高分子量蛋白質複合体の NMR 構造解析法の開発

2015 Fiscal Year Annual Research Report

• Project/Area Number: 26650018
• Research Institution: Osaka University
• Principal Investigator: 児嶋 長次郎 大阪大学, たんぱく質研究所, 准教授 (50333563)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Keywords: NMR / 蛋白質 / 立体構造

Outline of Annual Research Achievements

溶液NMRによる蛋白質の立体構造解析には分子量上限が存在し、分子量10万以上の蛋白質で構造決定に成功した例は無い。そこで本研究では分子量10万以上の蛋白質の立体構造決定を可能にする新規NMR構造解析法の開発を目的とする。具体的には、高感度13C直接観測法および13C観測1H常磁性緩和促進法を開発し、固体NMRで成功している13Cの帰属法や構造計算法を溶液NMR用に最適化する。最終的には、開発した手法で分子量約11万の「花成ホルモン/受容体/転写因子/DNA」４者複合体の構造解析を行い、花成ホルモンによる花成誘導機構の解明を目指す。
平成２７年度は、平成２６年度から引き続き、(1)高感度13C直接観測法の開発、(2)13C観測1H常磁性緩和促進法、13C帰属法、構造計算法の開発、(3)花成ホルモン受容体の構造解析、に取り組むとともに、(4)花成ホルモン受容体転写因子DNA４者複合体の立体構造解析、にも取り組んだ。具体的には、平成２６年度に開発に成功している１回の測定で２回データを取り込むDouble- acquisition法と13C直接観測と組み合わせて信号強度を２～４倍に増強する測定法など、最先端のNMR構造解析手法を花成ホルモン受容体および花成ホルモン受容体転写因子DNA４者複合体に適用した。その結果、花成ホルモン受容体では多くの主鎖信号の帰属に成功し、低分解能構造を得ることに成功した。４者複合体では信号の消失が激しく信号帰属には至らなかったものの、X線溶液散乱法など他の手法によって構造解析を進めることができた。

Research Products

• [Int'l Joint Research] IOCB AS CR(チェコ)
  • Country Name: CZECH
  • Counterpart Institution: IOCB AS CR
• [Journal Article] 1H, 15N and 13C resonance assignments of the conserved region in the middle domain of S. pombe Sin1 protein (2015)
  • Author(s): Saori Kataoka, Kyoko Furuita, Yoshikazu Hattori, Naohiro Kobayashi, Takahisa Ikegami, Kazuhiro Shiozaki, Toshimichi Fujiwara, and Chojiro Kojima
  • Journal Title: Biomol. NMR Assign. Volume: 9 Pages: 89-92
  • DOI: 10.1007/s12104-014-9550-6
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Direct Detection of the Mercury-Nitrogen Bond in the Thymine-HgII-Thymine Base-pair with 199Hg NMR Spectroscopy (2015)
  • Author(s): Takenori Dairaku, Kyoko Furuita, Hajime Sato, Jakub Sebera, Daichi Yamanaka, Hiroyuki Otaki, Shoko Kikkawa, Yoshinori Kondo, Ritsuko Katahira, F. Matthias Bickelhaupt, Celia Fonseca Guerra, Akira Ono, Vladimir Sychrovsky, Chojiro Kojima, and Yoshiyuki Tanaka
  • Journal Title: Chem. Commun. Volume: 51 Pages: 8488-8491
  • DOI: 10.1039/c5cc02423d
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Physicochemical natures of interfaces control activity of ferredoxin NADP+ reductase through its interprotein interactions with ferredoxin (2015)
  • Author(s): Misaki Kinoshita, Ju yaen Kim, Satoshi Kume, Yukiko Sakakibara, Toshihiko Sugiki, Chojiro Kojima, Genji Kurisu, Takahisa Ikegami, Toshiharu Hase, Yoko Kimata-Ariga, and Young-Ho Lee
  • Journal Title: BBA - Bioenergetics Volume: 1847 Pages: 1200-1211
  • DOI: 10.1016/j.bbabio.2015.05.023
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Exploring a DNA sequence for 3-dimensional structure determination of a silver(I)-mediated C-C base pair in a DNA duplex with 1H NMR spectroscopy (2015)
  • Author(s): Takenori Dairaku, Kyoko Furuita, Hajime Sato, Yoshinori Kondo, Chojiro Kojima, Akira Ono, and Yoshiyuki Tanaka
  • Journal Title: Nucleosides Nucleotides Nucleic Acids Volume: 34 Pages: 877-900
  • DOI: 10.1080/15257770.2015.1088160
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] PRE and PCS techniques utilized for high-resolution protein structure determination (2015)
  • Author(s): Chojiro Kojima
  • Organizer: The 2015 International Chemical Congress of Pacific Basin Societies
  • Place of Presentation: Sheraton Waikiki (Honolulu)
  • Year and Date: 2015-12-15 – 2015-12-20
  • Int'l Joint Research / Invited
• [Presentation] NMR tools developed for drug discovery (2015)
  • Author(s): Chojiro Kojima
  • Organizer: International Symposium on Structure and Folding of Disease Related Proteins
  • Place of Presentation: Seoul National University (Seoul)
  • Year and Date: 2015-12-04 – 2015-12-05
  • Int'l Joint Research / Invited
• [Presentation] Utilization of Paramagnetic NMR techniques for protein structure determination (2015)
  • Author(s): Chojiro Kojima
  • Organizer: The 6th Asia-Pacific NMR Symposium
  • Place of Presentation: The Hong Kong University of Science and Technology (Hong Kong)
  • Year and Date: 2015-08-13 – 2015-08-16
  • Int'l Joint Research / Invited
• [Presentation] Paramagnetic NMR techniques utilized for protein structure analysis (2015)
  • Author(s): Chojiro Kojima
  • Organizer: The 3rd Awaji International Workshop on “Electron Spin Science & Technology: Biological and Materials Science Oriented Applications”
  • Place of Presentation: The Awaji Yumebutai International Conference Center (Awaji)
  • Year and Date: 2015-06-14 – 2015-06-17
  • Int'l Joint Research / Invited
• [Presentation] Drug screening using NMR to regulate protein function (2015)
  • Author(s): Chojiro Kojima
  • Organizer: The First Trilateral Workshop For Frontier Protein Studies
  • Place of Presentation: Peking University (Beijing)
  • Year and Date: 2015-04-23 – 2015-04-25
  • Int'l Joint Research / Invited