2000 Fiscal Year Annual Research Report

自発的磁場配向膜中生理活性ペプチドの高分解能構造解析法の開発

Research Project

Project/Area Number	12034217
Research Institution	Himeji Institute of Technology
Principal Investigator	内藤晶姫路工業大学, 理学部, 助教授 (80172245)
Co-Investigator(Kenkyū-buntansha)	辻暁姫路工業大学, 理学部, 助手 (60227387) 斉藤肇姫路工業大学, 理学部, 教授 (30100150)
Keywords	生理活性ペプチド / 磁場配向膜 / 小胞体 / 膜分断 / 膜融合 / 化学シフトテンソル / 膜貫通ヘリックス / MAS
Research Abstract	本研究では生理活性ペプチドの膜結合構造を明らかにするため、自発的磁場配向膜を用いた新しい方法の開発を目的として研究を進め、以下の成果を得た。 1.これまで明らかにしてきた長楕円型小胞体を形成するメリチン-DMPC小胞体の膜分断における形態変化を微分干渉光学顕微鏡を用いて観測した。この結果、脂質二重膜は相転移点(Tm)以上の温度では、膜融合を繰り返し30μmにも達する巨大小胞体を形成することが判明した。 2.メリチン-DMPC脂質二重膜は磁場の存在下では、長軸が短軸に比べて、4倍以上長い楕円小胞体を形成し、その長軸を磁場に平行に向けて自発的に磁場配向することが明らかになった。 3.メリチンのカルボニル炭素を^<13>C同位体標識した試料を合成し、自発的磁場配向膜に組み込んだ。このカルボニル炭素の化学シフトテンソルを静止条件(磁場配向条件)、低速MAS条件、および高速MAS条件で測定した。この結果、カルボニル炭素の^<13>C等方化学シフト値から、メリチンは膜中でN端およびC端に渡ってα-ヘリックス構造をとっていることが判明した。 4.磁場配向状態のメリチンの化学シフト値の解析から、メリチンのヘリックスはヘリックス軸から傾いた軸の回りで歳差運動をしており、その回転軸は膜面に垂直であることが判明した。従って、メリチンは膜結合状態で膜貫通ヘリックスを形成しており、N-端のヘリックスは膜の垂線から30°傾き、C-端のヘリックスは膜の垂線から10°傾いた状態で、その方向を絶えず変化させて膜中を2次元拡散しているメリチンの動的構造が明らかになった。

Research Products
(13 results)

All Other

All Publications (13 results)

[Publications] A.Naito: "Conformation and dynamics of melittin bound to magnetically oriented lipid bilayers by solid-state ^<31>P and ^<13>C NMR spectroscopy"Biophys.J.. 78. 2405-2417 (2000)
[Publications] M.Kamihira: "Conformational transition and fibrillation mechanism of human calcitonin as studied by high-resolution solid-state ^<13>C NMR"Protein Science. 9. 867-877 (2000)
[Publications] S.Yamaguchi: "Irreversible conformational change or bacterio-opsin induced by binding of retinal during its reconstitution of bacterio-opsin, as studied by ^<13>C NMR"J.Biochem.. 127. 861-869 (2000)
[Publications] S.Nakatsuji: "Novel photo-responsive organic spin systems : preparation and properties of norbornadienes and spiropyrans with TEMPO radical substituents"J.Chem.Soc.,Perkin Trans.. 2. 1969-1975 (2000)
[Publications] H.Kimura: "Determination of N-H bond lengths of ^<15>N-labeled poly (L-alanines) by ^1H CRAMPS NMR"Macromolecules. 18. 6627-6629 (2000)
[Publications] J.Bolze: "Small angle X-ray scattering and ^<31>P NMR studies on the phase behavior of phospholipid bilayered mixed micelles"Chem.Phys.Lett.. 329. 215-220 (2000)
[Publications] Y.Kawase: "Alteration of conformation and dynamics of bacteriorhodopsin induced by protonation of Asp85 and deprotonation of Schiff base as studied by ^<13>C NMR"Biochemistry. 47. 14472-14480 (2000)
[Publications] H.Saito: "Conformation and backbone dynamics of bacteriorhodopsin revealed by ^<13>C NMR"Biochim.Biophys.Acta. 1460. 39-48 (2000)
[Publications] S.Kimura: "A ^<13>C NMR study on [3-^<13>C]-[1-^<13>C] Val labeled transmembrane peptides of bacteriorhodopsin in lipid bilayers : insertion, rigid-body motions at ambient temperature"Biopolymers. 58. 78-88 (2001)
[Publications] K.Nishimura: "Natural abundance ^<13>C RBDOR coupled to a singly ^<15>N-labeled nucleus : simulaneous determination of interatomic distances in crystalline ammonium [^<15>N] L-glutamate monohydrate"J.Mol.Struct.. 560. 29-38 (2001)
[Publications] S.Yamaguchi: "Surface dynamics of Bacteriorhodopsin as revealed by ^<13>C NMR studies on [^<13>C] Ala-labeled protein : Determination of millisecond or microsecond motions in interhelical loops and C-terminal α-helix"J.Biochem.. 129. 373-382 (2001)
[Publications] S.Yamaguchi: "Cytoplasmic surfacee structure of bacteriorhodopsin consisting of interhelical loops and C-terminal a-helices modified by a variety of enviromnental factors as studied by ^<13>C NMR"Eur.J.Biochem.. (in press). (2001)
[Publications] S.Kimura: "Characterizationof α-helix structures in polypeptides, revealed by ^<13>C=O…H-^<15>N hydrogen bond lengths determined by ^<13>C REDOR NMR"J.Mol.Struct.. (in press). (2001)

