2003 年度実績報告書

リボソーム再生系を標的としたNMRによる相互作用解析法と新規抗菌剤の開発

研究課題

研究課題/領域番号	14370756
研究機関	大阪大学
研究代表者	小林祐次大阪大学, 薬学研究科, 教授 (20127228)
研究分担者	吉田卓也大阪大学, 薬学研究科, 助手 (00294116) 大久保忠恭大阪大学, 薬学研究科, 助教授 (90272997)
キーワード	リボソーム / RRF / NMR / 相互作用 / 抗菌剤 / ドラッグデザイン
研究概要	昨年度本研究によってX線結晶構造解析を用い明らかとなった腸炎ビブリオ薗RRF、大腸菌RRFの立体構造に基き、その阻害剤候補をコンピューターを利用したデータベースからのバーチャルスクリーニングによって選別した。安定同位体標識したRRFと得られたライブラリー分子群との相互作用をNMR(緩和フィルター法と拡散フィルター法による候補分子の選別、及び飽和移動法とHSQC法を用いた結合部位の同定)によって検出することを試みた結果、RRFとの相互作用を示唆する分子が得られた。これらNMRスクリーニング系を評価するため、RRF阻害活性を簡便にin vivo及びin vitroで検出できる系を設計し、実際に使用可能なことを確認した。また、RRF阻害剤の設計に応用することを目的とし、NMRによるRRFの分子内運動の解析を行ったところ、RRFに特異的なドメイン運動がRRFの活性と相関していることを示す結果が得られた。このことはRRFのドメイン運動を抑制する低分子化合物によってRRFを阻害できる可能性を示している。またRRFの変異体を用いた相互作用実験並びにRRF-リボソーム複合体のクライオ電子顕微鏡解析を行い、RRFとリボソームの相互作用部位について多くの知見を得た。さらに、RRFの部分フラグメントとリボソームの相互作用解析を行い、リボソーム結合に必要十分なRRFの構造を明らかにしつつある。

研究成果
(20件)

すべてその他

すべて文献書誌 (20件)

