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New stable-isotope labeling methods for structural studies

Research Project

Project/Area Number 08558076
Research Category

Grant-in-Aid for Scientific Research (A)

Allocation TypeSingle-year Grants
Section展開研究
Research Field Biophysics
Research InstitutionThe University of Tokyo

Principal Investigator

YOKOYAMA Shigeyuki  The University of Tokyo, Graduate School of Science, Professor, 大学院・理学系研究科, 教授 (00159229)

Co-Investigator(Kenkyū-buntansha) ITO Yutaka  RIKEN (The Institute of Physical and Chemical Research) Laboratory of Cellular a, 遺伝生化学研究室, 研究員 (80261147)
KIGAWA Takanori  RIKEN (The Institute of Physical and Chemical Research) Cellular Signaling Labor, 細胞情報伝達研究室, 研究員 (20270598)
KAWAI Gota  Chiba Institute of Technology, Department of Industrial Chemistry, Associate Pro, 工学部, 助教授 (70211860)
MUTO Yutaka  The University of Tokyo, Graduate School of Science, Lecturer, 大学院・理学系研究科, 講師 (30192769)
岡田 務  日本酸素株式会社, 事業企画部, 専門部長(研究職)
Project Period (FY) 1996 – 1998
Project Status Completed (Fiscal Year 1998)
Budget Amount *help
¥19,700,000 (Direct Cost: ¥19,700,000)
Fiscal Year 1998: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 1997: ¥5,200,000 (Direct Cost: ¥5,200,000)
Fiscal Year 1996: ¥10,500,000 (Direct Cost: ¥10,500,000)
KeywordsNMR / protein / RNA / stable-isotope labeling / selective labeling / deuteration / tertiary structure / cell-free protein synthesis / 安定同定位体標識
Research Abstract

1. Stable-isotope labeling of proteins
Improvement of cell-free protein production method : The yield of Escherichia coli cell-free protein synthesis system has been improved to be about 0.3 mg/mL reaction mixture in a few hours for the batch method, and has further been increased to be about 6 mg/mL reaction mixture in 10-20 hours by using the improved batch system to the dialysis method. Using the dialysis method, we have synthesized uniformly ^<15>N or ^<13>C labeled proteins. On the other hand, by preparing an amber suppressor tRNA charged with ^<15>N or ^<13>C labeled tyrosine, we have synthesized site-specifically labeled proteins in the batch system. The global fold of a 30-kDa protein was analyzed by preparation of [Ile/Leu/Val-^1H/^<13>C, Phe/Tyr-^1H, u-^2H, u-^<l5>N]protein.
2. Stable-isotope labeling of RLNAs
[^<15>N]-, [^<13>C, ^<15>N]-, [^2H(30-70%), ^<13>C], and [^2H(50%), ^<13>C, ^<15>N]NTPs were synthesized, and used in T7 RNA polymerase transcription for preparation of uniformly labeled RNAs. By the random 2H labeling, NMR signals of large RNAs were well observed. On the other hand, [5-^2H]uridine and [3-^<15>N]uridine were incorporated in specific position(s). This site-directed labeling was found to be very useful for NMR stydies of polyuridine RNAs complexed with a protein. Furthermore, by ligation of [^<l3>C,^<l5>N]pGp with non-labeled RNA fragment(s), site-specifically labeled longer RNAs were prepared.

Report

(4 results)
  • 1998 Annual Research Report   Final Research Report Summary
  • 1997 Annual Research Report
  • 1996 Annual Research Report
  • Research Products

    (33 results)

All Other

All Publications (33 results)

