2002 年度実績報告書

膜蛋白質の動的構造と信号認識、物質輸送

研究課題

研究課題/領域番号	14580629
研究機関	姫路工業大学
研究代表者	斉藤肇姫路工業大学, 理学研究科, 教授 (30100150)
研究分担者	加茂直樹北海道大学, 大学院・薬学研究科, 教授 (10001976) 山口悟姫路工業大学, 大学院・理学研究科, 助手 (20347529) 辻暁姫路工業大学, 大学院・理学研究科, 助教授 (60227387)
キーワード	膜蛋白質 / バクテリオロドプシン / フォボロドプシン / ゆらぎ / ダイナミクス / 固体NMR
研究概要	バクテリオロドプシン(bR)、ファラオニス由来フォボロドプシン(ppR)は光をエネルギーとしそれぞれ光駆動プロトンポンプ、負の走光性をコントロールする膜蛋白質である。[3-^<13>C]Ala、[1-^<13>C]Val標識蛋白質を調製しその固体NMR測定を行うことにより以下の結果を得た。 1)二次元結晶化とダイナミクス二次元結晶を作らないbR変異体W12L、W80Lでは蛋白質-蛋白質、蛋白質-脂質間の相互作用が大きく変化していることがわかった。bR一分子当たりの脂質の数が大幅に増大し、膜貫通α-ヘリックスの揺らぎの周波数が10^2から10^4Hzに変化することがわかった。 2)細胞質側ループ構造 bRの細胞質側ループはカチオンを介してループ間でバンドルを作っていることがわかった。また膜から突出したα-ヘリックスの構造変化によりプロトン輸送の調整をしていることを見い出した。 3)ppRの構造と揺らぎ大腸菌発現したppRを脂質二重膜に再構成させた試料の固体NMR測定からppRはbRの動的構造の描像によく一致していることがわかった。しかし、ループ部位の[3-^<13>C]Ala標識信号の消失がみられ単量体でこの部分はデカップル周波数との干渉領域(10^5Hz)ていどの揺らぎを持つことがわかった。さらにトランスデューサーと結合することで揺らぎの周波数が一桁低下し、情報伝達への寄与が示唆された。

研究成果
(16件)

すべてその他

すべて文献書誌 (16件)

[文献書誌] S.Kimura: "Dynamics and Orientation of Transmembrane Peptide from Bacteriorhodopsin Incorporated into Lipid Bilayer as Revealed by Solid State ^<31>P and ^<13>C NMR Spectroscopy"Biopolymers. 63. 122-131 (2002)
[文献書誌] A.Naito: "Dynorphin Induced Magnetic Ordering in Lipid Bilayers as Studied by ^<31>P NMR Spectroscopy"Biochim. Biophys. Acta. 1558. 34-44 (2002)
[文献書誌] S.Kimura: "Dynamic Structure of Transmembrance α-helical Fragments of Bacteriorhodopsin Characterozed by ^<13>C Chemical Shift Tensor and Hydrogen Bond Distance by REDOR NMR"J. Mol. Struct. 602-603. 125-131 (2002)
[文献書誌] Y.Shindo: "Stepwise Conformational Transitions of Crystalline Disodium Adenosine 5'-Triphosphate with Relative Humidity as Studied by High Resolution Solid State ^<13>C and ^<31>P NMR"J. Mol. Struct. 602-603. 389-397 (2002)
[文献書誌] A.Fukutani: "Determination of Principal Components and Their Directions of ^<15>N Chemical Shift Tensor and N-H bond Lengths in Simple Peptides as Parameters for Characterization of N-H...O=C Hydrogen Bond"J. Mol. Struct. 602-603. 491-503 (2002)
[文献書誌] E.Alberti: "Study of Wheat High Molecular Weight 1D×5 Subunit by ^<13>C and ^1H Solid State NMR : II. The Roles of Non-Repctitive Terminal Domains and Length of Repetive Domain"Biopolymers (Biospectroscopy). 65. 158-168 (2002)
[文献書誌] H.Saito: "Residue-specific millisecond to microsecond fluctuations in bacteriorhodopsin induced by disrupted or disorganized two-dimensional crystalline lattice, through modified lipid-helix and helix-helix interactions, as revealed by ^<13>C NMR"Biochim. Biophys. Acta. 1565. 97-106 (2002)
[文献書誌] H.Saito: "Dynamic Structure of Bacteriorhodopsin Revealed by ^<13>C Solid-state NMR"J. Photosci. 9. 110-113 (2002)
[文献書誌] H.Saito: "Dynamic Aspect of Bacteriorhodopsin as Viewed from ^<13>C NMR : Conformational Elucidation, Surface Dynamics and Information Transfer from the Surface to Inner Residues"Spectroscopy. 16. 107-120 (2002)
[文献書誌] M.Kamihira: "Effect of Electrostatic Interaction on Fibril Formation of Human Calcitonin as Studied by High Resolution Solid-State ^<13>C NMR"J. Biol. Chem.. 278. 2859-2865 (2003)
[文献書誌] T.Arakawa: "Dynamic Structure of pharaonis Phoborhodopsin (Sensory Rhodopsin II) and Complex with a Cognate Truncated Transducer as Revealed by Site-directed ^<13>C Solid-state NMR"FEBS Lett.. 536. 237-240 (2003)
[文献書誌] K.Yonebayashi: "Cytoplasmic Surface Structures of Bacteriorhodopsin Modifed by Site-directed Mutations and Cation-bindings as Revealed by ^<13>C NMR"European Biophys. J.. 32. 1-11 (2003)
[文献書誌] H.Saito: "Dynamic Aspect of Bacteriorhodopsin as a Typical Membrane Protein as Revealed by Site-directred Solid-state ^<13>C NMR"Solid State NMR. (in press). (2003)
[文献書誌] H.Saito: "Site-directed ^<13>C Solid-state NMR Studies on Membrane Proteins : Strategy and Goals toward Revealing Conformation and Dynamics as Illustrated for ^<13>C-Labeled Bacteriorhodopsin"Magn. Reson. Chem.. (in press). (2003)
[文献書誌] H.Saito: "Annu. Rep. NMR Spectrosc."Academic Press, London. 39-108 (2002)
[文献書誌] A.Naito: "Encyclopedia of Nuclear Magnetic Resonance, Volume 9 : Advances in NMR"John-Wiley and Sons, Chichester. 283-291 (2002)

