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Rotary catalysis mechanism of H^+-ATP synthase investigated by novel NMR methodology

Research Project

Project/Area Number 14208082
Research Category

Grant-in-Aid for Scientific Research (A)

Allocation TypeSingle-year Grants
Section一般
Research Field Biophysics
Research InstitutionOsaka University

Principal Investigator

AKUTSU Hideo  Osaka University, Institute for Protein Research, Professor, 蛋白質研究所, 教授 (60029965)

Co-Investigator(Kenkyū-buntansha) FUJIWARA Toshimichi  Osaka University, Institute for Protein Research, Associate Professor, 蛋白質研究所, 助教授 (20242381)
YAGI Hiromasa  Osaka University, Institute for Protein Research, Instructor, 蛋白質研究所, 助手 (70332749)
Project Period (FY) 2002 – 2004
Project Status Completed (Fiscal Year 2004)
Budget Amount *help
¥48,490,000 (Direct Cost: ¥37,300,000、Indirect Cost: ¥11,190,000)
Fiscal Year 2004: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000)
Fiscal Year 2003: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000)
Fiscal Year 2002: ¥37,700,000 (Direct Cost: ¥29,000,000、Indirect Cost: ¥8,700,000)
Keywordssolution NMR / solid-state NMR / H^+-ATP synthase / segmental isotope labeling / β subunit / monitor protein / structural analysis / subunit c / 巨大タンパク質 / サブユニットC / F_1βサブユニット / 区分安定同位体標識法
Research Abstract

F_OF_1-ATP synthase is a multisubunit enzyme that catalyzes ATP synthesis using the electrochemical potential of proton gradient. F_1-ATPase has been shown to be a step-wise molecular motor. The rotation mechanism has been explained by the interaction of the γ axis with the open and closed forms of the β subunit. Although NMR should be a powerful method to elucidate its mechanism, the molecular size (473 amino acid residues, 52 kDa) was the major obstacle to analyze. We have applied the segmental labeling based on intein ligation to the β subunit, and succeeded in assigning 89% NH (402/451),89% C_α (417/473),83% C_β(357/431),90% CO (425/473) signals of the β subunit monomer. The secondary structures predicted by the chemical shifts of the main chain atoms and the relative orientations determined by residual dipolar couplings indicated that the subunit β monomer takes the open form in the absence of nucleotide. Furthermore, the chemical shift perturbation and the residual-dipolar-coupling changes induced by nucleotide binding have shown the conformational change from the open to closed forms takes place on nucleotide binding. The intrinsic conformational change of the β subunit monomer induced by nucleotide binding must be one of essential driving forces of the rotation of F_1-ATPase. In F_OF_1-ATP synthase, an oligomer ring of F_Oc subunits acts as a rotary proton channel of F_O-proton motor. We have determined the NMR structure of F_Oc from thermophilic Bacillus PS3, TF_Oc in an organic solvent. Our results showed that, independent of pH, the carboxyl group of the essential Glu56 of TF_Oc protrudes to the outside of the hairpin, a third orientation differed from either of two orientations in EF_Oc. A solid-state NMR analysis of solid subunit c was also carried out.

Report

(4 results)
  • 2004 Annual Research Report   Final Research Report Summary
  • 2003 Annual Research Report
  • 2002 Annual Research Report
  • Research Products

    (30 results)

All 2004 2003 2002 Other

All Journal Article (15 results) Book (1 results) Publications (14 results)

  • [Journal Article] Signal assignments and chemical-shift structural analysis of uniformly ^<13>C, ^<15>N-labeled peptide, Mastoparan-X, by multidimensional solid-state NMR under magic-angle spinning2004

    • Author(s)
      T.Fujiwara
    • Journal Title

      J.Biomol.NMR 28

      Pages: 311-352

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Annual Research Report 2004 Final Research Report Summary
  • [Journal Article] Precision ^1H-^1H distance measurement via ^<13>C NMR signals : Utilization of the ^1H-^1H double-zuantum dipolar interactions recoupled under MAS conditions2004

    • Author(s)
      Y.Matsuki
    • Journal Title

      Magn.Reson.Chem. 42

      Pages: 291-300

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Annual Research Report 2004 Final Research Report Summary
  • [Journal Article] A conformational change of H^+-ATPase β monomer revealed on segmental isotope labeling NMR spectroscopy2004

    • Author(s)
      H.Yagi
    • Journal Title

      J.Am.Chem.Soc. 126

      Pages: 16621-16638

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Annual Research Report 2004 Final Research Report Summary
  • [Journal Article] Signal assignments and chemical-shift structural analysis of uniformly ^<13>C, ^<15>N-labeled peptide, Mastoparan-X, by multidimensional solid-state NMR under magic-angle spinning2004

    • Author(s)
      T.Fujiwara
    • Journal Title

      J.Biomol.NMR Vol.28

      Pages: 311-352

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Precision ^1H-^1H distance measurement via ^<13>C NMR signals : Utilization of the ^1H-^1H double-quantum dipolar interactions recoupled under MAS conditions2004

    • Author(s)
      Y.Matsuki
    • Journal Title

      Magn.Reson.Chem. Vol.42

      Pages: 291-300

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] A conformational change of H^+-ATPase β monomer revealed on segmental isotope labeling NMR spectroscopy2004

