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 analogue for the purpose of developing a ratiometric pH sensor. The design relied on the electrostatic influence of a titratable amino acid side chain, which affects the absorption and, thus, the 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.