GTP'
荧光
生物物理学
细胞内
细胞生物学
鸟苷三磷酸
GTP酶
GTP结合蛋白调节剂
绿色荧光蛋白
生物化学
化学
G蛋白
生物
信号转导
酶
基因
量子力学
物理
作者
Anna Bianchi‐Smiraglia,Mitra S. Rana,Colleen E. Foley,Leslie M. Paul,Brittany C. Lipchick,Sudha Moparthy,Kalyana Moparthy,Emily E. Fink,Archis Bagati,Edward Hurley,Hayley C. Affronti,Andrei V. Bakin,Eugene Kandel,Dominic J. Smiraglia,M. Laura Feltri,Rui Sousa,Mikhail A. Nikiforov
出处
期刊:Nature Methods
[Springer Nature]
日期:2017-09-04
卷期号:14 (10): 1003-1009
被引量:46
摘要
GTP is a major regulator of multiple cellular processes, but tools for quantitative evaluation of GTP levels in live cells have not been available. We report the development and characterization of genetically encoded GTP sensors, which we constructed by inserting a circularly permuted yellow fluorescent protein (cpYFP) into a region of the bacterial G protein FeoB that undergoes a GTP-driven conformational change. GTP binding to these sensors results in a ratiometric change in their fluorescence, thereby providing an internally normalized response to changes in GTP levels while minimally perturbing those levels. Mutations introduced into FeoB to alter its affinity for GTP created a series of sensors with a wide dynamic range. Critically, in mammalian cells the sensors showed consistent changes in ratiometric signal upon depletion or restoration of GTP pools. We show that these GTP evaluators (GEVALs) are suitable for detection of spatiotemporal changes in GTP levels in living cells and for high-throughput screening of molecules that modulate GTP levels.
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