A rationally enhanced red fluorescent protein expands the utility of FRET biosensors

Nat Commun. 2020 Apr 15;11(1):1848. doi: 10.1038/s41467-020-15687-x.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Fluorescence Resonance Energy Transfer*
  • Humans
  • Luminescent Proteins / chemistry*
  • Mutagenesis / genetics
  • Mutagenesis / physiology
  • Red Fluorescent Protein
  • Signal Transduction / genetics
  • Signal Transduction / physiology


  • Luminescent Proteins