biostudies-literature00510Mo GCHNIDDK NIH HHSNCI NIH HHSNIGMS NIH HHS1848https://www.ebi.ac.uk/biostudies/studies/S-EPMC7160135biostudies-literatureUnknown11(1)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.Nature communicationsA rationally enhanced red fluorescent protein expands the utility of FRET biosensors.PMC7160135R01 DK073368R35 CA197622T32 GM008326R01 GM111665Zhang JPosner CSun TMo GCHRodriguez EA51falseA rationally enhanced red fluorescent protein expands the utility of FRET biosensors.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.2020-01-01T00:00:00Z2020 Apr2021-02-21T04:39:27Z2020-05-22T17:54:39ZS-EPMC71601353229606110.1038/s41467-020-15687-x