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

Gary C. H. Mo, Clara Posner, Erik A. Rodriguez, Tengqian Sun and Jin Zhang ()
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Gary C. H. Mo: University of California San Diego
Clara Posner: University of California San Diego
Erik A. Rodriguez: The George Washington University
Tengqian Sun: University of California San Diego
Jin Zhang: University of California San Diego

Nature Communications, 2020, vol. 11, issue 1, 1-9

Abstract: Abstract 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.

Date: 2020
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DOI: 10.1038/s41467-020-15687-x

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