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Development of material for electron microscopy analysis using protein complex with huge internal space

Research Project

Project/Area Number 19H02519
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Review Section Basic Section 27040:Biofunction and bioprocess engineering-related
Research InstitutionTohoku University

Principal Investigator

Tanaka Yoshikazu  東北大学, 生命科学研究科, 教授 (20374225)

Co-Investigator(Kenkyū-buntansha) 松井 崇  北里大学, 理学部, 講師 (30463582)
安部 聡  東京工業大学, 生命理工学院, 助教 (40508595)
Project Period (FY) 2019-04-01 – 2022-03-31
Project Status Completed (Fiscal Year 2021)
Budget Amount *help
¥17,550,000 (Direct Cost: ¥13,500,000、Indirect Cost: ¥4,050,000)
Fiscal Year 2021: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Fiscal Year 2020: ¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000)
Fiscal Year 2019: ¥7,930,000 (Direct Cost: ¥6,100,000、Indirect Cost: ¥1,830,000)
Keywordsヘモシアニン / 電子顕微鏡 / 構造解析 / クライオ電子顕微鏡 / タンパク質包摂
Outline of Research at the Start

近年,注目を集めているクライオ電子顕微鏡(クライオ電顕)による蛋白質の立体構造解析では,顕微鏡写真からピックアップした数万個以上の蛋白質の投影像から3次元構造を再構築する.そのため,形に特徴のない小さな蛋白質はノイズに埋もれてピックアップが難しく,解析の難度が高くなる.本研究では分子量4 MDaの巨大蛋白質会合体 ヘモシアニンの内部空隙に目的蛋白質を結合させ,これを丸ごと構造解析する手法を開発することにより,ピックアップの難しい小さな蛋白質でも構造決定できる技術を実現することを目指す.

Outline of Final Research Achievements

Method to encapsulate the guest protein inside the hemocyanin via various linkers was developed. The effect of guest binding on the decameric structure of the host differed depending on the linker. This was suggested to be due to the strength of the binding of the guest inside the host. Electron microscopic observation also showed that by improving the conditions, it was possible to maintain a stable decameric structure with the guest encapsulated inside. Evaluation using gold nanoparticles suggested the importance of improving the encapsulation ratio.

Academic Significance and Societal Importance of the Research Achievements

著しい発展を遂げているクライオ電顕単粒子解析であるが、100 kDa以下の小さな蛋白質や形状に際立った特徴のない蛋白質の解析は依然として難しく、あらゆる蛋白質の構造を容易に決定できる手法の開発を望む声は大きい。ヘモシアニンをゲストに用い、その開発を目指した本研究で得られた知見、すなわち、ホストの会合構造がゲストを結合させるリンカーの種類により異なることや、ゲストの包摂率を向上させることの重要性が、今後の技術開発に寄与することが期待される。

Report

(4 results)
  • 2021 Annual Research Report   Final Research Report ( PDF )
  • 2020 Annual Research Report
  • 2019 Annual Research Report
  • Research Products

    (34 results)

All 2022 2021 2020 2019

All Journal Article (10 results) (of which Int'l Joint Research: 2 results,  Peer Reviewed: 10 results,  Open Access: 6 results) Presentation (24 results) (of which Int'l Joint Research: 3 results,  Invited: 9 results)

  • [Journal Article] Chimeric mutants of staphylococcal hemolysin, which act as both one‐component and two‐component hemolysin, created by grafting the stem domain2022

    • Author(s)
      Ghanem Nouran、Kanagami Natsuki、Matsui Takashi、Takeda Kein、Kaneko Jun、Shiraishi Yasuyuki、Choe Christian A.、Uchikubo‐Kamo Tomomi、Shirouzu Mikako、Hashimoto Tsubasa、Ogawa Tomohisa、Matsuura Tomoaki、Huang Po‐Ssu、Yokoyama Takeshi、Tanaka Yoshikazu
    • Journal Title

      The FEBS Journal

      Volume: 11 Issue: 12 Pages: 11-20

    • DOI

      10.1111/febs.16354

    • Related Report
      2021 Annual Research Report
    • Peer Reviewed / Int'l Joint Research
  • [Journal Article] Structural insights reveal the second base catalyst of isomaltose glucohydrolase2021

