1999 年度実績報告書

バクテリオロドプシンのプロトンポンプ機構

研究課題

研究課題/領域番号	10044092
研究機関	姫路工業大学
研究代表者	斉藤肇姫路工業大学, 理学部, 教授 (30100150)
研究分担者	片岡幹雄奈良先端技術大学院大学, 物質創生科学研究科, 教授 (30150254) 辻暁姫路工業大学, 理学部, 助手 (60227387) 内藤晶姫路工業大学, 理学部, 助教授 (80172245) 神取秀樹京都大学, 大学院・理学研究科, 講師 (70202033)
キーワード	膜蛋白質 / バクテリオロドプシン / 固体NMR / 赤外分光学 / X線回折 / 光異性化反応 / プロトンポンプ
研究概要	バクテリオロドプシン(bR)のプロトンポンプ活性機構について、米国カリホルニア大学Lanyi教授、ウエイン州立大学Needleman教授との位置特異的変異蛋白質のデザインおよび調整を軸に、固体高分解能NMR、赤外分光学、X線回折の三つの面からの日米共同研究を行い、日米共同研究により以下の結果を得た。 (1)固体NMR プロトンポンプは、光吸収によるレチナールの異性化、細胞外側でのプロトン放出、細胞質側でのプロトンのとりこみ、シッフ塩基の脱プロトン化によるAsp85へのプロトン移動によって生じる。この間の蛋白質骨格構造の変化を知るために、[1-^<13>C]Val-標識bRおよびD85N変異株のスペクトルを高アルカリ状態で調べた。後者の変異株はAsp85のプロトン付加に対応しM中間体様構造において、ヘリックスBおよびB-Cループに大きな構造変化をもたらすことを明らかにするとともに、蛋白構造変化への寄与の重要性を指摘することができた。 (2)赤外分光学レチナールの光異性化反応における蛋白質側の構造変化を詳細に知るために、偏光赤外分光学測定を行った。光異性化にともなう蛋白質構造変化の解析を行い、重水置換可能なO-H伸縮振動から、89位のスレオニン残基において、異性化反応にともなって水素結合強度が強まることを観測することができた。 (3)X線回折時分割測定の可能性を拡張するために、紫膜の光反応の時間分解X線小角回折データに対して、特異値分解法を適用した。その結果、光吸収後に生成するシッフ塩基の脱プロトン化からの戻り反応において、2っつの異なる構造をとることが明らかになった。すなわち、速い成分はMに、遅い成分はN中間体の構造の特長を示すことがわかった。なお、米国側では、単結晶のX線回折により基底状態およびM中間体における高分解能の3次元構造の解析に成功している。

研究成果

(20件)

すべてその他

すべて文献書誌 (20件)

[文献書誌] S.Tuzi: "Localization of a cation binding site in the loop between helices F and G of bacteriorhodopsin as studied by ^<13>C NMR"Biophysical Journal. 76. 1523-1531
[文献書誌] M.Kamihira: "Phenyl ring dynamics of enkephalin mo************* solventss in the crystalline state by ^2H NMR spectroscopy"Journal of Physical Chemistry,A. 103. 3356-3363
[文献書誌] M.Tanio: "Long-distance effects of site-directed mutations on backbone conformation in bacteriorhodopsin from solid state NMR of [1-^<13>C]Val-labeled proteins"Biophysical Journal. 77. 431-442
[文献書誌] M.Tanio: "Conformational changes of bacteriorhodopsin along the proton-conductance chain as studied with ^<13>C NMR of [3-^<13>C]Ala-labeled protein :Arg 82 may function as information mediator"Biophysical Journal. 77. 1577-1584
[文献書誌] K.Nishimura: "Analysis of dipolar dephasing pattern in I-S_n multispin system for obtaining the information of molecular packing and its application to crystalline N-acetyl-Pro-Gly-Phe by REDOR solid state NMR"Journal of Physical Chemistry B. 103. 8398-8408
[文献書誌] S.Yamaguchi: "Irreversible conformational change of bacteri-opsin induced by binding of the retinal in the in vitro folding to bacteriorhodopsin as studied by ^<13>C NMR"Journal of Biochemistry(Tokyo). (in press).
[文献書誌] M.Kamihira: "Conformational transitions and fibrillaation mechanism of human carcitonin as studied high resolution solid state ^<13>C NMR"Protein Science,. (in Press). (2000)
[文献書誌] A Naito: "Conformation and dynamics of mellitin bound to magnetically oriented lipid bilayers solid state ^<13>C NMR"Biophysical Journal. (2000)
[文献書誌] H,Saito: "Conformation and backbone dynamics ofbacteriorhodopsin Revealed by ^<13>C NMR"Biochimica et Biophysica Acta. (in press). (2000)
[文献書誌] M.Kataoka: "Low energy dynamics of globular proteins studied by inelastic neutron scattering"Journal of Physical Chemistry of Solid. 1285-1289 (1999)
[文献書誌] M.Kataoka: "Dynamical and structural modifications of staphylococcal nyclease on C-terninal truncation,"Physica B. 226. 20-26 (1999)
[文献書誌] T.Oka: "Conformational change of helix G in the bacteriorhodopsin photocycle:Investigation with heavy atom labeling and X-ray diffraction"Biophysical Journal. 76. 1018-1023 (1999)
[文献書誌] D.Hamada: "Evidence for the nature of rate-limiting steps in protein folding by analysis of the effects of 2,2,2-trifluoroethanol,"Nature Structural Biology. 7. 58-61 (2000)
[文献書誌] M.Kataoka: "Structures of photointermediates and their implication to the proton pump mechanism"Biochimica Biophysica Acta. (in press). (2000)
[文献書誌] Y.Yamazaki: "Fourier Transform Infrared Studies on Conformation Changes of bd-type Ubiquinol Oxidase from Escherichia coli upon Photoreduction of the Redox Metal Centers"Journal of Biochemistry. 125. 1131-1136 (1999)
[文献書誌] Young-Shin Chon: "Existence of Two L Photointermediates of Halorhodopsin from Halobacterium salinarum,Different in their Protein and Water FTIR Bands"Biochemistry. 38. 9449-9455 (1999)
[文献書誌] H.Kandori: "Structural Change of Threonine 89 upon Photoisomerization in Bacteriorhodopsin as Revealed by Polarized FTIR Spectroscopy"Biochemistry. 38. 9676-9683 (1999)
[文献書誌] H.Kandori: "Role of Internal Water Molecules in Bacteriorhodopsin"Biochimica et Biophysica Acta. (in press). (2000)
[文献書誌] M.Kamihira: "Peptide Science 1998"Protein Research Foundation. 337-340
[文献書誌] M.Kamihira: "Peptide Science 1998"Protein Research Foundation. 85-88

