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AnaIysis of dynamic behavior of ribonuclease upon ligand binding using high resolution NMR

Research Project

Project/Area Number 12672088
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Physical pharmacy
Research InstitutionKyushu University

Principal Investigator

UEDA Tadashi  Kyushu Univ., Grad. School Pharm. Sci., Assoc. Prof., 薬学研究院, 助教授 (90184928)

Co-Investigator(Kenkyū-buntansha) ABE Yoshito  Kyushu Univ., Grad. School Pharm. Sci., Assistant. Prof., 薬学研究院, 助手 (60315091)
HASHIMOTO Yoshio  Kyushu Univ., Grad. School Pharm. Sci., Assistant. Prof., 薬学研究院, 助手 (50253472)
IMOTO Taiji  Kyushu Univ., Grad. School Pharm. Sci., Prof., 薬学研究院, 教授 (90038282)
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: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2000: ¥3,000,000 (Direct Cost: ¥3,000,000)
KeywordsNMR / Pichia pastoris / order parameter / ribonulease / ^<15>N labeling / induced fit / 酵素誘導適合 / コドンユーセージ / ヒトリボクレアーゼ3 / Pichia pastoris
Research Abstract

The idea that the internal motions in enzymes were restricted upon ligand binding has been accepted. Recently, it was reported that internal motions in some enzymes such 4-oxalocrotonate tautomerase, hen and human lysozymes increased upon ligand binding. Now, it is controversial whether internal motions in enzymes increase or not upon ligand binding. Therefore, in this research, in order elucidate whether the increased internal motions in enzymes upon binding its ligand is in general or not, we prepared ^<15>N uniformly labeled ribonuclease T1 from Pichia pastoris and measured the relaxation time (T_1 and T_2) of nitrogen atoms and NOEs between ^1H and ^<15>N in them in the presence or absence of 3'-GMP. Order parameters in every residues of ^<15>N uniformly labeled ribonuclease T1 was calculated by model free analysis, of the relaxation time (T_1 and T_2) of nitrogen atoms and NOEs between ^1H and ^<15>N. As the results, it was elucidated that some residues in ribonuclease T1 had the smaller order parameters, indicating that the internal motions in ribonuclease T1 molecule increased upon binding its ligand.

Report

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

    (13 results)

All Other

All Publications (13 results)

  • [Publications] Mine S, Ueda T et al.: "Analysis of the internal motion of free and ligand-bound human lysozyme by use of N-15 NMR relaxation measurement : A comparison with those of hen lysozyme"Protein Science. 9. 1669-1684 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Obita T, Ueda T et al.: "Assignment of ^1H and ^<15>N resonances of mouse lysozyme M"J. Biomolecular NMR. 18. 361-362 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Ueda T et al.: "Aggregation and chemical reaction in hen lysozyme caused by heating at pH 6 are depressed by osmolytes, sucrose and trehalose"J. Biochem.. 130. 491-496 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Ohmura T, Ueda T et al.: "Stabilization of hen egg white Iysozyme by a cavity-filling mutation"Protein Science. 10. 313-320 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Mine S. Ueda T. Hashimoto Y. Imoto T.: "Analysis of the internal motion of free and ligand-bound human lysozyme by use of N-15 NMR relaxation measurement: A comparison with those of hen lysozyme"Protein Sci. 9. 1669-1684 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Obita T. Ueda T. Tanaka Y. Hashimoto Y. Imoto T.: "Assignment of ^1H and ^<15>N resonances of mouse lysozyme M"J. Biomol. NMR. 18. 361-362 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Ohmura T. Ueda T. Ootsuka K. Saito M. Imoto T.: "Stabilization of hen egg white lysozyme by a cavity-filling mutation"Protein Sci.. 10. 313-320 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] UedaT. Nagata M. ImotoT.: "Aggregation and chemical reaction in hen lysozyme caused by heating at pH 6 are depressed by osmolytes, sucrose and trehalose"J. Biochem.. 130. 491-496 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Ohmura T., Ueda T., Hashimoto Y., Imoto T.: "Tolerance of point substitution of methionine for isoleucine in hen egg white lysozyme"Protein Eng.. 14・6. 421-425 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Ueda T., Nagata M., Imoto T.: "Aggregation and chemical reaction in hen lysozyme caused by heating at pH 6 are depressed by osmolytes, sucrose and trehalose"J. Biochem.. 130・4. 491-496 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Ohkuri T., Ueda T., Tsurumaru M., Imoto T.: "Evidence for an initiation site for hen lysozyme folding from reduced form using its dissected peptide fragments"Protein Eng.. 14・11. 829-833 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Mine et al.: "Analysis of the internal motion of free and ligand-bound human lysozyme by use of N-15 NMR relaxation measurement : A comparison with those of hen lysozyme"Protein Science. 9・9. 1669-1684 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Obita et al.: "Assignment of ^1H and ^<15>N resonances of mouse lysozyme M"Journal of Biomolecular NMR. 18・4. 361-362 (2000)

    • Related Report
      2000 Annual Research Report

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

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