Development of an NMR strategy for the structural analysis of themolecular recognition in the membrane protein complex in lipidbilayers

• Project/Area Number: 19590037
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Physical pharmacy
• Research Institution: The University of Tokyo
• Principal Investigator: OSAWA Masanori The University of Tokyo, 大学院・薬学系研究科, 助教 (60361606)
• Co-Investigator(Kenkyū-buntansha): SHIMADA Ichio 東京大学, 大学院・薬学系研究科, 教授 (70196476)
• Project Period (FY): 2007 – 2008
• Project Status: Completed (Fiscal Year 2008)
• Budget Amount: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2008: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000); Fiscal Year 2007: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
• Keywords: 膜蛋白質 / 分子認識 / NMR / 構造生物学 / 相互作用解析

Research Abstract

脂質二重膜中の膜蛋白質はビーズに固定化することにより、安定性が劇的に向上するが、不溶性成分の共存によりNMRシグナルの広幅化・感度の低下が問題となっていた。そこで、マジック角回転(MAS)による高感度化・高分解能化を目指した。
本研究では、高速MASに耐えることができ、広い固・液界面が確保可能な固定化担体を幅広く比較検討した結果、CM-シリカビーズを見出した。
次に、ユビキチン(Ub)と酵母Ub水酸化酵素(YUH)の相互作用系において、YUHをCMシリカビーズに固定化し、不均一系超分子を模倣した系を作成した。このYUH固定化Zn-NTAシリカビーズを均一2H15N標識したUbの溶液に懸濁し、MAS 条件下で転移交差飽和法の測定を試みたところ、従来法では解析が困難であった固体と液体が共存した超分子系において、Ub 上のYUH 結合界面残基の同定に成功した。