[文献書誌] Nakano, H. et al.: "Crystallization and preliminary X-ray crystallographic studies of a mutant of ribosome recycling factor from Escherichia coli, Arg132G1y"Acta Crystallogr. Sect. D. 58. 124-126 (2002)
[文献書誌] Yoshida, T. et al.: "Backbone NMR assignments of Ribosome Recycling Factors (RRFs) from Escherichia coli and Thermotoga maritime"J.Biomol.NMR. 22. 195-196 (2002)
[文献書誌] Uchiyama, S. et al.: "Thermodynamic characterization of variants of mesophilic cytochrome c and its thermophilic counterpart"Protein Eng.. 15. 455-461 (2002)
[文献書誌] Shimizu, T. et al.: "Interaction among silkworm ribosomal proteins P1, P2 and PO required for functional protein binding to the GTPase-associated domain of 28S rRNA"Nucleic Acids Research. 30. 2620-2627 (2002)
[文献書誌] Hemmi, H. et al.: "Solution Structure of Ascidian Trypsin Inhibitor Determined by Nuclear Magnetic Resonance Spectroscopy"Biochemistry. 41. 10657-10664 (2002)
[文献書誌] Otaka, A. et al.: "Remodeling of gp41-C34 Peptide Leads to Highly Effective Inhibitors of the Fusion of HIV-1 with Target Cells"Angewante Chemie. 41. 2937-2940 (2002)
[文献書誌] Doi, M.et al.: "Simple and efficient syntheses of Boc- and Fmoc-protected 4(R)-and 4(S)-fluoroproline solely from 4(R)-hydroxyproline"Tetrahedron. 58. 8453-8459 (2002)
[文献書誌] Yamamoto, Y. et al.: "Influence of Amino Acid Side Chain Packing on Fe-Methionine Coordination in Thermostable Cytochrome c"J.Am.Chem.Soc.. 124. 11574-11575 (2002)
[文献書誌] Okajima, T. et al.: "Novel Trimeric Adenylate Kinase from an Extremely Thermoacidophilic Archaeon, Sulfolobus solfataricus : Molecular Cloning, Nucleotide Sequencing, Expression in Escherichia coli, and Characterization of the Recombinant Enzyme"Biosci.Biotechnol.Biochem.. 66. 2112-2124 (2002)
[文献書誌] Yamamoto, Y. et al.: "Influence of Amino Acid Side Chain Packing on Fe-Methionine Coordination in Thermostable Cytochrome c"J.Am.Chem.Soc.. 124. 11574-11575 (2002)
[文献書誌] Sambongi, Y.: "Cytochrome c from a thermophilic bacterium has provided insights into the mechanisms of protein maturation, folding, and stability"Eur.J.Biochem.. 269. 3355-3361 (2002)
[文献書誌] Nakano, H. et al.: "Structure and Binding Mode of a Ribosome Recycling Factor (RRF) from Mesophilic Bacterium"J.Biol.Chem.. 278. 3427-3436 (2003)
[文献書誌] Minamino, T. et al.: "Effect of Intracellular pH on Rotational Speed of Bacterial Flagellar Motors"J.Bacteriol.. 185. 1190-1194 (2003)
[文献書誌] Yoshida, T. et al.: "Characteristic domain motion in the ribosome recycling factor revealed by 15N NMR relaxation experiments and molecular dynamics simulations"Biochemistry. 42. 4101-4107 (2003)
[文献書誌] Hemmi, H. et al.: "Inhibittory Specificity Change of the Ovomucoid Third Domain of the Silver Pheasant upon Introduction of an Engineered Cys14-Cys39 Bond"Biochemistry. 42. 2524-2534 (2003)
[文献書誌] Doi, M.et al.: "Characterization of Collagen Model Peptides Containing 4-Fluoroproline ; (4(S)-Fluoroproline-Pro-Gly)10 Forms a Triple Helix, but (4(R)-Fluoroproline-Pro-Gly)10 Dose Not"J.Am.Chem.Soc.. 125. 9922-9923 (2003)
[文献書誌] Terui, N. et al.: "Relationship between Redox Function and Protein Stability of Cytochromes c"J.Am.Chem.Soc.. 125. 13650-13651 (2003)
[文献書誌] Ohwada, T. et al.: "α,α-Disubstituted Glycines Bearing a Large Hydrocarbon Ring Peptide Self-Assembly through Hydrophobic Recognition"Chem.Eur.J.. 10. 617-626 (2003)
[文献書誌] Takashima, K. et al.: "Distribution Computing and NMR Constraint Based High Resolution Structure Determination: Applied for Bioactive Peptide Endothelin-1 to Determine C-terminal Folding"Chem.Eur.J.. (in press). (2004)
[文献書誌] Takinowaki, H. et al.: "1H, 13C and 15N resonance assignments of the N-terminal 16kDa domain of Escherichia coli Ada protein."J.Biomol.NMR. (in press). (2004)

2003 年度 実績報告書

リボソーム再生系を標的としたNMRによる相互作用解析法と新規抗菌剤の開発

研究代表者

小林 祐次 大阪大学, 薬学研究科, 教授 (20127228)

研究成果

[文献書誌] Nakano, H. et al.: "Crystallization and preliminary X-ray crystallographic studies of a mutant of ribosome recycling factor from Escherichia coli, Arg132G1y"Acta Crystallogr. Sect. D. 58. 124-126 (2002)

[文献書誌] Yoshida, T. et al.: "Backbone NMR assignments of Ribosome Recycling Factors (RRFs) from Escherichia coli and Thermotoga maritime"J.Biomol.NMR. 22. 195-196 (2002)

[文献書誌] Uchiyama, S. et al.: "Thermodynamic characterization of variants of mesophilic cytochrome c and its thermophilic counterpart"Protein Eng.. 15. 455-461 (2002)

