| 配分額 *注記 |
6,630千円 (直接経費: 5,100千円、間接経費: 1,530千円)
2023年度: 3,380千円 (直接経費: 2,600千円、間接経費: 780千円)
2022年度: 3,250千円 (直接経費: 2,500千円、間接経費: 750千円)
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| 研究実績の概要 |
The original aim of this project was to obtain mechanistic insight into the dynamic response of genetically encoded biosensors. Working towards this goal, we have been building upon our expertise in biosensor engineering, chemical biology, and photochemistry to reveal the mechanism of lactate biosensors developed in our lab. In FY2023 our primary focus was on the characterization and application of a photocaged lactate (MNI-L-lac). This work has been made publicly available since January 2024, when we posted a preprint posted to bioRxiv. This manuscript described the synthesis, in vitro characterization, and cell-based characterization of the photocaged MNI-L-lac. This manuscript will be submitted to a peer reviewed journal as soon as a few additional cell-based imaging experiments are completed.
With MNI-L-lac in hand, we are now well positioned to undertake the rapid uncaging of lactate in crystals in order to perform time-lapse structural studies of the biosensor response. Unfortunately, we have continued to struggle to produce diffracting crystals of the apo state. Work towards this goal is continuing in collaboration with the Nureki lab. In the meantime, we have succeeded in obtaining a cryo-EM structure of a lactate-bound lactate biosensor. While this structure can not reveal the full dynamic mechanism that we were hoping for, it does provide important insights into the key interactions chromophore-protein interactions responsible for the fluorescence response. Further efforts to interpret and validate the mechanistic insights are ongoing.
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