Abstract
Förster Resonance Energy Transfer (FRET) between two fluorescent proteins can be exploited to create fully genetically encoded and thus subcellularly targetable sensors. FRET sensors report changes in energy transfer between a donor and an acceptor fluorescent protein that occur when an attached sensor domain undergoes a change in conformation in response to ligand binding. The design of sensitive FRET sensors remains challenging as there are few generally applicable design rules and each sensor must be optimized anew. In this review we discuss various strategies that address this shortcoming, including rational design approaches that exploit self-associating fluorescent domains and the directed evolution of FRET sensors using high-throughput screening.
Highlights
Förster Resonance Energy Transfer (FRET) between two fluorescent proteins can be exploited to create fully genetically encoded and subcellularly targetable sensors
Encoded FRET sensors for intracellular ligands offer a number of benefits including modular sensor design, a sensor concentration-independent output signal and accurate subcellular targeting
Genetic encoding allows convenient distribution among cell biologists, as an increasing number of FRET sensors have become available through depositories such as AddGene
Summary
Encoded intracellular fluorescent sensors have been developed to image a range of intracellular parameters including the concentration of many chemical species [1,2,3]. A FRET sensor for Ca2+, Cameleon, was one of the first sensors to be constructed using the principle of FRET between two fluorescent domains (Figure 1B) This construct is a single polypeptide chain consisting of ECFP, calmodulin (CaM), M13 and EYFP. As Cameleon’s output signal was emission ratiometric, measurements were not sensitive to fluctuations in sensor concentration, optical path length or excitation intensity [33].The dynamic range (DR) (ΔR/Rmin) of this sensor was a very reasonable 70% Achieving this DR required extensive testing of different designs. Technologies such as FlAsH [35] and SNAP-tag [36] have made site-specific incorporation of synthetic dyes in natural protein domains straightforward These strategies allow novel sensor approaches to be developed [37,38] and allow sensitive monitoring of changes in protein conformation [39]. The graph on the right shows the typical emission spectra in presence and absence of Ca2+ seen with Cameleon and related sensors
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