  • [Publications] D.-M.Kim: "A Highly Efficient Cell-free Protein Synthesis System from Escherichia coli" Eur.J.Biochem.239. 881-886 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] B.O.Smith: "An Approach to Global Fold Determination Using Limited NMR Data from Larger Proteins Selectively Protonated at Specific Residue Types" J.Biomol.NMR. 8. 360-368 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] I.Kim: "NMR Analysis of the Hydrogen Bonding Interactions of the RNA-binding Domains of the Drosophila Sex-lethal Protein with Target RNA Fragments with Site-specific[3-^<15>N]Uridine Substitutions" Nucleic Acids Res.25. 1565-1569 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] S.Koshiba: "The Solution Structure of the Pleckstrin Homology Domain of Mouse Son-of-Sevenless 1(mSos1)" J.Mol.Biol.269. 579-591 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Y.Ito,: "The “Regional Polysterism" in the GTP-bound Form of the Human c-Ha-Ras Protein" Biochemistry,. 36. 9109-9119 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] M.Inoue,: "A Characteristic Arrangement of Aromatic Amino Acid Residues in the Solution Structure of the Amino-terminal RNA-binding Domain of Drosophila Sex-lethal" J.Mol.Biol.,. 272. 82-94 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] J.Iwahara,: "A Helix-turn-helix Structure Unit in Human Centromere Protein B(CENP-B)" EMBO J.,. 17. 827-837 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] T.Yabuki,: "Dual Amino Acid-selective and Site-directed Stable-isotope Labeling of the Human c-Ha-Ras Protein by Cell-free Synthesis" J.Biomol.NMR,. 11. 295-306 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] T.Kigawa,: "Solution Structure of the Ras-binding Domain of RGL" FEBS Lett.,. 441. 413-418 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] S.-W.Chi: "Chemical Shift Perturbation Studies of the Interactions of the Second RNA-binding Domain of the Drosophila Sex-lethal Protein with the transformer pre-mRNA Polyuridine Tract and 3'Splice Site Sequences" Eur.J.Biochem.,. 259. 1-13 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] T.Terada,: "Nuclear Magnetic Resonance and Molecular Dynamics Studies on the Interactions of the Ras-Binding Domain of Raf-1 with the Wild-type and Mutant Ras Proteins" J.Mol.Biol.,. 286. 219-232 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] D.-M.Kin, T.Kigawa, C.-Y.Choi and S.Yokoyama: "A Highly Efficient Cell-free Protein Synthesis System from Escherichia coli" Eur.J.Biochem.239. 881-886 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] B.O.Smith, Y.Ito, A.Raine, S.Teichmann, L.Ben-Tovim, D.Nietlispach, R.W.Broadhurst, T.Terada, M.Kelly, H.Oschkinat, T.Shibata, S.Yokoyama and E.D.Laue: "An Approach to Global Fold Determination Using Limited NMR Data from Larger Proteins Selectively Protonated at Specific Residue Types" J.Biomol.NMR. 8. 360-368 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] I.Kim, Y.Muto, M.Inoue, S.Watanabe, A.Kitamura, S.Yokoyama, K.Hosono, H.Takaku, A.Ono, M.Kainosho, H.Sakamoto and Y.Shimura: "NMR Analysis of the Hydrogen Bonding Interactions of the RNA-binding Domains of the Drosophila Sex-lethal Protein with Target RNA Fragments with Site-specific [3-^<15>N] Uridine Substitutions" Nucleic Acids Res.25. 1565-1569 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] S.Koshiba, T.Kigawa, J.-H.Kim, M.Shirouzu, D.Bowtell and S.Yokoyama: "The Solution Structure of the Pleckstrin Homology Domain of Mouse Son-of-Sevenless 1 (mSos1)" J.Mol.Biol.269. 579-591 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Y.Ito, K.Yamasaki, J.Iwahara, T.Terada, A.Kamiya, M.Shirouzu, Y.Muto, G.Kawai, S.Yokoyama, E.D.Laue, M.Walchli, T.Shibata, S.Nishimura and T.Miyazawa: "The "Regional Polysterism" in the GTP-bound Form of the Human c-Ha-Ras Protein" Biochemistry. 36. 9109-9119 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] M.Inoue, Y.Muto, H.Sakamoto, T.Kigawa, K.Takio, Y.Shimura and S.Yokoyama: "A Characteristic Arrangement of Aromatic Amino Acid Residues in the Solution Structure of the Amino-terminal RNA-binding Domain of Drosophila Sex-lethal" J.Mol.Biol.272. 