[文献書誌] Shimizu, T. et al.: "Interaction among silkworm ribosomal proteins P1, P2 and PO required for functional protein binding to the GTPase-associated domain of 28S rRNA"Nucleic Acids Research. 30. 2620-2627 (2002)

[文献書誌] Hemmi, H. et al.: "Solution Structure of Ascidian Trypsin Inhibitor Determined by Nuclear Magnetic Resonance Spectroscopy"Biochemistry. 41. 10657-10664 (2002)

[文献書誌] Otaka, A. et al.: "Remodeling of gp41-C34 Peptide Leads to Highly Effective Inhibitors of the Fusion of HIV-1 with Target Cells"Angewante Chemie. 41. 2937-2940 (2002)

[文献書誌] Doi, M.et al.: "Simple and efficient syntheses of Boc- and Fmoc-protected 4(R)-and 4(S)-fluoroproline solely from 4(R)-hydroxyproline"Tetrahedron. 58. 8453-8459 (2002)

[文献書誌] Yamamoto, Y. et al.: "Influence of Amino Acid Side Chain Packing on Fe-Methionine Coordination in Thermostable Cytochrome c"J.Am.Chem.Soc.. 124. 11574-11575 (2002)

[文献書誌] Yamamoto, Y. et al.: "Influence of Amino Acid Side Chain Packing on Fe-Methionine Coordination in Thermostable Cytochrome c"J.Am.Chem.Soc.. 124. 11574-11575 (2002)

[文献書誌] Sambongi, Y.: "Cytochrome c from a thermophilic bacterium has provided insights into the mechanisms of protein maturation, folding, and stability"Eur.J.Biochem.. 269. 3355-3361 (2002)

[文献書誌] Nakano, H. et al.: "Structure and Binding Mode of a Ribosome Recycling Factor (RRF) from Mesophilic Bacterium"J.Biol.Chem.. 278. 3427-3436 (2003)

[文献書誌] Minamino, T. et al.: "Effect of Intracellular pH on Rotational Speed of Bacterial Flagellar Motors"J.Bacteriol.. 185. 1190-1194 (2003)

[文献書誌] Yoshida, T. et al.: "Characteristic domain motion in the ribosome recycling factor revealed by 15N NMR relaxation experiments and molecular dynamics simulations"Biochemistry. 42. 4101-4107 (2003)

[文献書誌] Hemmi, H. et al.: "Inhibittory Specificity Change of the Ovomucoid Third Domain of the Silver Pheasant upon Introduction of an Engineered Cys14-Cys39 Bond"Biochemistry. 42. 2524-2534 (2003)

[文献書誌] Doi, M.et al.: "Characterization of Collagen Model Peptides Containing 4-Fluoroproline ; (4(S)-Fluoroproline-Pro-Gly)10 Forms a Triple Helix, but (4(R)-Fluoroproline-Pro-Gly)10 Dose Not"J.Am.Chem.Soc.. 125. 9922-9923 (2003)

[文献書誌] Terui, N. et al.: "Relationship between Redox Function and Protein Stability of Cytochromes c"J.Am.Chem.Soc.. 125. 13650-13651 (2003)

[文献書誌] Ohwada, T. et al.: "α,α-Disubstituted Glycines Bearing a Large Hydrocarbon Ring Peptide Self-Assembly through Hydrophobic Recognition"Chem.Eur.J.. 10. 617-626 (2003)

[文献書誌] Takashima, K. et al.: "Distribution Computing and NMR Constraint Based High Resolution Structure Determination: Applied for Bioactive Peptide Endothelin-1 to Determine C-terminal Folding"Chem.Eur.J.. (in press). (2004)

[文献書誌] Takinowaki, H. et al.: "1H, 13C and 15N resonance assignments of the N-terminal 16kDa domain of Escherichia coli Ada protein."J.Biomol.NMR. (in press). (2004)

2003 年度実績報告書

小林祐次大阪大学, 薬学研究科, 教授 (20127228)