Signal amplification system to detect divalent manganese with a unique pair of enzyme variant and the fluorescent substrate

2023 Fiscal Year Final Research Report

• Project/Area Number: 21K15246
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 47020:Pharmaceutical analytical chemistry and physicochemistry-related
• Research Institution: Tohoku University (2022-2023); Kobe Pharmaceutical University (2021)
• Principal Investigator: Takashima Ippei 東北大学, 多元物質科学研究所, 助教 (50769742)
• Project Period (FY): 2021-04-01 – 2024-03-31
• Keywords: シグナル増強 / タンパク質工学 / 酵素-基質ペア / マンガン / 蛍光プローブ

Outline of Final Research Achievements

Herein we developed a signal-amplification method to detect manganese via an enzymatic reaction driven by a pair of an enzyme mutant and its substrate. Although the mutant coordinates various metals, only manganese was found to activate the enzymatic reaction at the μM order. Furthermore, several mutants prepared here showed different selectivity and affinity for manganese, giving information beneficial for the design. Sterically bulky substrate synthesized here selectively reacts with the mutant and does not show undesired reaction with endogenous enzymes. We also sought the designed molecule which is immobilized on the neighboring protein after the reaction, resulting in the accumuration of the signal molecules in cells. In the future, we will perform intracellular imaging using these molecular tools.

Free Research Field

ケミカルバイオロジー

Academic Significance and Societal Importance of the Research Achievements

細胞内マンガンの詳細解析において、選択的なリガンドが皆無であり、マンガンの常磁性に伴う蛍光消光の問題もあって、実用性の高い蛍光プローブはほとんど開発されていない。本研究では、生体内に存在するマンガン選択的な酵素反応に着目し、タンパク質-合成分子ペアによる新たな検出機構を提案すると共に、世界で初めて細胞内小器官内でのマンガンの詳細な局在を可視化する技術を開発できる。