Abstract
Two-state ratiometric biosensors change conformation and spectral properties in response to specific biochemical inputs. Much effort over the past two decades has been devoted to engineering biosensors specific for ions, nucleotides, amino acids, and biochemical potentials. The utility of these biosensors is diminished by empirical errors in fluorescence-ratio signal measurement, which reduce the range of input values biosensors can measure accurately. Here, we present a formal framework and a web-based tool, the SensorOverlord, that predicts the input range of two-state ratiometric biosensors given the experimental error in measuring their signal. We demonstrate the utility of this tool by predicting the range of values that can be measured accurately by biosensors that detect pH, NAD+, NADH, NADPH, histidine, and glutathione redox potential. The SensorOverlord enables users to compare the predicted accuracy of biochemical measurements made with different biosensors, and subsequently select biosensors that are best suited for their experimental needs.
Highlights
Two-state ratiometric biosensors change conformation and spectral properties in response to specific biochemical inputs
We set out to determine how the precision of our fluorescence-ratio microscopy influenced the range of EGSH values we could measure accurately
The SensorOverlord toolkit enables users to predict the accuracy of concentrations and chemical potentials derived from fluorescence ratio measurements with two-state biosensors
Summary
Two-state ratiometric biosensors change conformation and spectral properties in response to specific biochemical inputs. The mechanisms that regulate EGSH in vivo remained largely unexplored until the development of the EGSH-specific, reduction–oxidation-sensitive Green Fluorescent Protein (roGFP) family of genetically-encoded biosensors[21,22,23] These GFP-derived biosensors include two cysteines that form a (reversible) intramolecular disulfide bond upon oxidation, resulting in spectral changes that can be quantified via fluorescence-ratio microscopy (Fig. 1b)[8]. We extend that framework to determine how the precision of our fluorescence-ratio signal measurements with the roGFP1-R12 biosensor constrains the range of EGSH values that can be measured accurately We generalize this extended framework for all two-state ratiometric biosensors with known spectral and biochemical properties.
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