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Nat Methods. 2017 Oct;14(10):1003-1009. doi: 10.1038/nmeth.4404. Epub 2017 Sep 4.

Internally ratiometric fluorescent sensors for evaluation of intracellular GTP levels and distribution.

Author information

1
Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, New York, USA.
2
Department of Biochemistry and Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.
3
Department of Biochemistry and Neurology, Hunter James Kelly Research Institute, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York, USA.
4
Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York, USA.

Abstract

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.

PMID:
28869758
PMCID:
PMC5636219
DOI:
10.1038/nmeth.4404
[Indexed for MEDLINE]
Free PMC Article

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