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

Abstract

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.

Illuminating Cellular Biochemistry: Fluorogenic Chemogenetic Biosensors for Biological Imaging

“…Engineering of hCRPBII combined with the use of a julolidine retinal analog further allowed the development of a ratiometric pH sensor. [80] The sensor takes advantage of the sensitivity of the bound chromophore to the electrostatic environment within the binding cavity. The absorption profile changes as a function of the protonation state of a carboxylic side chain in proximity of the bound iminium.…”

Engineering Glowing Chemogenetic Hybrids for Spying on Cells

“…The presence of a titratable functional group close to the chromophore binding pocket renders both the absorption and fluorescence emission sensitive to pH change. [97] Fluorescent chemogenetic reporters can also be used creatively to generate new types of biosensors. By coupling chemogenetic reporters with a sensing domain, it is possible to condition the fluorogen binding (and thus fluorescence) to the recognition of the analyte.…”

Human Cellular Retinol Binding Protein II Forms a Domain‐Swapped Trimer Representing a Novel Fold and a New Template for Protein Engineering

“…Human cellular retinol binding protein II (hCRBPII) chaperones both retinol and retinal within the cell. Our group has engineered these templates to create rhodopsin mimics used to understand protein‐based absorption tuning, protein‐directed photoisomerization pathways, [35–37] new classes of fluorescent and photoswitchable fluorescent protein tags, [38] and pH sensors [39,40] . We recently reported that some variants of hCRBPII give rise to domain‐swapped (DS) dimers [41] .…”

“…Our group has engineered these templates to create rhodopsin mimics used to understand protein-based absorption tuning, protein-directed photoisomerization pathways, [35][36][37] new classes of fluorescent and photoswitchable fluorescent protein tags, [38] and pH sensors. [39,40] We recently reported that some variants of hCRBPII give rise to domain-swapped (DS) dimers. [41] We then exploited the hCRBPII DS dimer as a design template to engineer a new class of protein conformational switches, activated by ligand binding or environmental reduction potential, characteristics lacking in the monomeric fold of the protein.…”