1999 年度 実績報告書

バクテリオロドプシンのプロトンポンプ機構

研究代表者

斉藤 肇 姫路工業大学, 理学部, 教授 (30100150)

研究成果

[文献書誌] S.Tuzi: "Localization of a cation binding site in the loop between helices F and G of bacteriorhodopsin as studied by ^<13>C NMR"Biophysical Journal. 76. 1523-1531

[文献書誌] M.Kamihira: "Phenyl ring dynamics of enkephalin mo************* solventss in the crystalline state by ^2H NMR spectroscopy"Journal of Physical Chemistry,A. 103. 3356-3363

[文献書誌] M.Tanio: "Long-distance effects of site-directed mutations on backbone conformation in bacteriorhodopsin from solid state NMR of [1-^<13>C]Val-labeled proteins"Biophysical Journal. 77. 431-442

[文献書誌] M.Tanio: "Conformational changes of bacteriorhodopsin along the proton-conductance chain as studied with ^<13>C NMR of [3-^<13>C]Ala-labeled protein :Arg 82 may function as information mediator"Biophysical Journal. 77. 1577-1584

[文献書誌] K.Nishimura: "Analysis of dipolar dephasing pattern in I-S_n multispin system for obtaining the information of molecular packing and its application to crystalline N-acetyl-Pro-Gly-Phe by REDOR solid state NMR"Journal of Physical Chemistry B. 103. 8398-8408

[文献書誌] S.Yamaguchi: "Irreversible conformational change of bacteri-opsin induced by binding of the retinal in the in vitro folding to bacteriorhodopsin as studied by ^<13>C NMR"Journal of Biochemistry(Tokyo). (in press).

[文献書誌] M.Kamihira: "Conformational transitions and fibrillaation mechanism of human carcitonin as studied high resolution solid state ^<13>C NMR"Protein Science,. (in Press). (2000)

[文献書誌] A Naito: "Conformation and dynamics of mellitin bound to magnetically oriented lipid bilayers solid state ^<13>C NMR"Biophysical Journal. (2000)

[文献書誌] H,Saito: "Conformation and backbone dynamics ofbacteriorhodopsin Revealed by ^<13>C NMR"Biochimica et Biophysica Acta. (in press). (2000)

[文献書誌] M.Kataoka: "Low energy dynamics of globular proteins studied by inelastic neutron scattering"Journal of Physical Chemistry of Solid. 1285-1289 (1999)

[文献書誌] M.Kataoka: "Dynamical and structural modifications of staphylococcal nyclease on C-terninal truncation,"Physica B. 226. 20-26 (1999)

[文献書誌] T.Oka: "Conformational change of helix G in the bacteriorhodopsin photocycle:Investigation with heavy atom labeling and X-ray diffraction"Biophysical Journal. 76. 1018-1023 (1999)

[文献書誌] D.Hamada: "Evidence for the nature of rate-limiting steps in protein folding by analysis of the effects of 2,2,2-trifluoroethanol,"Nature Structural Biology. 7. 58-61 (2000)

[文献書誌] M.Kataoka: "Structures of photointermediates and their implication to the proton pump mechanism"Biochimica Biophysica Acta. (in press). (2000)

[文献書誌] Y.Yamazaki: "Fourier Transform Infrared Studies on Conformation Changes of bd-type Ubiquinol Oxidase from Escherichia coli upon Photoreduction of the Redox Metal Centers"Journal of Biochemistry. 125. 1131-1136 (1999)

[文献書誌] Young-Shin Chon: "Existence of Two L Photointermediates of Halorhodopsin from Halobacterium salinarum,Different in their Protein and Water FTIR Bands"Biochemistry. 38. 9449-9455 (1999)

[文献書誌] H.Kandori: "Structural Change of Threonine 89 upon Photoisomerization in Bacteriorhodopsin as Revealed by Polarized FTIR Spectroscopy"Biochemistry. 38. 9676-9683 (1999)

[文献書誌] H.Kandori: "Role of Internal Water Molecules in Bacteriorhodopsin"Biochimica et Biophysica Acta. (in press). (2000)

[文献書誌] M.Kamihira: "Peptide Science 1998"Protein Research Foundation. 337-340

[文献書誌] M.Kamihira: "Peptide Science 1998"Protein Research Foundation. 85-88

1999 年度実績報告書

斉藤肇姫路工業大学, 理学部, 教授 (30100150)