Fig. 2

Fluorescent protein biosensors. A–C. The mode of action of three redox biosensors as well as their fluorescence spectra are shown as examples. A rxYFP is an intensiometric biosensor with a single excitation and emission peak. It was generated by introducing two cysteines in β-strands 7 and 11 (N149C, S202C) of YFP. rxYFP fluorescence decreases when a disulfide bridge forms between the two cysteines under oxidizing conditions. B roGFP2 is a ratiometric probe that was generated similarly as rxYFP. While the reduced form of roGFP2 has a single excitation peak at 488 nm, the oxidized form has two, at 405 and 488 nm. Ratio measurement provides a readout of the parameter of interest. C ECFP-RL7-EYFP is a ratiometric FRET sensor where ECFP and EYFP have been coupled by a small linker carrying 4 cysteines. Only ECFP is excited at 434 nm. Under reducing conditions, only ECFP emits fluorescence at 477 nm. Under oxidizing conditions, two disulfide bridges form in the linker, bringing both FPs in close proximity and allowing energy transfer from ECFP to EYFP, thus shifting the emission peak to 527 nm. D A non-exhaustive list of FP biosensors according to the physiological parameter they measure. The FPs constituting these sensors and the sensor type are indicated. Additional FP biosensors for small molecules and protein activity can be found at https://www.addgene.org/fluorescent-proteins/biosensors/. ^a already expressed in Plasmodium; ^b YFP-based sensors are especially sensitive to pH and must be used in parallel with a pH control probe sharing similar pKa and properties when employed to measure parameters other than pH