Fluorescent, Reagentless Biosensors for Phosphate: Altering Properties, Expanding Usefulness

Abstract

Two forms of biosensor for inorganic phosphate (Pi) have been developed, based on the phosphate binding protein of Escherichia coli, and are now widely used for time-resolved measurements of enzymes that produce Pi. One form is an adduct with a single coumarin fluorophore. For the second version, two cysteine mutations were introduced and labeled with a rhodamine. When physically close to each other and appropriately orientated, two rhodamine dyes can interact to form a non-covalent dimer. In this state they have little or no fluorescence, unlike the high fluorescence intensity of monomeric rhodamine. The labeling sites were so placed that the distance and orientation between the rhodamines changes with the conformation change associated with Pi binding. This movement alters the extent of interaction between rhodamines and gives a large fluorescence increase as Pi binds. In both these forms, Pi binds rapidly and tightly, making them good probes for rapid reaction assays, where all Pi can be bound to the biosensor: that is, the probe must be at least stoichiometric with the Pi. The rhodamine version has a large advantage over coumarin in terms of sensitivity and photobleaching. However, to make the probe more useful for different assay formats, such as high-throughput, the biosensor would best be used sub-stoichiometrically. This is more efficient in terms of usage amounts and has the potential of measuring over a wider range of Pi concentrations. To achieve this, several strategies were examined to locate mutations on this two domain protein to weaken the Pi binding while maintaining the fluorescence response.