Research Products

• [Journal Article] Backbone resonance assignment for the cytoplasmic regions of G protein-activated inwardly rectifying potassium channel 1(GIRKl) (2009)
  • Author(s): Yokogawa M.
  • Journal Title: Biomolecular NMR Assiglnments (In press)
  • Peer Reviewed
• [Journal Article] Stable isotope labeling of proteinby Kluyveromyces lactis for NMR study (2008)
  • Author(s): Sugiki T., Shimada I., Takahashi H.
  • Journal Title: J Biomol NMR 42 Pages: 159-162
  • Peer Reviewed
• [Journal Article] Carbohydrate bindingmechanism of the macrophagegalactose-type C-type lectin 1 revealedby saturation transfer experiments (2008)
  • Author(s): Sakakura M., Oo-Puthinan S., MoriyamaC., Kimura T., Moriya J., Irimura T., Shimada I.
  • Journal Title: J Biol Chem 283 Pages: 33665-33673
  • Peer Reviewed
• [Journal Article] Chondroitin sulfate E fragmentsenhance CD44 cleavage andCD44-dependent motility in tumor cells (2008)
  • Author(s): Sugahara K. N., Hirata T., Tanaka T., Ogino S., Takeda M., Terasawa H., Shimada I, Tamura J, ten Dam GB, vanKuppevelt TH, Miyasaka M.
  • Journal Title: Cancer Res. 68 Pages: 7191-7199
  • Peer Reviewed
• [Journal Article] Amino acidselective cross-saturation method foridentification of proximal residue pairsin a protein-protein complex (2008)
  • Author(s): Igarashi S., Osawa M., Takeuchi K., Ozawa S., Shimada I.
  • Journal Title: J Am Chem Soc. 130 Pages: 12168-12176
  • Peer Reviewed
• [Journal Article] Stable isotope labeling of protein by Kluyveromyces lactis for NMR study. (2008)
  • Author(s): Sugiki T.
  • Journal Title: J Biomol NMR. 42 Pages: 159-162
  • Peer Reviewed
• [Journal Article] Carbohydrate binding mechanism of the macrophage galactose-type C-type lectin 1 revealed by saturation tranfer experiments. (2008)
  • Author(s): Sakakura M.
  • Journal Title: J Biol Chem. 238 Pages: 33665-33673
  • Peer Reviewed
• [Journal Article] Chondroitin sulfate E fragments enhance CD44 cleavage and CD44-dependent motility in tumor cells. (2008)
  • Author(s): Sugahara K. N.
  • Journal Title: Cancer Res. 68 Pages: 7191-7199
  • Peer Reviewed
• [Journal Article] Amino acid selective cross-saturation method for identification of proximal residue pairs in a protein-protein complex (2008)
  • Author(s): Igarashi S.
  • Journal Title: J. Am. Chem. Soc. 130 Pages: 12168-12176
  • Peer Reviewed
• [Journal Article] The amino acids involved in the distinct carbohydrate specific ities between macrophage galactose-type C-type lectins 1 and 2(CD301a and b)of mice (2008)
  • Author(s): Oo-puthinan S
  • Journal Title: Biochim Biophys Acta 1780 Pages: 133-147
  • Peer Reviewed
• [Journal Article] Structural insights into activation ofphosphatidylinositol 4-kinase (Pik1) byyeast frequenin (Frq1) (2007)
  • Author(s): Strahl T, Huttner IG, Lusin JD, OsawaM, King D, Thorner J, Ames JB.
  • Journal Title: J Biol Chem. 282(42) Pages: 30949-59
  • Peer Reviewed
• [Journal Article] Structuralbasis of the collagen-binding mode ofdiscoidin domain receptor 2 (2007)
  • Author(s): Ichikawa O, Osawa M, Nishida N,Goshima N, and Shimada I.
  • Journal Title: EMBO J 26 Pages: 4168-4176
• [Journal Article] Mechanism of mRNA deadenylation : evidence for a molecular interplaybetween translation termination factoreRF3 and mRNA deadenylases (2007)
  • Author(s): Funakoshi Y, Doi Y, Hosoda N, Uchida N,Osawa M, Shimada I, Tsujimoto M,Suzuki T, Katada T, Hoshino S.
  • Journal Title: Genes Dev. 21(23) Pages: 3135-48
  • Peer Reviewed
• [Journal Article] Nickel binding to NikA : anadditional binding site reconcilesspectroscopy, calorimetry andcrystallography (2007)
  • Author(s): Addy C, Ohara M, Kawai F, Kidera A,Ikeguchi M, Fuchigami S, Osawa M,Shimada I, Park SY, Tame JR andHeddle JG.
  • Journal Title: Acta Crystallogr D BiolCrystallogr 63 Pages: 221-229
  • Peer Reviewed
• [Journal Article] Bovin NV and Irimura T. The aminoacids involved in the distinctcarbohydrate specificities betweenmacrophage galactose-type C-typelectins 1 and 2 (CD301a and b) of mice (2007)
  • Author(s): Oo-puthinan S, Maenuma K, SakakuraM, Denda-Nagai K, Tsuiji M, Shimada I, Nakamura-Tsuruta S, and Hirabayashi S.
  • Journal Title: Biochim Biophys Acta 1780(2) Pages: 89-100
  • Peer Reviewed
• [Journal Article] Structural basisof the transition-state stabilization inantibody-catalyzed hydrolysis (2007)
  • Author(s): Sakakura M, Takahashi H, Shimba N,Fujii I, and Shimada I.
  • Journal Title: J Mol Biol 367 Pages: 133-147
  • Peer Reviewed
• [Journal Article] PairwiseNMR Experiments for theDetermination of Protein BackboneDihedral Angle Based onCross-Correlated Spin Relaxation. (2007)
  • Author(s): Takahashi H and Shimada I.
  • Journal Title: JBiomol NMR 37 Pages: 179-185
  • Peer Reviewed
• [Journal Article] Backboneresonance assignment for the outermembrane lipoprotein receptor LolBfrom Escherichia coli (2007)
