Gohil Khyati, Wu Sheng-Yi, Takahashi-Yamashiro Kei, Shen Yi, Campbell Robert E
Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada.
Department of Chemistry, The University of Tokyo, Tokyo 113-0033, Japan.
ACS Sens. 2023 Feb 24;8(2):587-597. doi: 10.1021/acssensors.2c01730. Epub 2023 Jan 24.
Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are indispensable tools for monitoring biochemical changes in cells. Green and red fluorescent protein-based FRET pairs offer advantages over the classically employed cyan and yellow fluorescent protein pairs, such as better spectral separation, lower phototoxicity, and less autofluorescence. Here, we describe the development of an mScarlet-derived green fluorescent protein (designated as mWatermelon) and its use as a FRET donor to the red fluorescent protein mScarlet-I as a FRET acceptor. We tested the functionality of this FRET pair by engineering biosensors for the detection of protease activity, Ca, and K. Furthermore, we described a strategy to enhance the FRET efficiency of these biosensors by modulating the intramolecular association between mWatermelon and mScarlet-I.
基于Förster共振能量转移（FRET）的基因编码生物传感器是监测细胞生化变化的不可或缺的工具。基于绿色和红色荧光蛋白的FRET对，相较于传统使用的青色和黄色荧光蛋白对具有诸多优势，比如更好的光谱分离、更低的光毒性以及更少的自发荧光。在此，我们描述了一种源自mScarlet的绿色荧光蛋白（命名为mWatermelon）的开发，并将其用作红色荧光蛋白mScarlet-I作为FRET受体时的FRET供体。我们通过构建用于检测蛋白酶活性、Ca和K的生物传感器来测试该FRET对的功能。此外，我们还描述了一种通过调节mWatermelon和mScarlet-I之间的分子内缔合来提高这些生物传感器FRET效率的策略。