    • Author(s)
      Tagami Takayoshi、Chen Minghao、Furunaga Yuta、Kikuchi Asako、Sadahiro Juri、Lang Weeranuch、Okuyama Masayuki、Tanaka Yoshikazu、Iwasaki Tomohito、Yao Min、Kimura Atsuo
    • Journal Title

      The FEBS Journal

      Volume: 289 Issue: 4 Pages: 1118-1134

    • DOI

      10.1111/febs.16237

    • Related Report
      2021 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Biochemical properties of CumA multicopper oxidase from plant pathogen, <i>Pseudomonas syringae</i>2021

    • Author(s)
      Ishida Konan、Tsukamoto Yuya、Horitani Masaki、Ogawa Tomohisa、Tanaka Yoshikazu
    • Journal Title

      Bioscience, Biotechnology, and Biochemistry

      Volume: 85 Issue: 9 Pages: 1995-2002

    • DOI

      10.1093/bbb/zbab123

    • Related Report
      2021 Annual Research Report
    • Peer Reviewed
  • [Journal Article] LC?MS peak assignment based on unanimous selection by six machine learning algorithms2021

    • Author(s)
      Ito Hiroaki、Matsui Takashi、Konno Ryo、Itakura Makoto、Kodera Yoshio
    • Journal Title

      Scientific Reports

      Volume: 11 Issue: 1 Pages: 1-1

    • DOI

      10.1038/s41598-021-02899-4

    • Related Report
      2021 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Anti-EGFR antibody 528 binds to domain III of EGFR at a site shifted from the cetuximab epitope2021

    • Author(s)
      Makabe Koki、Yokoyama Takeshi、Uehara Shiro、Uchikubo-Kamo Tomomi、Shirouzu Mikako、Kimura Kouki、Tsumoto Kouhei、Asano Ryutaro、Tanaka Yoshikazu、Kumagai Izumi
    • Journal Title

      Scientific Reports

      Volume: 11 Issue: 1 Pages: 5790-5790

    • DOI

      10.1038/s41598-021-84171-3

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Design of in‐cell protein crystal for environmentally responsive construction of a supramolecular filament2021

    • Author(s)
      Abe Satoshi、Pham Thuc Toan、Negishi Hashiru、Yamashita Keitaro、Hirata Kunio、Ueno Takafumi
    • Journal Title

      Angewandte Chemie International Edition

      Volume: - Issue: 22 Pages: 12341-12345

    • DOI

      10.1002/anie.202102039

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Highly accurate and precise quantification strategy using stable isotope dimethyl labeling coupled with GeLC-MS/MS2021

    • Author(s)
      Ryo Konno, Takashi Matsui, Hiroaki Ito, Yusuke Kawashima, Makoto Itakura, Yoshio Kodera
    • Journal Title

      Biochemical and Biophysical Research Communications

      Volume: 550 Pages: 37-42

    • DOI

      10.1016/j.bbrc.2021.02.101

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] A highly efficient method for extracting peptides from a single mouse hypothalamus2021

    • Author(s)
      Yuzuru Nakagawa, Takashi Matsui, Ryo Konno, Yusuke Kawashima, Toshiya Sato, Makoto Itakura, Yoshio Kodera
    • Journal Title

      Biochemical and Biophysical Research Communications

      Volume: 548 Pages: 155-160

    • DOI

      10.1016/j.bbrc.2021.02.041

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Protein encapsulation in the hollow space of hemocyanin crystals containing a covalently conjugated ligand2019

    • Author(s)
      Tsubasa Hashimoto, Yuxin Ye, Mihoko Ui, Tohohisa Ogawa, Takashi Matsui, and Yoshikazu Tanaka
    • Journal Title

      Biochem. Biophys. Res. Commun.

