2003 Fiscal Year Annual Research Report

膜タンパク質の動的構造と信号認識、物質輸送

Research Project

Project/Area Number	14580629
Research Institution	Himeji Institute of Technology
Principal Investigator	山口悟姫路工業大学, 大学院・理学研究科, 助手 (20347529)
Co-Investigator(Kenkyū-buntansha)	加茂直樹北海道大学, 大学院・薬学研究科, 教授 (10001976) 辻暁姫路工業大学, 大学院・理学研究科, 助教授 (60227387)
Keywords	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
Research Abstract	光をエネルギーとしバクテリオロドプシン(bR)は光駆動プロトンポンプ、ファラオニス由来フォボロドプシン(ppR)はトランスデューサー(pHtrII)を介して負の走光性をコントロールする膜蛋白質である。これらの[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)bRの揺らぎ bRは紫膜中で二次元結晶を作るが、その膜表面部位は50μs以下の比較的遅い揺らぎを持っていることがわかった。 (2)bR安定性に対するGlu残基の寄与 bRの細胞膜外側に位置するGlu^9、Glu^<74>、Glu^<194>、Glu^<204>のミュータントを用いた実験からこれらがbRの二次構造の安定性に寄与していることがわかった。特にGlu^<194>、Glu^<204>はプロトン放出基であり、ポンプ機構と揺らぎの相関を示唆する結果が得られたことは興味深い。 (3)ppR-pHtrII(1-195)複合体形成による膜外部位の揺らぎの変化 pHtrII(1-195)は脂質二重膜中のみならず膜外部位にα-ヘリックス構造を持つことがわかった。このヘリックスはppRと複合体を形成すると揺らぎが上昇することが明らかになり、情報伝達への寄与が示唆された。以上のことから固体高分解能NMRは一般には困難とされている膜蛋白質の構造解析に非常に有用な解析方法であることが示された。

Research Products
(11 results)

All Other

All Publications (11 results)

[Publications] P.Barre, S.Yamaguchi, H.Saito, D.Huster: "Backbone dynamics of bacteriorhodopsin as studied by ^<13>C solid-state NMR spectroscopy"Europian Biophys.J.. 32. 578-584 (2003)
[Publications] S.Tuzi, N.Uekama, M.Okada, S.Yamaguchi, H.Saito, H.Yagisawa: "Structure and dynamics of the phospholipase C-delta 1 PH domain located at the lipid bilayer surface"J Biol Chem. 278. 28019-28025 (2003)
[Publications] H.Saito, S.Yamaguchi, K.Ogawa, M.Marquez, C.Sanz, E.Padros: "Glutamic Acid Residue(s) of Bacteriorhdopsin at the extracellular Surface as Determinants for Conformation and Dynamics as Revealed by Site-directed Solid-state ^<13>C NMR"Biophys.J. 278. 28019-28025 (2003)
[Publications] S.Tuzi, A.Naito, H.Saito: "Local Protein Structure and Dynamics at Kinked Transmembrane α-Helices of [1- ^<13>C]Pro-labeled Bacteriorhodopsin as Revealed by Site-directed Solid-state ^<13>C NMR"J.Mol.Struct. 654. 205-214 (2003)
[Publications] H.Saito, K.Yamamoto, S.Tuzi, S.Yamaguchi: "Backbone Dynamics of Membrane Proteins in Lipid Bilayers : The Effect of Two Dimensional Array Formation as Revealed by Site-directed Solid-state ^<13>C NMR Studies on [3- ^<13>C]Ala- and [1- ^<13>C]Val-labeled Bacteriorhodopsin"Biochim Biophys Acta. 86. 1673-1681 (2004)
[Publications] H.Saito, S.Yamaguchi, H.Okuda, A.Shiraishi, S.Tuzi: "Dynamic Structure of Bacteriorhodopsin as a Typical Membrane Protein as Revealed by Site-directed Solid-state ^<13>C NMR, Solid State NMR"Solid State NMR. 25. 5-14 (2004)
[Publications] H.Saito, J.Mikami, S.Yamaguchi, M.Tanio, A.Kira, T.Arakawa, K.Yamamoto, S.Tuzi: "Site-directed ^<13>C solid-state NMR studies on membrane proteins : strategy and goals toward revealing conformation and dynamics as illustrated for bacteriorhodopsin labeled with [1- ^<13>C]amino acid residues"Magn Reson Chem. 42. 218-230 (2004)
[Publications] A.Naito, M.Kamihira, R.Inoue, H.Saito: "Structural Diversity of Amyloid Fibril Formed in Human Calcitonin as Viewed from Site-directed ^<13>C Solid State NMR Spectroscopy"Magn.Reson.Chem. 42. 247-257 (2004)
[Publications] S.Yamaguchi, S.Tuzi, J.U.Bowie, H.Saito: "Secondary Structure and Backbone Dynamics of Eschelicia coli Diacylglycerol Kinase, as Revealed by Site-directed Solid-state ^<13>C NMR"Biochim Biophys Acta. (in press). (2004)
[Publications] S.Yamaguchi, K.Shimono, Y.Sudo, S.TuZi, A.Naito, H.Saito: "Conformation and Dynamics of [3- ^<13>C]Ala-, [1- ^<13>C]Val-labeled Truncated pharaonis Transducer, pHtrII(1-159) as Revealed by Site-directed ^<13>C Solid-state NMR : Their Changes due to Association with Phoborhodopsin (Sensory Rhodopsin II)"Biophys.J. (in press). (2004)
[Publications] A.Kira, M.Tanio, S.Tuzi, H.Saito: "Significance of low-frequency local fluctuation motions in the transmembrane B and a helices of bacteriorhodopsir to facilitate efficient proton uptake from thecytoplasmic surface, as revealed by site-directed solid-state ^<13>C NMR"Eur.Biophys.J. (in press). (2004)

