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

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 ch

SUBMITTER: Bianchi-Smiraglia A 

PROVIDER: S-EPMC5636219 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature