FRET-based biosensors

Intramolecular FRET-based indicators are often referred to as biosensors of biochemical activities6. The emission spectrum of the donor fluorophore should overlap the excitation spectrum of the acceptor fluorophore, but both excitation spectra should be well enough separated to allow independent excitation5. When the stoichiometry of the donor and the acceptor is fixed, as it is when they are fused in a single polypeptide chain, then the experimentally most convenient readout of FRET is the ratio of acceptor to donor fluorescence. Many reporters that are designed on the basis of intramolecular FRET changes have been developed for measuring biochemical events in cells. Early applications used BFP as the donor and GFP as the acceptor, but the dimness and ability of BFP to be bleached soon led to its replacement by CFP, whereupon GFP had to be replaced by YFP to maintain spectral separation5.

Examples: indicators of...

...protease activity

Sensors responding to specific protease activity contain a protease-sensitive linker, recognized by protease, and dequench fluorescence from the donor FP3,5. These sensors represent an essentially irreversible class of biosensors3.

The FRET-based biosensor shown in figure uses CFP as donor and YFP as acceptor.

[From Ref. 3] Sensors responding to specific protease activity contain a protease-sensitive linker, recognized by protease, and dequench fluorescence from the donor FP. These sensors represent an essentially irreversible class of biosensors. The FRET-based biosensor shown in figure uses CFP as donor and YFP as acceptor.

...kinase activity

Kinase biosensors report the phosphorylation status of a model kinase substrate; phosphorylation of an appropriate peptide motif promotes intramolecular binding to a phosphopeptide-binding domain3.

The FRET change is usually reversed by phosphatases, so the fluorescent substrates report continuously the balance between kinase and phosphatase activities, with time resolution in the order of a few seconds and a spatial resolution of micrometres (far better than conventional assays)5.

The FRET-based biosensor shown in figure uses CFP as donor and YFP as acceptor.

[From Ref. 3] Kinase biosensors report the phosphorylation status of a model kinase substrate; phosphorylation of an appropriate peptide motif promotes intramolecular binding to a phosphopeptide-binding domain. The FRET-based biosensor shown in figure uses CFP as donor and YFP as acceptor.

...Ca2+

[From Ref. 21] Cyan fluorescence protein labeled calmodulin and yellow fluorescence protein labeled calmodulin binding peptide (M13-YFP) were coexpressed. High Ca2+ levels (right) lead to binding and FRET emission of YFP (pseudo color red); low Ca2+ levels (left) lead to little FRET and mostly blue emission (pseudocolor green).

FRET-based biosensors provide indicators of Ca2+ concentrations in whole organisms, tissues, organelles and submicroscopic environments, previously out of reach for quantitative monitoring2. Genetically encoded Ca2+ indicators were constructed by sandwiching CaM, a peptide linker and M13 between CFP and YFP5,22,42. Increased levels of intracellular Ca2+ switches on the affinity of CaM for the adjacent M13 sequence, which results in a change in orientation or distance between the two fluorescent proteins and a large increase in FRET efficiency. In this way, the FRET signal can be calibrated to the intracellular concentration of Ca2+. These biosensors are called cameleons, because they readily change colour and retract and extend a long tongue (M13) into and out of the mouth of the calmodulin22.

The replacement of M13 by a peptide that is derived from CaM-dependent kinase kinase produces a cameleon analogue, with a larger, although somewhat slower, response than the traditional cameleons43.

[From Ref. 43]

Levels of free intracellular CaM–Ca2+ complex can be sensed by reporters in which the donor and acceptor are connected by just a CaM-binding peptide (the calmodulin-binding domain from smooth muscle myosin light chain kinase44), but in this case the binding of CaM straightens the peptide linker and so decreases FRET.

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