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A rationally enhanced red fluorescent protein expands the utility of FRET biosensors.
Nature Communications ( IF 14.7 ) Pub Date : 2020-04-15 , DOI: 10.1038/s41467-020-15687-x
Gary C H Mo 1, 2 , Clara Posner 1, 3 , Erik A Rodriguez 4 , Tengqian Sun 1 , Jin Zhang 1, 3, 5
Affiliation  

Genetically encoded Förster Resonance Energy Transfer (FRET)-based biosensors are powerful tools to illuminate spatiotemporal regulation of cell signaling in living cells, but the utility of the red spectrum for biosensing was limited due to a lack of bright and stable red fluorescent proteins. Here, we rationally improve the photophysical characteristics of the coral-derived fluorescent protein TagRFP-T. We show that a new single-residue mutant, super-TagRFP (stagRFP) has nearly twice the molecular brightness of TagRFP-T and negligible photoactivation. stagRFP facilitates significant improvements on multiple green-red biosensors as a FRET acceptor and is an efficient FRET donor that supports red/far-red FRET biosensing. Capitalizing on the ability of stagRFP to couple with multiple FRET partners, we develop a novel multiplex method to examine the confluence of signaling activities from three kinases simultaneously in single living cells, providing evidence for a role of Src family kinases in regulating growth factor induced Akt and ERK activities.

中文翻译:


合理增强的红色荧光蛋白扩展了 FRET 生物传感器的用途。



基于基因编码的福斯特共振能量转移(FRET)的生物传感器是阐明活细胞中细胞信号传导时空调节的强大工具,但由于缺乏明亮且稳定的红色荧光蛋白,红色光谱在生物传感中的效用受到限制。在这里,我们合理改善了珊瑚源荧光蛋白TagRFP-T的光物理特性。我们发现一种新的单残基突变体 super-TagRFP (stagRFP) 的分子亮度几乎是 TagRFP-T 的两倍,并且光活化可以忽略不计。 stagRFP 作为 FRET 受体促进了多种绿-红生物传感器的显着改进,并且是支持红色/远红 FRET 生物传感的高效 FRET 供体。利用 stagRFP 与多个 FRET 伴侣偶联的能力,我们开发了一种新颖的多重方法来检查单个活细胞中三种激酶信号活动的汇合情况,为 Src 家族激酶在调节生长因子诱导的 Akt 中的作用提供证据和 ERK 活动。
更新日期:2020-04-24
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