Supplementary File 1.pdf

The SensorOverlord predicts the accuracy of measurements with ratiometric biosensors. Researchers derived a map from roGFP-biosensor fluorescence-ratio measurements into glutathione redox potential values. They reviewed the general chemistry of roGFP biosensors, derived the map between the fluorescence ratio and the fraction of oxidized biosensors, and derived the map between the fluorescence ratio and the glutathione redox potential. The roGFP biosensors have two cysteines that form a reversible intramolecular disulfide bond, and both reduced and oxidized species exhibit green fluorescence. The degree of chromophore protonation is influenced by the oxidation state of the two roGFP cysteines. The fraction of oxidized biosensors provides a chemically interpretable description of the biosensor's oxidation and can be calculated by recording the ratio of the biosensor's green fluorescent emission at two different wavelengths. The researchers also described how to calculate the biosensor's redox potential given the fraction of oxidized biosensors. The glutathione redox couple mediates the indirect effects of oxidants and reductants on the thiol-disulfide balance of many proteins, including roGFPs. Measuring the glutathione redox potential enables predictions about the oxidation state of the network of cysteines trading electrons with glutathione. The map between the fluorescence ratio and the fraction of oxidized biosensors is derived using dual-excitation single-emission biosensors and dual-emission single-excitation biosensors. The resulting equation relates the fluorescence ratio to the fraction of oxidized biosensors, allowing for the calculation of the glutathione redox potential.