Bianchi-Smiraglia Anna, Rana Mitra S, Foley Colleen E, Paul Leslie M, Lipchick Brittany C, Moparthy Sudha, Moparthy Kalyana, Fink Emily E, Bagati Archis, Hurley Edward, Affronti Hayley C, Bakin Andrei V, Kandel Eugene S, Smiraglia Dominic J, Feltri Maria Laura, Sousa Rui, Nikiforov Mikhail A
Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, New York, USA.
Department of Biochemistry and Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.
Nat Methods. 2017 Oct;14(10):1003-1009. doi: 10.1038/nmeth.4404. Epub 2017 Sep 4.
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.
GTP是多种细胞过程的主要调节因子，但用于定量评估活细胞中GTP水平的工具尚不存在。我们报告了基因编码的GTP传感器的开发和特性，这些传感器是通过将环状排列的黄色荧光蛋白（cpYFP）插入细菌G蛋白FeoB中经历GTP驱动构象变化的区域而构建的。GTP与这些传感器的结合导致其荧光的比例变化，从而在对GTP水平变化提供内部归一化响应的同时，对这些水平的干扰最小。引入FeoB以改变其对GTP亲和力的突变产生了一系列具有宽动态范围的传感器。至关重要的是，在哺乳动物细胞中，当GTP池耗尽或恢复时，传感器的比例信号显示出一致的变化。我们表明，这些GTP评估器（GEVALs）适用于检测活细胞中GTP水平的时空变化，以及用于高通量筛选调节GTP水平的分子。