      Volume: 印刷中 Issue: 1 Pages: 31-36

    • DOI

      10.1016/j.bbrc.2019.04.062

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Crystal structure of the catalytic unit of GH87-type α-1,3-glucanase Agl-KA from Bacillus circulans2019

    • Author(s)
      S. Yano, W. Suyotha, N. Oruro, T. Matsui, S. Shiga, T. Itoh, T. Hibi, Y. Tanaka, M. Wakayama, K. Makabe
    • Journal Title

      Scientific Reports

      Volume: 9 Issue: 1 Pages: 15259-15259

    • DOI

      10.1038/s41598-019-51822-5

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Presentation] クライオ電子顕微鏡単粒子解析法によるウサギ赤血球ライセートに内在する80Sリボソーム構造解析の検討2021

    • Author(s)
      千足啄馬、横山武司、田中良和
    • Organizer
      第44回日本分子生物学会年会
    • Related Report
      2021 Annual Research Report
  • [Presentation] クライオ電子顕微鏡単粒子解析によるリボソームを標的としたアミノ配糖体抗菌薬の新規作用機序の解明2021

    • Author(s)
      伴野詢太、浅野航佑、鈴木仁人、横山武司、田中良和
    • Organizer
      第44回日本分子生物学会年会
    • Related Report
      2021 Annual Research Report
  • [Presentation] 薬剤耐性獲得機構の解明に向けた、クライオ電子顕微鏡単粒子解析による非結核性抗酸菌Mycobacterium abscessusリボソームへのマクロライド結合様式の解明2021

    • Author(s)
      橋本翼、高田希美、千足啄馬、深野華子、山本健太郎、鈴木仁人、星野仁彦、横山武司、田中良和
    • Organizer
      第44回日本分子生物学会年会
    • Related Report
      2021 Annual Research Report
  • [Presentation] Cryo-EM visualizes ribosomes in mammalian cell free translation system2021

    • Author(s)
      Takuma Chiashi, Takeshi Yokoyama, Yoshikazu Tanaka
    • Organizer
      日本・ウクライナ・タイ二国間交流事業合同シンポジウム
    • Related Report
      2021 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Elucidation of the binding mode of a macrolide antibiotic to NTM ribosome for understanding drug resistance mechanism by using cryo-EM2021

    • Author(s)
      Tsubasa Hashimoto, Nozomi Takada, Takuma Chiashi, Hanako Fukano, Kentaro Yamamoto, Masato Suzuki, Yoshihiko Hoshino, Takeshi Yokoyama, Yoshikazu Tanaka
    • Organizer
      第59回日本生物物理学会年会
    • Related Report
      2021 Annual Research Report
  • [Presentation] Elucidation of new action mechanism of aminoglycoside antibiotics on ribosomes using single particle cryo-electron microscopy2021

    • Author(s)
      Junta Tomono,Kosuke Asano,Masato Suzuki, Takeshi Yokoyama,Yoshikazu Tanaka
    • Organizer
      第59回日本生物物理学会年会
    • Related Report
      2021 Annual Research Report
  • [Presentation] 非結核性抗酸菌Mycobacterium abscessusリボソームにおけるマクロライド耐性機構の解明を目指したクライオ電子顕微鏡構造解析2021

    • Author(s)
      橋本翼、高田希美、千足啄馬、深野華子、山本健太郎、鈴木仁人、星野仁彦、横山武司、田中良和
    • Organizer
      2021年度生物物理学会、北海道支部・東北支部合同例会
    • Related Report
      2021 Annual Research Report
  • [Presentation] Design of In-cell Protein Crystals for Development of Bio-solid Materials2021

    • Author(s)
      Satoshi Abe and Takafumi Ueno
    • Organizer
      22nd American Conference on Crystal Growth and Epitaxy (ACCGE-22)
    • Related Report
      2021 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 迅速タンパク質結晶化とサブミクロン結晶構造解析2021

    • Author(s)
      安部 聡、田中潤子、上野隆史
    • Organizer
      第15回バイオ関連化学シンポジウム
    • Related Report
      2021 Annual Research Report
  • [Presentation] 無細胞タンパク質合成を利用した結晶化とサブミクロン結晶構造解析2021