2002 年度 実績報告書

膜蛋白質の動的構造と信号認識、物質輸送

研究代表者

斉藤 肇 姫路工業大学, 理学研究科, 教授 (30100150)

研究成果

[文献書誌] S.Kimura: "Dynamics and Orientation of Transmembrane Peptide from Bacteriorhodopsin Incorporated into Lipid Bilayer as Revealed by Solid State ^<31>P and ^<13>C NMR Spectroscopy"Biopolymers. 63. 122-131 (2002)

[文献書誌] A.Naito: "Dynorphin Induced Magnetic Ordering in Lipid Bilayers as Studied by ^<31>P NMR Spectroscopy"Biochim. Biophys. Acta. 1558. 34-44 (2002)

[文献書誌] S.Kimura: "Dynamic Structure of Transmembrance α-helical Fragments of Bacteriorhodopsin Characterozed by ^<13>C Chemical Shift Tensor and Hydrogen Bond Distance by REDOR NMR"J. Mol. Struct. 602-603. 125-131 (2002)

[文献書誌] Y.Shindo: "Stepwise Conformational Transitions of Crystalline Disodium Adenosine 5'-Triphosphate with Relative Humidity as Studied by High Resolution Solid State ^<13>C and ^<31>P NMR"J. Mol. Struct. 602-603. 389-397 (2002)

[文献書誌] A.Fukutani: "Determination of Principal Components and Their Directions of ^<15>N Chemical Shift Tensor and N-H bond Lengths in Simple Peptides as Parameters for Characterization of N-H...O=C Hydrogen Bond"J. Mol. Struct. 602-603. 491-503 (2002)

[文献書誌] E.Alberti: "Study of Wheat High Molecular Weight 1D×5 Subunit by ^<13>C and ^1H Solid State NMR : II. The Roles of Non-Repctitive Terminal Domains and Length of Repetive Domain"Biopolymers (Biospectroscopy). 65. 158-168 (2002)

[文献書誌] H.Saito: "Dynamic Structure of Bacteriorhodopsin Revealed by ^<13>C Solid-state NMR"J. Photosci. 9. 110-113 (2002)

[文献書誌] H.Saito: "Dynamic Aspect of Bacteriorhodopsin as Viewed from ^<13>C NMR : Conformational Elucidation, Surface Dynamics and Information Transfer from the Surface to Inner Residues"Spectroscopy. 16. 107-120 (2002)

[文献書誌] M.Kamihira: "Effect of Electrostatic Interaction on Fibril Formation of Human Calcitonin as Studied by High Resolution Solid-State ^<13>C NMR"J. Biol. Chem.. 278. 2859-2865 (2003)

[文献書誌] T.Arakawa: "Dynamic Structure of pharaonis Phoborhodopsin (Sensory Rhodopsin II) and Complex with a Cognate Truncated Transducer as Revealed by Site-directed ^<13>C Solid-state NMR"FEBS Lett.. 536. 237-240 (2003)

[文献書誌] K.Yonebayashi: "Cytoplasmic Surface Structures of Bacteriorhodopsin Modifed by Site-directed Mutations and Cation-bindings as Revealed by ^<13>C NMR"European Biophys. J.. 32. 1-11 (2003)

[文献書誌] H.Saito: "Dynamic Aspect of Bacteriorhodopsin as a Typical Membrane Protein as Revealed by Site-directred Solid-state ^<13>C NMR"Solid State NMR. (in press). (2003)

[文献書誌] H.Saito: "Site-directed ^<13>C Solid-state NMR Studies on Membrane Proteins : Strategy and Goals toward Revealing Conformation and Dynamics as Illustrated for ^<13>C-Labeled Bacteriorhodopsin"Magn. Reson. Chem.. (in press). (2003)

[文献書誌] H.Saito: "Annu. Rep. NMR Spectrosc."Academic Press, London. 39-108 (2002)

[文献書誌] A.Naito: "Encyclopedia of Nuclear Magnetic Resonance, Volume 9 : Advances in NMR"John-Wiley and Sons, Chichester. 283-291 (2002)

2002 年度実績報告書

斉藤肇姫路工業大学, 理学研究科, 教授 (30100150)