2001 Fiscal Year Annual Research Report

異方的磁気相互作用を利用した膜結合生理活性ペプチド構造解析法の開発

Research Project

Project/Area Number	13024263
Research Category	Grant-in-Aid for Scientific Research on Priority Areas (A)
Research Institution	Yokohama National University
Principal Investigator	内藤晶横浜国立大学, 大学院・工学研究院, 教授 (80172245)
Co-Investigator(Kenkyū-buntansha)	西村勝之横浜国立大学, 大学院・工学研究院, 助手 (00334631) 辻暁姫路工業大学, 理学部, 助教授 (60227387) 斉藤肇姫路工業大学, 理学部, 教授 (30100150)
Keywords	固体NMR / オピオイドペプチド / リン脂質 / 磁場配向 / ダイノルフィン / α-helix / 生体膜 / 光学顕微鏡
Research Abstract	本年度は生体膜に結合した状態で生理活性機能を示す生理活性ペプチドの立体構造と運動性固体NMRの手法によって解析する方法の開発を行い、以下の成果を得た。自発磁場配向膜の開発オピオイドペプチドダイノルフィンとリン脂質Dimyristoyl-phosphatidylcholine (DMPC)をモル比で1:10に混合して調製した脂質2分子膜はゲル-液晶相転移点(Tc)以上において膜面を磁場に平行に向けて磁場に自発的に配向する性質を観測した。さらに光学顕微鏡で2分子膜の形態を観測したところ、Tc以上の温度で放置すると、長い円筒状の小胞を形成することが分かった。これらの結果から、ダイノルフィン-DMPC2分子膜小胞は磁場中で楕円体を形成し、楕円長軸を磁場に平行に向けて、自発的に磁場配向する性質を示すことが分かった。膜結合ダイノルフィンの構造決定ダイノルフィン-DMPC2分子膜が自発的に磁場配向する性質を利用して、ダイノルフィンの膜結合構造を解析することを試みた。ダイノルフィンのカルボニル炭素を^<13>Cで標識したダイノルフィンをDMPCリン脂質膜に再構成し、磁場配向状態におけるカルボニル炭素の異方的化学シフト相互作用の解析を行った。この結果、ダイノルフィンのN-末端はα-helixを形成しており、このhelixは膜法線と一定の傾き角を保ちながら膜中に挿入した構造を持つことが判明した。一方、中央部からC-末端にかけてはペプチドは特定の構造をとらないことから、膜の表面に位置していることが判明した。さらにこの状態のペプチドは膜中で10^5Hz以上の速度で向きを変える動的な構造であることが分かった。

Research Products

(13 results)

All Other

All Publications (13 results)

