: "Imaging and Nano-Manipulation of Single Bio-Molecules." Biophysical Chemistry,. (in press).

[Publications] Y.Harada,et al.: "Single Molecule Imaging and Nano-Manipulation of Biomolecules." Methods in Cell Biology. 55. 117-128 (1997)

[Publications] H,Kojima,et al.: "Mechanics of Single Kinesin Molecules Measured by Optical Trapping Nanometry." Biophys. J.73. 2012-2022 (1997)

[Publications] M.Tokunaga,et al.: "Subpiconewton Intermolecular Force Microscopy." Biochem.and Biophys.Res.Commun.231. 565-569 (1997)

[Publications] A.H.Iwane,et al.: "Myosin Subfragment-1 is Fully Equipped with Factors Essential for Motor Function." Biochem.and Biophys.Res.Commun.230. 76-80 (1997)

[Publications] A.H.Iwane,et al.: "Single Molecular Assay of the Individual ATP Turnovers by a Myosin GFP Fusion Protein Expressed in vitro." FEBS Letters. 407. 235-258 (1997)

[Publications] Y.Inoue,et al.: "Movements of Truncated Kinesin Fragments with a Short or an Artificial Flexible Neck." Proc.Natl.Acad.Sci.U.S.A.94. 7275-7280 (1997)

[Publications] H.Higuchi,et al.: "Kinetics of Force Generation by Single Kinesin Molecules Activated by Laser Photolysis of Caged ATP." Proc.Natl.Acad.Sci.U.S.A.94. 4395-4400 (1997)

[Publications] M.Anson,and K.Oiwa: "Singnal-to-noise ratio,spatial and time resolution of measurements with single fluorescent molecules." Biophysical Journal. 74. A185 (1998)

[Publications] K.Oiwa,et al.: "Isolation and characterization of 2'-0- and 3'-0-isomers of Cy3-EDA-ATP:kinetics and single molecule studies." Biophysical Journal.74. A260 (1998)

[Publications] K.Oiwa,et al.: "Direct observation of a central bare zone in a native thick filament isolated from the anterior byssus retractor muscle of Mytilus edulis using fluorescent ATP analogue" Journal of Biochemistry,. (in press).

[Publications] A.Yamada,et al.: "Unidirectional movement of fluorescent microtubules on rows" Journal of Cell Science. 111. 93-98 (1998)

[Publications] H.Suzuki,et al.: "Control of actin moving trajectory by patterned poly(methylmethacrylate)tracks." Biophysical Journal. 72. 1997-2001 (1997)

[Publications] T.Tani,and S.Kamimura: "Reactivation of sea-urchin sperm flagella induced rapid Photolysis of caged ATP." J.Exp.Biol.,. (in press).

[Publications] S.Yumura,et al.: "Transport of myosin II to the equatorial region without its own motor activity in mitotic Dictyostelium cells." Molecular Biology of the Cell. 8. 2089-2099 (1997)

[Publications] H.Nagai,and N.Shimamoto: "Most conserved regions of the E.coli primary sigma factor are involved in interaction with RNA polymerase core enzyme." Gene.Cells 2,. (in press).

[Publications] T.Kinebuchi,et al.: "Functional domain of Esherichia coli Single-stranded DNA binding protein as assessed by analysis of the deletion mutants." Biochemistry. 36. 6732-6738 (1997)

[Publications] T.Kubori,et al.: "Physical interference between Esherichia cole RNA polymerase molecules transcribing in tandem enhances abortive synthesis and misincorporation." Nuc.Acids Res.25. 2640-2647 (1997)

[Publications] R.Sen,et al.: "Reduction in abortive transcription from the lambdaPR promoter by mutations in region 3 of the sigma-70 subunit of E.cole RNA Polymerase." J.Biol.Chem.273(in press). (1998)

[Publications] 加畑博幸、嶋本伸雄: "DNA・蛋白質相互作用の可視化とスライディング運動" 蛋白質・核酸・酵素増刊. 42. 1181-1186 (1997)

[Publications] 久堀智子、嶋本伸雄: "転写開始とその調節の新モデル" 生物物理. 214. 249-253 (1997)

[Publications] 柳田俊雄、石渡信一: "見る"生物物理から見た生命像3" 吉岡書店, (1997)

[Publications] T.Q.P.Uyeda,et al.: "In Dictyostelium-A model system for cell and development biology." Universal Academy Press,Tokyo, 12 (1997)

[Publications] 嶋本伸雄、郷通子: "遺伝子の構造生物学Vol,2 196" 共立出版、東京, (1998)

木下一彦慶應義塾大学, 理工学部, 教授 (30124366)