A Genetically Encoded Ratiometric pH-Probe: Wavelength Regulation-Inspired Design of pH Indicators.

Mutants of human Cellular Retinol Binding Protein II (hCRBPII) were engineered to bind a julolidine retinal analog for the purpose of developing a ratiometric pH sensor. The design relied on the electrostatic influence of a titratable amino acid side chain, affecting the absorption, and thus emission of the protein/fluorophore complex. The ratio of emissions obtained at two excitation wavelengths that correspond to the absorption of the two forms of the protein/fluorophore complex, leads to a concentration independent measure of pH.