1997 年度実績報告書

生物分子モーターの1分子計測と1分子操作

研究課題

研究課題/領域番号	09279104
研究種目	重点領域研究
研究機関	慶応義塾大学
研究代表者	木下一彦慶應義塾大学, 理工学部, 教授 (30124366)
研究分担者	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)
キーワード	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
研究概要	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)

研究成果
(32件)

すべてその他

すべて文献書誌 (32件)

[文献書誌] K,Akashi,et al.: "Formation of Giant Liposomes Promoted by Divalent Alkali Cations : Critical Role of Electrostatic Repulsion." Biophys. J.(in press).
[文献書誌] 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)
[文献書誌] I.Sase,et al.: "Axial Rotation of Sliding Actin Filaments Revealed by Single-Fluorophore Imaging." Proc.Natl.Acad.Sci.USA. 94. 5646-5650 (1997)
[文献書誌] H.Noji,et al.: "Ditect Observation of the Rotation of Fl-ATPase." Nature. 386. 299-302 (1997)
[文献書誌] 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)
[文献書誌] 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)
[文献書誌] T.Aoki,et al.: "Non-Contact Scanning probe Microscopy with Sub-Piconewton Force Sensitivitiy." Ultramicroscopy. 70. 45-55 (1997)
[文献書誌] 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)
[文献書誌] T.Funatsu,et al.: "Imaging and Nano-Manipulation of Single Bio-Molecules." Biophysical Chemistry,. (in press).
[文献書誌] Y.Harada,et al.: "Single Molecule Imaging and Nano-Manipulation of Biomolecules." Methods in Cell Biology. 55. 117-128 (1997)
[文献書誌] H,Kojima,et al.: "Mechanics of Single Kinesin Molecules Measured by Optical Trapping Nanometry." Biophys. J.73. 2012-2022 (1997)
[文献書誌] M.Tokunaga,et al.: "Subpiconewton Intermolecular Force Microscopy." Biochem.and Biophys.Res.Commun.231. 565-569 (1997)
[文献書誌] 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)
[文献書誌] 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)
[文献書誌] 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)
[文献書誌] 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)
[文献書誌] M.Anson,and K.Oiwa: "Singnal-to-noise ratio,spatial and time resolution of measurements with single fluorescent molecules." Biophysical Journal. 74. A185 (1998)
[文献書誌] 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)
[文献書誌] 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).
[文献書誌] A.Yamada,et al.: "Unidirectional movement of fluorescent microtubules on rows" Journal of Cell Science. 111. 93-98 (1998)
[文献書誌] H.Suzuki,et al.: "Control of actin moving trajectory by patterned poly(methylmethacrylate)tracks." Biophysical Journal. 72. 1997-2001 (1997)
[文献書誌] T.Tani,and S.Kamimura: "Reactivation of sea-urchin sperm flagella induced rapid Photolysis of caged ATP." J.Exp.Biol.,. (in press).
[文献書誌] 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)
[文献書誌] 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).
[文献書誌] 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)
[文献書誌] 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)
[文献書誌] 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)
[文献書誌] 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)
[文献書誌] 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)
[文献書誌] 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)
[文献書誌] T.Q.P.Uyeda,et al.: "In Dictyostelium-A model system for cell and development biology." Universal Academy Press,Tokyo, 12 (1997)
[文献書誌] 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 年度 実績報告書

生物分子モーターの1分子計測と1分子操作

研究代表者

木下 一彦 慶應義塾大学, 理工学部, 教授 (30124366)

研究成果

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

[文献書誌] 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)

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

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

[文献書誌] 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)

[文献書誌] 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)

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

[文献書誌] 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)

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

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

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

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

[文献書誌] 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)

[文献書誌] 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)

[文献書誌] 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)

[文献書誌] 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)

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

[文献書誌] 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)

[文献書誌] 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).

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

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

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

[文献書誌] 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)

[文献書誌] 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).

[文献書誌] 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)

[文献書誌] 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)

[文献書誌] 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)

[文献書誌] 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)

[文献書誌] 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)

[文献書誌] 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)

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

[文献書誌] 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 年度実績報告書

木下一彦慶應義塾大学, 理工学部, 教授 (30124366)