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1998 Fiscal Year Final Research Report Summary

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)
Project Period (FY) 1996 – 1998
KeywordsNMR / protein / RNA / stable-isotope labeling / selective labeling / deuteration / tertiary structure / cell-free protein synthesis
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.

  • Research Products

    (22 results)

All Other

All Publications (22 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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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
  • [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

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Published: 1999-12-08  

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