    • Author(s)
      H.Yagi
    • Journal Title

      J.Am.Chem.Soc. Vol.126

      Pages: 16632-16638

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] X-ray induced reduction of the crystal of high-molecular weight cytochrome c revealed by microspectrometry2004

    • Author(s)
      M.Sato
    • Journal Title

      J.Synchrotron Rad. 11

      Pages: 113-116

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Correlation between the g tensors and the nonplanarity of porphyrin rings in Desulfovibrio vulgaris Miyazaki F cytochrome c_3 studied by single crystal EPR2004

    • Author(s)
      T.Saitoh
    • Journal Title

      Bull.Chem.Soc.Jpn. 77

      Pages: 357-363

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Roles of non-coordinated aromatic residues in redox regulation of cytochrome c3 from Desulfovibrio vulgaris Miyazaki F2004

    • Author(s)
      Y.Takayama
    • Journal Title

      Biochemistry 43

      Pages: 10859-10866

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Band-selective recoupling of homonuclear double-quantum dipolar interaction with a generalized composite degrees pulse : application to ^<13>C aliphatic region-selective magnetization transfer in solids2003

    • Author(s)
      Y.Matsuki
    • Journal Title

      J.Magn.Reson. 162

      Pages: 54-66

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Phase-modulated heteronuclear decoupling in NMR of solids2003

    • Author(s)
      A.K.Khitrin
    • Journal Title

      J.Magn.Reson. 162

      Pages: 46-53

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Band-selective recoupling of homonuclear double-quantum dipolar interaction with a generalized composite degrees pulse : application to ^<13>C aliphatic region-selective magnetization transfer in solids2003

    • Author(s)
      M.Matsuki
    • Journal Title

      J.Magn.Reson. Vol.162

      Pages: 54-66

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Phase-modulated heteronuclear decoupling in NMR of solids2003

    • Author(s)
      A.K.Khitrin
    • Journal Title

      J.Magn.Reson. Vol.162

      Pages: 46-53

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Synthesis of a membrane protein with two transmembrane regions2002

    • Author(s)
      T.Sato
    • Journal Title

      J.Peptide Sci. 8

      Pages: 172-180

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Synthesis of a membrane protein with two transmembrane regions2002

    • Author(s)
      T.Sato
    • Journal Title

      J.Peptide Sci. Vol.8

      Pages: 172-180

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Book] NMR分光法-原理から応用まで-(日本分光学会測定法シリーズ41)2003

    • Author(s)
      阿久津秀雄
    • Total Pages
      260
    • Publisher
      学会出版センター
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Publications] A.K.Khitrin: "Phase-modulated heteronuclear decoupling in NMR of solids"J Magn.Reson.. 162. 46-53 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Y.Matsuki: "Band-selective recoupling of homonuclear double-quantum dipolar interaction with a generalized composite degrees pulse : application to ^<13>C aliphatic resion-selective magnetization transfer in solids"J.Magn.Reson.. 162. 54-66 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Y.Matsuki: "Precision 1H-^1H distance measurement via ^<13>C NMR signals: Utilization of the ^1H-^1H double-quantum dipolar interactions recoupled under MAS conditions"Magn.Reson.Chem.. 42. 291-300 (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] T.Fujiwara: "Signal assignments and chemical-shift structural analysis of uniformly ^<13>C,^<15>N-labeled peptide, Mastparan X, by multidimensional solid-state NMR under magic angle spinning"J.Biomol.NMR. (in press).

    • Related Report
      2003 Annual Research Report
  • [Publications] M.Sato: "X-ray induced reduction of the crystal of high-molecular-weight cytochrome c revealed by microspectrometry"J.Synchrotron Rad.. 11. 113-116 (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] T.Saitoh: "Correlation between the g tensors and the nonplanarity of porphyrin rings in Desulfovibrio vulgaris Miyazaki F cytochrome c3, studied by single crystal EPR"Bull.Chem.Soc.Jpn.. 77. 357-363 (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] 阿久津秀雄 他3名編: "NMR分光法-原理から応用まで- (日本分光学会測定法シリーズ41)"学会出版センター. 260 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] T.Sato 他7名: "Synthesis of a membrane protein with two transmembrane regions"J.Peptide Sci.. 8. 172-180 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] A.Y.Nosaka 他4名: "Characterization of water in TiO_2 photocatalytic systems by solid state ^1H-NMR spectrescopy"Chem.Lett.. 200L. 420-421 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] K.Sugase 他7名: "Designing analogs of mini atrial ratriuretic paptide based on structural analysis by NMR and restrained molecular dynamics"J.Med.Chem.. 45. 881-887 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] E.Harada 他6名: "Redox-coupled confermational althernations in cytochrome C_3 from D.unlgaris Miyazaki F on the basis of its neduced solution structure"J.Mol.Biol.. 319. 767-778 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] E.Hanada 他8名: "A dinectional electron transfer regulation based on heme-chain anchitecture in the small tetraheme cytochrome c fine Shenauel Dueidens"FEBS Cett.. 532. 333-337 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Y.-H.Lee 他6名: "Backbone NMR assiginments of the metal-free UneE from Bacillus pasteurii"J.Biomol.NMR. 24. 361-362 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 田之倉優, 村松知成, 阿久津秀雄訳: "生化学キーノート"シュプリンガー・フェアラーク東京. 460 (2002)

    • Related Report
      2002 Annual Research Report

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Published: 2002-04-01   Modified: 2016-04-21  

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