82-94 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] J.Iwahara, T.Kigawa, K.Kitagawa, H.Masumoto, T.Okazaki and S.Yokoyama: "A Helix-turn-helix Structure Unit in Human Centromere Protein B (CENP-B)" EMBO J.17. 827-837 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] T.Yabuki, T.Kigawa, N.Dohmae, K.Takio, T.Terada, Y.Ito, E.D.Laue, J.A.Copper, M.Kainosho, and S.Yokoyama: "Dual Amino Acid-selective and Site-directed Stable-isotope Labeling of the Human c-Ha-Ras Protein by Cell-free Synthesis" J.Biomol.NMR. 11. 295-306 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] T.Kigawa, M.Endo, Y.Ito, M.Shirouzu, A.Kikuchi, and S.Yokoyama: "Solution Structure of the Ras-binding Domain of RGL" FEBS Lett.441. 413-418 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] S.-W.Chi, Y.Muto, M.Inoue, I.Kim, H.Sakamoto, Y.Shimura, S.Yokoyama, B.-S.Choi, and H.Kim: "Chemical Shift Perturbation Studies of the Interactions of the Second RNA-binding Domain of the Drosophila Sex-lethal Protein with the transformer pre-mRNA Polyuridine Tract and 3'Splice Site Sequences" Eur.J.Biochem.259. 1-13 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] T.Terada, Y.Ito, M.Shirouzu, M.Tateno, K.Hashimoto, T.Kigawa, T.Ebisuzaki, K.Takio, T.Shibata, S.Yokoyama, B.O.Smith, E.D.Laue, and J.A.Cooper: "Nuclear Magnetic Resonance and Molecular Dynamics Studies on the Interactions of the Ras-Binding Domain of Raf-1 with the Wild-type and Mutant Ras Proteins" J.Mol.Biol.286. 219-232 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Kigawa,T.: "Solution structure of the Ras-binding domain of RGL" FEBS Lett.441. 413-418 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Chi,Seung-Wook: "Chemical shift perturbation studies of the interactions of the second RNA-binding domain of the drosophila Sex-lethal proteinwith tra PPT and 3'splice-site sequence" Eur.J.Biochemistry. 259. 1-13 (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] Terada,T.: "Nuclear Magnetic Resonance and Molecular Dynamics Studies on the Interactions of the Ras-binding Domain of Raf-1 with Wild-type and Mutant Ras Proteins" J.Mol.Biol.286. 219-232 (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] I.Kim: "NMR analysis of the hydrogen bonding interactions of the RNA-binding domains of the Drosophila Sex-lethal protein with target RNA fragments with site-specofic[3-15N]uridine substinitions" Nucleic Acids Research. 25. 1565-1569 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] M.Inoue: "A Characteristic Arrangement of Aromatic Amino Acid Residues in the Solution Structure of the" J.Mol.Biol.272. 82-94 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Y.Ito: "Regional Polysterism in the GTP-Bound Form of the Human c-Ha-Ras Protein" Biochemistry. 36. 9109-9119 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] J.Iwahara: "A Helix-turn-helix Structure Unit in the Human Centromere Protein B(CENP-B)" EMBO J.17. 827-837 (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] S.Koshiba: "The Solution Structure of the Pleckstrin Homology Domain of Mouse Son-of Sevenless 1(mSosl)" J.Mol.Biol.269. 579-591 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] T.Yabuki: "Dual amino acid-selective and site-directed stable-isotope labeling of the human c-Ha-Ras protein by cell-free synthesis" J.Biomol.NMR.,. (in press). (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] Smith,B.O.: "An approach to global fold determination using limited NMR data from larger proteins selectively protonated at specific residue types" J.Biomol.NMR. 8. 360-368 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Kim,D.-M.: "A highly efficient cell-free protein synthesis system from Escherichia coli" Biochem.239. 881-886 (1996)

    • Related Report
      1996 Annual Research Report

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Published: 1996-04-01   Modified: 2016-04-21  

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