2000 Fiscal Year Annual Research Report

自発的磁場配向膜中生理活性ペプチドの高分解能構造解析法の開発

Principal Investigator

内藤 晶 姫路工業大学, 理学部, 助教授 (80172245)

Research Products

[Publications] A.Naito: "Conformation and dynamics of melittin bound to magnetically oriented lipid bilayers by solid-state ^<31>P and ^<13>C NMR spectroscopy"Biophys.J.. 78. 2405-2417 (2000)

[Publications] M.Kamihira: "Conformational transition and fibrillation mechanism of human calcitonin as studied by high-resolution solid-state ^<13>C NMR"Protein Science. 9. 867-877 (2000)

[Publications] S.Yamaguchi: "Irreversible conformational change or bacterio-opsin induced by binding of retinal during its reconstitution of bacterio-opsin, as studied by ^<13>C NMR"J.Biochem.. 127. 861-869 (2000)

[Publications] S.Nakatsuji: "Novel photo-responsive organic spin systems : preparation and properties of norbornadienes and spiropyrans with TEMPO radical substituents"J.Chem.Soc.,Perkin Trans.. 2. 1969-1975 (2000)

[Publications] H.Kimura: "Determination of N-H bond lengths of ^<15>N-labeled poly (L-alanines) by ^1H CRAMPS NMR"Macromolecules. 18. 6627-6629 (2000)

[Publications] J.Bolze: "Small angle X-ray scattering and ^<31>P NMR studies on the phase behavior of phospholipid bilayered mixed micelles"Chem.Phys.Lett.. 329. 215-220 (2000)

[Publications] Y.Kawase: "Alteration of conformation and dynamics of bacteriorhodopsin induced by protonation of Asp85 and deprotonation of Schiff base as studied by ^<13>C NMR"Biochemistry. 47. 14472-14480 (2000)

[Publications] H.Saito: "Conformation and backbone dynamics of bacteriorhodopsin revealed by ^<13>C NMR"Biochim.Biophys.Acta. 1460. 39-48 (2000)

[Publications] S.Kimura: "A ^<13>C NMR study on [3-^<13>C]-[1-^<13>C] Val labeled transmembrane peptides of bacteriorhodopsin in lipid bilayers : insertion, rigid-body motions at ambient temperature"Biopolymers. 58. 78-88 (2001)

[Publications] K.Nishimura: "Natural abundance ^<13>C RBDOR coupled to a singly ^<15>N-labeled nucleus : simulaneous determination of interatomic distances in crystalline ammonium [^<15>N] L-glutamate monohydrate"J.Mol.Struct.. 560. 29-38 (2001)

[Publications] S.Yamaguchi: "Surface dynamics of Bacteriorhodopsin as revealed by ^<13>C NMR studies on [^<13>C] Ala-labeled protein : Determination of millisecond or microsecond motions in interhelical loops and C-terminal α-helix"J.Biochem.. 129. 373-382 (2001)

[Publications] S.Yamaguchi: "Cytoplasmic surfacee structure of bacteriorhodopsin consisting of interhelical loops and C-terminal a-helices modified by a variety of enviromnental factors as studied by ^<13>C NMR"Eur.J.Biochem.. (in press). (2001)

[Publications] S.Kimura: "Characterizationof α-helix structures in polypeptides, revealed by ^<13>C=O…H-^<15>N hydrogen bond lengths determined by ^<13>C REDOR NMR"J.Mol.Struct.. (in press). (2001)

内藤晶姫路工業大学, 理学部, 助教授 (80172245)