[Publications] S.Yamaguchi: "Surface dynamics of Bacteriorhodopsin as revealed by ^<13>C NMR syudies on [^<13>C]Ala-labeled protein : Determination of millisecond or microsecond motions in interhelical loops and C-terminal a-helix"J.Biochem.. 129. 373-382 (2001)
[Publications] S.Kimura: "A ^<13>C NMR syudy on [3-^<13>C]-, [1-^<13>C]Ala-, or [1-^<13>C]Val-labeled transmembrane peptides of bacteriorhodopsin in lipid bilayers : Insertion, rigid-body motions, and local conformational fluctuations at ambient temperature"Biopolymers. 58. 78-88 (2001)
[Publications] S.Yamaguchi: "Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C-terminal α-helix, modified by a variety of environmental factors as studied by ^<13>C-NMR"Eur.J.Biochem.. 268. 2218-2228 (2001)
[Publications] 内藤晶: "自発配向膜を用いた生理活性ペプチドの高次構造、配向、ダイナミックスの解析"生物物理. 41. 258-261 (2001)
[Publications] S.Tuzi: "Regio-selective detection of dynamic structure of transmembrane α-helicies as revealed from ^<13>C NMR spectra of [3-^<13>C]Ala-labeled bacteriorhodopsin in the presence of Mn^<2+> ion"Biophys.J.. 81. 425-434 (2001)
[Publications] K.Nishimura: "Natural abundance ^<13>C REDOR coupled to a singly ^<15>N-labeled nucleus : Simultaneous determination of interatomic distances in crystalline ammonium [^<15>N]L-glutamate monohydrate"J.Mol.Struct.. 560. 29-38 (2001)
[Publications] H.Kimura: "Characterization of 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.. 562. 197-203 (2001)
[Publications] S.Kimura: "Dynamics and orientation of transmembrane peptide from bacterirhodopsin incorporated into lipid bilayer as revealed by solid state ^<31>P and ^<13>C NMR spectroscopy"Biopolymers. (in press). (2002)
[Publications] S.Kimura: "Dynamic structure of transmembrane α-helical fragments of bacteriorhodopsin in lipid bilayer characterized by ^<13>C chemical shift tensor and hydrogen bond distance by REDOR NMR"J.Mol.Struct.. 602-603. 125-131 (2002)
[Publications] Y.Shindo: "Stepwise conformational transition of crystalline disodium adenosin 5'-triphosphate with relative humidity as studied by high resolutin solid state ^<13>C and ^<31>P NMR"J.Mol.Struct.. 602-603. 389-397 (2002)
[Publications] 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 bonds"J.Mol.Struct.. 602-603. 491-503 (2002)
[Publications] A.Naito: "Dynorphin induced magnetic ordering in lipid bilayers as studied by ^<31>P NMR spectroscopy"Biochim.Bionhys.Acta. 1558. 34-44 (2002)
[Publications] A.Naito: "Solis state NMR of biomolecules in Handbook of Thin film Materials"Academic Press. 773 (2002)

2001 Fiscal Year Annual Research Report

異方的磁気相互作用を利用した膜結合生理活性ペプチド構造解析法の開発

Principal Investigator

内藤 晶 横浜国立大学, 大学院・工学研究院, 教授 (80172245)

Research Products

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

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

[Publications] S.Yamaguchi: "Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C-terminal α-helix, modified by a variety of environmental factors as studied by ^<13>C-NMR"Eur.J.Biochem.. 268. 2218-2228 (2001)

[Publications] 内藤 晶: "自発配向膜を用いた生理活性ペプチドの高次構造、配向、ダイナミックスの解析"生物物理. 41. 258-261 (2001)

[Publications] S.Tuzi: "Regio-selective detection of dynamic structure of transmembrane α-helicies as revealed from ^<13>C NMR spectra of [3-^<13>C]Ala-labeled bacteriorhodopsin in the presence of Mn^<2+> ion"Biophys.J.. 81. 425-434 (2001)

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

[Publications] H.Kimura: "Characterization of 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.. 562. 197-203 (2001)

[Publications] S.Kimura: "Dynamics and orientation of transmembrane peptide from bacterirhodopsin incorporated into lipid bilayer as revealed by solid state ^<31>P and ^<13>C NMR spectroscopy"Biopolymers. (in press). (2002)

[Publications] S.Kimura: "Dynamic structure of transmembrane α-helical fragments of bacteriorhodopsin in lipid bilayer characterized by ^<13>C chemical shift tensor and hydrogen bond distance by REDOR NMR"J.Mol.Struct.. 602-603. 125-131 (2002)

[Publications] Y.Shindo: "Stepwise conformational transition of crystalline disodium adenosin 5'-triphosphate with relative humidity as studied by high resolutin solid state ^<13>C and ^<31>P NMR"J.Mol.Struct.. 602-603. 389-397 (2002)

[Publications] 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 bonds"J.Mol.Struct.. 602-603. 491-503 (2002)

[Publications] A.Naito: "Dynorphin induced magnetic ordering in lipid bilayers as studied by ^<31>P NMR spectroscopy"Biochim.Bionhys.Acta. 1558. 34-44 (2002)

[Publications] A.Naito: "Solis state NMR of biomolecules in Handbook of Thin film Materials"Academic Press. 773 (2002)

内藤晶横浜国立大学, 大学院・工学研究院, 教授 (80172245)

[Publications] 内藤晶: "自発配向膜を用いた生理活性ペプチドの高次構造、配向、ダイナミックスの解析"生物物理. 41. 258-261 (2001)