  • Author(s): Nakada S, Sakakura M, Takahashi H,Tokuda H and Shimada I.
  • Journal Title: BiomolecularNMR Assignments 1 Pages: 121-123
  • Peer Reviewed
• [Journal Article] Backboneresonance assignments for theperiplasmic chaperone LolA fromEscherichia coli (2007)
  • Author(s): Nakada S, Takahashi H, Sakakura M,Kurono M and Shimada I.
  • Journal Title: Biomolecular NMRAssignments 1 Pages: 125-127
  • Peer Reviewed
• [Journal Article] Structural insights into activation of phosphatidylinositol 4-kinase(Pikl) by yeast frequenin(Frql). (2007)
  • Author(s): Strahl T.
  • Journal Title: J Biol Chem. 282 Pages: 30949-30959
  • Peer Reviewed
• [Journal Article] Structural basis of the collagen-binding mose of discoidin domain receptor 2 (2007)
  • Author(s): Ichikawa O.
  • Journal Title: EMBO J. 26 Pages: 4168-4176
  • Peer Reviewed
• [Journal Article] Mechanism of mRNA deadenylation: evidence for a molecular interplay between translation termination factor eRF3 and mRNA deadenylases. (2007)
  • Author(s): Funakoshi Y
  • Journal Title: Genes Dev. 21 Pages: 3135-3148
  • Peer Reviewed
• [Journal Article] Nickl binding to NikA: an additional binding site reconciles spectroscopy. calorimetry and crystallography (2007)
  • Author(s): Addy C
  • Journal Title: Acta Crystallogr D Biol Crystallogr. 63 Pages: 221-229
  • Peer Reviewed
• [Journal Article] Pairwise NMR Experiments for the Determination of Protein Backbone Dihedral Angle Based on Cross-Correlated Spin Relaxation (2007)
  • Author(s): Takahashi H
  • Journal Title: J Biomol NMR 37 Pages: 179-185
  • Peer Reviewed
• [Journal Article] Backbone resonance assignment for the outer membrane lipop rotein receptor LoIB from Escherichia coli. (2007)
  • Author(s): Nakada S
  • Journal Title: Biomolecular NMR Assignments 1 Pages: 121-123
  • Peer Reviewed
• [Journal Article] Backbone resonance assignments for the periplasmic chaperone LolA from Escherichia coli. (2007)
  • Author(s): Nakada S
  • Journal Title: Biomolecular NMR Assignments 1 Pages: 125-127
  • Peer Reviewed
• [Journal Article] Structural basis of the transition-state stabilization in antibody-catalyzed hydrolysis. (2007)
  • Author(s): Sakakura M
  • Journal Title: J Mol Biol. 367 Pages: 133-147
  • Peer Reviewed
• [Presentation] 生体超分子における分子認識機構解明に向けたHR-MASの適用 (2008)
  • Author(s): 豊永 翔
  • Organizer: 第47回NMR 討論会
  • Place of Presentation: 茨城県つくば市
  • Year and Date: 2008-11-13
• [Presentation] 巨大タンパク質複合体のモデル構築を目的とした残基選択的交差飽和法の開発 (2008)
  • Author(s): 小澤新一郎
  • Organizer: 第47回NMR 討論会
  • Place of Presentation: 茨城県つくば市
  • Year and Date: 2008-11-13
• [Presentation] 生体超分子における分子認識機構解明に向けたHR-MASの適用 (2008)
  • Author(s): 豊永翔
  • Organizer: 第47回NMR討論会
  • Place of Presentation: 茨城県つくば市
  • Year and Date: 2008-11-13
• [Presentation] 巨大タンパク質複合体のモデル構築を目的とした残基選択的交差飽和法の開発 (2008)
  • Author(s): 小澤新一郎
  • Organizer: 第47回NMR討論会
  • Place of Presentation: 茨城県つくば市
  • Year and Date: 2008-11-13
• [Presentation] Structural studies onthe interaction of spermine withinwardly rectifying potassium channel (2008)
  • Author(s): Osawa M. et.al.
  • Organizer: XXIIIrd International Conference onMagnetic Resonance in BiologicalSystems
  • Place of Presentation: San Diego, CA, USA
  • Year and Date: 2008-08-24
• [Presentation] Structural basis of the gating of G protein-activatedinwardly rectifying potassium channelby Gbg (2008)
  • Author(s): Yokogawa M. et.al.
  • Organizer: XXIIIrd InternationalConference on Magnetic Resonance in Biological Systems
  • Place of Presentation: San Diego, CA, USA
  • Year and Date: 2008-08-24
• [Presentation] Structura .studies on the interaction of sperrmine with inwardly rectifying potassium channel (2008)
  • Author(s): Osawa M.
  • Organizer: XXIIIrd International Conference on Magnetic Resonance in Biological Systems
  • Place of Presentation: San Diego, CA, USA
  • Year and Date: 2008-08-24
• [Presentation] Structural basis of the gating of G protein-activated inwafdly rectifying potassium channel by Gbg (2008)
  • Author(s): Yokogawa M.
  • Organizer: XXIIIrd International Conference on Magnetic Resonance in Biological Systems
  • Place of Presentation: San Diego, CA, USA
  • Year and Date: 2008-08-24
• [Presentation] HR-MASを用いた生体超分子における分子認識機構を解明する新規NMR手法の開発 (2007)
  • Author(s): 豊永 翔
  • Organizer: 第46回NMR討論会
  • Place of Presentation: 北海道札幌市
  • Year and Date: 2007-09-10
• [Book] NMRによるソフトな分子間相互作用解析法の開発 (2008)
  • Author(s): 松本昌彦、上田卓見、嶋田一夫
  • Publisher: 生化学
• [Book] コラーゲン結合タンパク質を介した生命プロセスの活性化機構 (2008)
  • Author(s): 西田紀貴、嶋田一夫
  • Publisher: 生化学
• [Book] 「等温滴定型カロリメトリー(ITC)-熱量測定による相互作用の定量的解析-」実験医学別冊「生命科学のための機器分析実験ハンドブック (2007)
  • Author(s): 大澤匡範、嶋田一夫
  • Publisher: 羊土社
• [Book] 交換系利用NMRによるリガンド分子の標的分子結合状態の構造解析 高橋栄夫・嶋田一夫 (2007)
  • Author(s): 高橋栄夫、嶋田一夫
  • Publisher: 蛋白質・核酸・酵素
• [Book] 実験医学別冊「生命科学のための機器分析実験ハンドブック」 (2007)
  • Author(s): 大澤匡範
  • Publisher: 羊土社
• [Book] 蛋白質・核酸・酵素 (2007)
  • Author(s): 高橋栄夫
  • Publisher: 共立出版