2003 Fiscal Year Annual Research Report

膜タンパク質の動的構造と信号認識、物質輸送

Principal Investigator

山口 悟 姫路工業大学, 大学院・理学研究科, 助手 (20347529)

Research Products

[Publications] P.Barre, S.Yamaguchi, H.Saito, D.Huster: "Backbone dynamics of bacteriorhodopsin as studied by ^<13>C solid-state NMR spectroscopy"Europian Biophys.J.. 32. 578-584 (2003)

[Publications] S.Tuzi, N.Uekama, M.Okada, S.Yamaguchi, H.Saito, H.Yagisawa: "Structure and dynamics of the phospholipase C-delta 1 PH domain located at the lipid bilayer surface"J Biol Chem. 278. 28019-28025 (2003)

[Publications] H.Saito, S.Yamaguchi, K.Ogawa, M.Marquez, C.Sanz, E.Padros: "Glutamic Acid Residue(s) of Bacteriorhdopsin at the extracellular Surface as Determinants for Conformation and Dynamics as Revealed by Site-directed Solid-state ^<13>C NMR"Biophys.J. 278. 28019-28025 (2003)

[Publications] S.Tuzi, A.Naito, H.Saito: "Local Protein Structure and Dynamics at Kinked Transmembrane α-Helices of [1- ^<13>C]Pro-labeled Bacteriorhodopsin as Revealed by Site-directed Solid-state ^<13>C NMR"J.Mol.Struct. 654. 205-214 (2003)

[Publications] H.Saito, S.Yamaguchi, H.Okuda, A.Shiraishi, S.Tuzi: "Dynamic Structure of Bacteriorhodopsin as a Typical Membrane Protein as Revealed by Site-directed Solid-state ^<13>C NMR, Solid State NMR"Solid State NMR. 25. 5-14 (2004)

[Publications] A.Naito, M.Kamihira, R.Inoue, H.Saito: "Structural Diversity of Amyloid Fibril Formed in Human Calcitonin as Viewed from Site-directed ^<13>C Solid State NMR Spectroscopy"Magn.Reson.Chem. 42. 247-257 (2004)

[Publications] S.Yamaguchi, S.Tuzi, J.U.Bowie, H.Saito: "Secondary Structure and Backbone Dynamics of Eschelicia coli Diacylglycerol Kinase, as Revealed by Site-directed Solid-state ^<13>C NMR"Biochim Biophys Acta. (in press). (2004)

山口悟姫路工業大学, 大学院・理学研究科, 助手 (20347529)