    • Author(s)
      安部 聡、上野隆史
    • Organizer
      第70回高分子討論会
    • Related Report
      2021 Annual Research Report
  • [Presentation] 細胞内タンパク質結晶の分子設計による生体固体材料の開発2021

    • Author(s)
      安部 聡
    • Organizer
      酵素工学研究会 第86回講演会
    • Related Report
      2021 Annual Research Report
    • Invited
  • [Presentation] 微量組織を対象とした高感度ペプチドミクス2021

    • Author(s)
      小寺義男, 中川譲, 板倉誠, 紺野亮, 紺野亮, 大橋潤子, 佐藤俊哉, 川島祐介, 松井崇
    • Organizer
      第46回日本医用マススペクトル学会年会
    • Related Report
      2021 Annual Research Report
  • [Presentation] SI-GeLC-MS/MSを用いた蛋白質切断状態の高精度比較分析2021

    • Author(s)
      小寺義男, 紺野亮, 伊東大晃, 松井崇
    • Organizer
      第69回質量分析総合討論会
    • Related Report
      2021 Annual Research Report
  • [Presentation] 巨大タンパク質会合体ヘモシアニンの構造解析2021

    • Author(s)
      田中良和
    • Organizer
      農芸化学会
    • Related Report
      2020 Annual Research Report
    • Invited
  • [Presentation] 巨大タンパク質会合体ヘモシアニンの結晶中の空隙への生体分子の包摂2020

    • Author(s)
      田中良和
    • Organizer
      化学工学会第51回秋季大会
    • Related Report
      2020 Annual Research Report
    • Invited
  • [Presentation] 蛋白質の立体構造解析 -X線結晶構造解析とクライオ電顕単粒子解析-2020

    • Author(s)
      田中良和
    • Organizer
      酵素工学研究会第84回講演会
    • Related Report
      2020 Annual Research Report
    • Invited
  • [Presentation] クライオ電子顕微鏡単粒子解析による百日咳壊死毒の構造解析2020

    • Author(s)
      次田篤史、松井崇、堀口安彦、横山武司、田中良和
    • Organizer
      日本生物物理学会
    • Related Report
      2020 Annual Research Report
  • [Presentation] 細胞内タンパク質結晶化の最適化と迅速構造解析2020

    • Author(s)
      安部 聡、小島摩利子、小暮遼河、上野隆史
    • Organizer
      第14回バイオ関連化学シンポジウム
    • Related Report
      2020 Annual Research Report
  • [Presentation] タンパク質の迅速結晶化とサブミクロン結晶の構造解析2020

    • Author(s)
      安部 聡、小島摩利子、上野隆史
    • Organizer
      第69回高分子討論会
    • Related Report
      2020 Annual Research Report
  • [Presentation] 細胞内結晶工学によるタンパク質結晶の機能化2020

    • Author(s)
      安部 聡
    • Organizer
      日本化学会東北支部講演会
    • Related Report
      2020 Annual Research Report
    • Invited
  • [Presentation] 巨大タンパク質会合体ヘモシアニンの構造解析 ―結晶構造と電顕構造―2019

    • Author(s)
      田中良和
    • Organizer
      2019年度 日本生化学会九州支部例会
    • Related Report
      2019 Annual Research Report
    • Invited
  • [Presentation] X線結晶構造解析とクライオ電子顕微鏡の相関構造解析の有用性ーヘモシアニンを例としてー2019

    • Author(s)
      田中良和
    • Organizer
      日本蛋白質科学会 ランチョンセミナー
    • Related Report
      2019 Annual Research Report
    • Invited
  • [Presentation] Structure analysis of squid hemocyanin using both X-ray crystallography and cryo-EM2019

    • Author(s)
      田中良和,松井崇,Stefan Raunser,Gatsogiannis Christos
    • Organizer
      化学系学協会東北大会
    • Related Report
      2019 Annual Research Report
    • Invited
  • [Presentation] tRNA thiolation mechanism involving [4Fe-4S] cluster2019

    • Author(s)
      Yoshikazu Tanaka
    • Organizer
      International Symposium on Nucleic Acids Chemistry
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research

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Published: 2019-04-18   Modified: 2023-01-30  

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