Development of high-speed AFM/single-molecule FRET to visualize synthesis and degradation of carbohydrate chain
| Project/Area Number |
17K19519
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| Research Category |
Grant-in-Aid for Challenging Research (Exploratory)
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| Allocation Type | Multi-year Fund |
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| Research Field |
Biomedical structure and function and related fields
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| Research Institution | Nagoya University |
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Principal Investigator |
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| Research Collaborator |
IINO RYOTA 大学共同利用機関法人自然科学研究機構, 分子科学研究所, 教授 (70403003)
YAGI HIROKAZU 名古屋市立大学, 講師 (70565423)
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| Project Period (FY) |
2017-06-30 – 2019-03-31
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| Project Status |
Completed (Fiscal Year 2018)
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| Budget Amount *help |
¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000)
Fiscal Year 2018: ¥3,120,000 (Direct Cost: ¥2,400,000、Indirect Cost: ¥720,000)
Fiscal Year 2017: ¥3,120,000 (Direct Cost: ¥2,400,000、Indirect Cost: ¥720,000)
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| Keywords | 高速原子間力顕微鏡 / 一分子FRET / 複合計測 / 糖鎖合成酵素 / 多糖分解酵素 / 蛍光共鳴エネルギー移動 / タンパク質 / 構造変化 / 一分子計測 / １分子計測(SMD) / 蛋白質 / 生物物理 / 走査プローブ顕微鏡 |
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| Outline of Final Research Achievements |
We have incorporated an image-splitting optical system into a combined high-speed AFM/ total-reflection fluorescent microscopy system which enables us to perform high-speed AFM imaging and single-molecule FRET simultaneously. We prepared the fluorescent labeling sample for single-molecule FRET measurements for bacterial chondroitin polymerase K4CP and glycoside hydrolase cellulase, TrCel6A, and succeeded in measuring changes of the FRET efficiently due to the extension of the chondroitin chain and the structural change of the cellulase. Although we tried to observe these molecule with the combined high-speed AFM and single-molecule FRET system, in the face of various problems, we have not reached the success within the period. However we are now establishing the optimum measurement conditions for the simultaneous observation and thus assume that we can gain novel insights about molecular mechanisms with detailed single-molecule analysis using the combined system.
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| Academic Significance and Societal Importance of the Research Achievements |
個々のタンパク質の構造とそのダイナミクス計測は、タンパク質の機能発現のメカニズムの直接的理解を可能にすることから、生命現象を理解する上で必須である。高速原子間力顕微鏡と一分子FRET計測の融合は、両手法の欠点を補いタンパク質一分子のダイナミクスの詳細が定量的に解析できる。この手法でこれまで困難であった、タンパク質のダイナミクス現象を計測できるようになり、生命科学分野の発展に大きなインパクトを与える手法になると考えられる。本研究成果は、複合計測の実現可能性と萌芽を示すものであり、学術的意義は大きいと考える。
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Report
(3 results)
Research Products
(17 results)
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[Journal Article] Rate constants, processivity, and productive binding ratio of chitinase A revealed by single- molecule analysis2018
Author(s)
Akihiko Nakamura, Tomoyuki Tasaki, Yasuko Okuni, Chihong Song, Kazuyoshi Murata, Toshiya Kozai, Mayu Hara, Hayuki Sugimoto, Kazushi Suzuki, Takeshi Watanabe, Takayuki Uchihashi, Hiroyuki Noji and Ryota Iino
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Journal Title
Phys. Chem. Chem. Phys.
Volume: 20
Issue: 5
Pages: 3010-3018
DOI
Related Report
Peer Reviewed / Open Access
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