Next Generation Biosensors Enabled by High-speed Visualization of Dynamic Mechanisms
Publicly Offered Research
| Project Area | Non-equilibrium-state molecular movies and their applications |
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| Project/Area Number |
22H04743
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| Research Category |
Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
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| Allocation Type | Single-year Grants |
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| Review Section |
Complex systems
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| Research Institution | The University of Tokyo |
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Principal Investigator |
キャンベル ロバート.アール 東京大学, 大学院理学系研究科(理学部), 教授 (40831318)
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| Project Period (FY) |
2022-04-01 – 2024-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥6,630,000 (Direct Cost: ¥5,100,000、Indirect Cost: ¥1,530,000)
Fiscal Year 2023: ¥3,380,000 (Direct Cost: ¥2,600,000、Indirect Cost: ¥780,000)
Fiscal Year 2022: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000)
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| Keywords | 高速分子動画 / 蛍光タンパク質 / イメージング / バイオセンサー / ケージド化合物 |
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| Outline of Research at the Start |
蛍光タンパク質を用いたバイオセンサーは、細胞中での目的分子の振舞いを解析する有用な研究ツールとして生物学研究に広く利用されている。研究代表者らは長年にわたり、世界に先駆けて様々なバイオセンサーを開発してきた。しかし、センサーの開発には現状多くの試行錯誤が必要であり、論理的な設計が難しい。本研究ではX線自由電子レーザーを活用した高速分子動画法によってセンサータンパク質の実際の動きに迫ることで、この課題を克服する。
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| Outline of Annual Research Achievements |
FY2022 was the first year of our Molecular Movies grant, which is being used to support a new project aimed at uncovering the mechanisms of genetically encoded biosensors. To achieve this goal we have proposed to use X-Ray Free-Electron Laser (XFEL) approaches to observe dynamic ligand binding-induced changes in biosensor structure in the context of protein crystals. In 2022 we had set three major goals for ourselves, and we are happy to achieved all three goals. The first goal was to establish collaborations with other Molecular Movies researchers with expertise in molecular dynamics and protein structural analysis. Following an introductory presentation at the Molecular Movies International Symposium in early 2022, we were able to establish three critical collaborations. Specifically, we initiated collaborations with computational experts Prof. Osamu Miyashita (RIKEN) and Prof. Shigehiko Hayashi (Kyoto University). We also established a critical collaboration with Prof. Nureki that is also in the Faculty of Science at the University of Tokyo. Through the collaboration with Prof. Nureki, we have accomplished the second major goal, which was crystallization of a lactate biosensor protein in the ligand-free state. Finally, the third major goal of FY2022 was to synthesize a photocaged version of lactate. This goal was recently achieved by Master’s student Ikumi Miyazaki.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The overall aim of this project is to obtain mechanistic insight into the the dynamic response of genetically encoded biosensors. To achieve this goal, we have proposed to build upon our existing expertise in biosensor engineering, chemical biology, and photochemistry to uncover the mechanism of the high performance lactate biosensors recently developed in our lab. In FY2022 we made excellent progress towards Aim 1 (crystallization of biosensors in their apo states), and Aim 2 (development of photo caged ligands). Biosensor crystallization (Aim 1) was achieved in collaboration with the Nureki lab. Aim 2 was achieved in our laboratory and we now have developed an effective and efficient synthetic route to photocaged lactate.
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| Strategy for Future Research Activity |
In FY2023, our efforts will be focussed on refining the crystallization conditions and the synthetic route, and then testing the photouncaging of caged lactate that has been soaked into crystals. Once we have confirmed that lactate can be uncaged in crystals, and that the crystallized biosensor can respond to binding of lactate, we will move on to performing time-lapse XFEL studies (Aim 3). By late in FY2023 we hope to have the results of these studies in hand and be performing mutagenic, biophysical, and photophysical follow up experiments to validate the results of the timelapse studies.
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Report
(1 results)
Research Products
(41 results)
