| 研究領域 | 高速分子動画法によるタンパク質非平衡状態構造解析と分子制御への応用 |
|---|
| 研究課題/領域番号 |
22H04743
|
|---|
| 研究機関 | 東京大学 |
|---|
研究代表者 |
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)
|
|---|
| 研究期間 (年度) |
2022-04-01 – 2024-03-31
|
| キーワード | 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 |
|---|
| 研究実績の概要 |
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.
|
| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
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.
|
| 今後の研究の推進方策 |
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.
|
