Publicly Offered Research
Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
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。
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.
令和5年度が最終年度であるため、記入しない。
All 2024 2023 2022 Other
All Journal Article (18 results) (of which Int'l Joint Research: 14 results, Open Access: 13 results, Peer Reviewed: 16 results) Presentation (55 results) (of which Int'l Joint Research: 27 results, Invited: 17 results) Book (1 results) Remarks (1 results)
Folia Pharmacologica Japonica
Volume: 159 Issue: 1 Pages: 25-30
10.1254/fpj.23036
ACS Central Science
Volume: 10 Issue: 2 Pages: 402-416
10.1021/acscentsci.3c01250
bioRxiv
Volume: 2024.01.30. Pages: 577898-577898
10.1101/2024.01.30.577898
Engineering Proceedings
Volume: 35 Pages: 20-20
10.3390/iecb2023-14563
Biochemical Society Transactions
Volume: 51 Issue: 4 Pages: 1585-1595
10.1042/bst20221413
ACS Sensors
Volume: 8 Issue: 8 Pages: 3014-3022
10.1021/acssensors.3c00484
RSC Advances
Volume: 13 Issue: 42 Pages: 29584-29593
10.1039/d3ra06108f
Nature Communications
Volume: 14 Issue: 1 Pages: 6598-6598
10.1038/s41467-023-42230-5
Biochemistry
Volume: 63 Issue: 1 Pages: 171-180
10.1021/acs.biochem.3c00451
Cell Reports
Volume: 42 Issue: 1 Pages: 111899-111899
10.1016/j.celrep.2022.111899
Volume: 8 Issue: 2 Pages: 587-597
10.1021/acssensors.2c01730
Volume: 13 Issue: 1 Pages: 1850-1850
10.1038/s41467-022-29508-w
Frontiers in Cell and Developmental Biology
Volume: 10 Pages: 885440-885440
10.3389/fcell.2022.885440
Protein Engineering, Design and Selection
Volume: 35
10.1093/protein/gzac004
Neurophotonics
Volume: 9 Issue: S1 Pages: 013001-013001
10.1117/1.nph.9.s1.013001
PLOS Biology
Volume: 20 Issue: 9 Pages: e3001772-e3001772
10.1371/journal.pbio.3001772
Protein Science
Volume: 31 Issue: 10
10.1002/pro.4440
Nature Chemical Biology
Volume: 19 Issue: 1 Pages: 38-44
10.1038/s41589-022-01134-z
https://www.chem.s.u-tokyo.ac.jp/campbell/
