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

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 coralderived 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.

An Overview on SARS-CoV-2 (COVID-19) and Other Human Coronaviruses and Their Detection Capability via Amplification Assay, Chemical Sensing, Biosensing, Immunosensing, and Clinical Assays

“…Biosensing assays can be categorized according to the types of transducers and bioreceptors. Enzymes [ 100 , 101 ], nucleic acids (DNA or RNA) [ 102 ], antibodies (monoclonal, polyclonal, and recombinant) [ 103 ], proteins [ 104 , 105 ], biomimetics (aptamers and molecularly imprinted polymers (MIPs)) [ 102 , 104 , 106 ], and microbial cells [ 106 ] are the main groups of bioreceptors that can be employed to detect various pathogens, especially human coronaviruses. Moreover, another significant part of a biosensor is the transducer, which efficiently transforms the physical and chemical alterations related to the bio-recognition event into quantifiable electrical responses.…”

Proton wires mediate the optical signal for ArcLight-type Genetically Encoded Voltage Indicators

“…There must be a route for the external environmental conditions to affect the chromophore. Indeed, a recently developed red fluorescent protein, stagRFP, was rationally designed by neutralizing an external acidic residue on the -barrel (35). This led to a reduction in the blinking to the dark state which improved the fluorescence of the FP.…”

Local cyclic adenosine monophosphate signalling cascades—Roles and targets in chronic kidney disease

“…51 Indeed, it is beginning to emerge that cAMP-mediated signalling occurs within defined intracellular nano-scale compartments, [52][53][54] enabling even multiple cellular responses to occur simultaneously. [55][56][57][58] The interplay of cAMP synthesis and degradation by the constitutively active and strategically positioned PDEs establishes gradients and local pools of cAMP that are sensed by cAMP effectors located at defined cellular compartments. The formation of the gradients involves the "buffering" of cAMP, that is, binding to targets to slow diffusion; otherwise synthesis would exceed PDEs' hydrolysis capacity and cAMP would evenly flood the cells.…”

“…Adrenomedullin acts through a phospholipase C pathway involving protein kinase C. A likely explanation for these opposite effects is that different pools of cAMP are activated by the two receptors 51 . Indeed, it is beginning to emerge that cAMP‐mediated signalling occurs within defined intracellular nano‐scale compartments, 52‐54 enabling even multiple cellular responses to occur simultaneously 55‐58 …”