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DESIGN OF SEQUENSE-SELECTIVE DNA-BINDING SMALL PROTEINS

Research Project

Project/Area Number 12680590
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Bioorganic chemistry
Research InstitutionKYOTO UNIVERSITY

Principal Investigator

MORII Takashi  INST. OF ADVANCED ENERGY, KYOTO UNIV., RES.ASSIST., エネルギー理工学研究所, 助手 (90222348)

Co-Investigator(Kenkyū-buntansha) MAKINO Keisuke  INT. INNOVETION CENTER, KYOTO UNIV., PROFESSOR, 国際融合創造センター, 教授 (50159141)
Project Period (FY) 2000 – 2001
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2000: ¥2,500,000 (Direct Cost: ¥2,500,000)
KeywordsDNA binding / DNA RECOGNITION / MOLECULAR RECOGNITION / ALPHA HELIX / DIMERIZATION / STRUCTURE-BASED DESIGN OF PROTEIN / METAL ION / FOLDING / タンパク質デザイン / 高次構造 / ロイシンジッパー
Research Abstract

We have employed a structure-based design to construct a small folding domain from the F-actin bundling protein villin that contains amino acids necessary for the DNA binding of the basic leucine zipper protein GCN4, and have compared its DNA binding with GCN4. The monomeric motif folds into a stable domain, and binds DNA in a rigid-body mechanism, while its affinity is not higher than that of the basic region peptide. Addition of the leucine zipper region to the folded domain restored its sequence-specific DNA binding comparable to that of GCN4. Unlike the monomeric folded domain, its leucine zipper derivative undergoes a conformational change upon the DNA binding. CD spectral and thermodynamic studies indicate that the DNA -contacting region is folded in the presence or absence of DNA, and suggest that the junction between the DNA-contacting and the leucine zipper regions transits to a helix in the presence of DNA. These results demonstrate that the structural transition outside the direct-contacting region, which adjusts the precise location of the DNA-contacting region, plays a critical role in the specific complex formation of the basic leucine zipper proteins.
Another design strategy utilized a well-folded small domain of C2H2 zinc finger motif as scaffold for the DNA binding α-helix. Appropriate substitution of the α-helical portion of the C2H2 zinc finger motif with amino acid residues necessary for the sequence-selective binding of GCN4 would afford a novel DNA binding domain with a folded structure. Studies on struacural aspects and the sequence-specific DNA binding of the novel protein in the presence of various metal ions are currently underway.

Report

(3 results)
  • 2001 Annual Research Report   Final Research Report Summary
  • 2000 Annual Research Report
  • Research Products

    (20 results)

All Other

All Publications (20 results)

  • [Publications] S.Sato, K.Makino, T.Morii: "DNA Binding of a Basic Leucine-Zipper Protein with Novel Folding Domain"Nucleic Acids Res. Symp. Ser.. 44. 13-14 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] M.Hagihara, T.Morii, K.: "Recognition of small molecules by a ribonucleopeptide"Nucleic Acids Res. Suppl.. 1. 7-8 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T.Morii, T.Tanaka, S.Sato, M.Hagihara, Y.Aizawa, K.Maino: "A General Strategy to Determine a Target DNA Sequence of Short Peptide : Application to a D-Peptide"J. Am. Chem. Soc.. 124. 180-181 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T.Morii, K.Sugimoto, K.Makino, M.Otsuka.K.Imoto, Y.Mori: "A New Fluorescent Biosensor for Inositol Trisphosphate"J. Am. Chem. Soc.. 124. 1139-1140 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T.Morii, S.Sato, M.Hagihara, Y.Mori, K.Imoto, K.Makino: "Structure-Based Design of a Leucine Zipper Protein with New DNA Contacting Region"Biochemistry. 41. 2177-2183 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T.Morii, M.Hagihara, S.Sato, K.Makino: "In Vitro Selection of ATP-binding Receptors Using a Ribonucleopeptide Complex"J. Am. Chem. Soc.. 124(印刷中). (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] S. Sato, K. Makino, and T. Morii: "DNA Binding of a Basic Leucine-Zipper Protein with Novel Folding Domain"Nucleic Acids Res. Symp. Ser.. 44. 13-14 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] M. Hagihara, T. Morii and K. Makino: "Recognition of small molecules by a ribonucleopeptide"Nucleic Acids Res. Suppl.. 1. 7-8 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T. Morii, T. Tanaka, S. Sato, M. Hagihara, Y. Aizawa and K. Makino: "A General Strategy to Determine a Target DNA Sequence of Short Peptide : Application to a D-Peptide"J. Am. Chem. Soc.. 124. 180-181 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T. Morii, K. Sugimoto, K. Makino, M. Otsuka, K. Imoto, and Y. Mori: "A New Fluorescent Biosensor for Inositol Trisphosphate"J. Am. Chem. Soc.. 124. 1139-1140 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T. Morii, S. Sato. M. Hagihara, Y. Mori, K. Imoto and K. Makino: "Structure-Based Design of a Leucine Zipper Protein with New DNA Contacting Region"Biochemistry. 41. 2177-2183 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T. Morii, M. Hagihara, S. Sato and K. Makino: "In Vitro Selection of ATP-binding Receptors Using a Ribonucleopeptide Complex"J. Am. Chem. Soc.. 124 (in press). (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] S.Sato, K.Makino, T.Morii: "DNA Binding of a Basic Leucine-Zipper Protein with Novel Folding Domain"Nucleic Acids Res. Symp. Ser.. 44. 13-14 (2000)

    • Related Report
      2001 Annual Research Report
  • [Publications] M.Hagihara, T.Morii, K.Makino: "Recognition of small molecules by a ribonucleopeptide"Nucleic Acids Res. Suppl.. 1. 7-8 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] T.Morii, T.Tanaka, S.Sato, M.Hagihara, Y.Aizawa, K.Makino: "A General Strategy to Determine a Target DNA Sequence of Short Peptide : Application to a D-Peptide"J. Am. Chem. Soc.. 124. 180-181 (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] T.Morii, K.Sugimoto, K.Makino, M.Otsuka, K.Imoto, Y.Mori: "A New Fluorescent Biosensor for Inositol Trisphosphate"J. Am. Chem. Soc.. 124. 1139-1140 (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] T.Morii, S.Sato, M.Hagihara, Y.Mori, K.Imoto, K.Makino: "Structure-Based Design of a Leucine Zipper Protein with New DNA Contacting Region"Biochemistry. 41. 2177-2183 (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] T.Morii, M.Hagihara, S.Sato, K.Makino: "In Vitro Selection of ATP-binding Receptors Using a Ribonucleopeptide Complex"J. Am. Chem. Soc.. 124(印刷中). (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] S.Sato,K.Makino,T.Morii: "DNA Binding of a Basic Leucine-Zipper Protein with Novel Folding Domain "Nucleic Acids Res.Symp.Ser.. 44. 13-14 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] T.Morii: "Design of Sequence Specific DNA Binding Peptide Dimers."Protein Science. 9. 148-148 (2000)

    • Related Report
      2000 Annual Research Report

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Published: 2000-04-01   Modified: 2016-04-21  

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