The pH-dependent photophysical and spectral properties of pH-sensing green fluorescent proteins

Protonation and deprotonation reactions significantly influence biological function, with buffers and transporters maintaining stable pH levels in the cytosol and various organs. However, pH can vary considerably in organ lumens, such as the airway surface liquid (ASL), which serves as a defense barrier for airways. Traditional microelectrodes, while precise, face limitations prompting the use of fluorescent pH sensors. Although many green fluorescent protein (GFP)-based pH sensors have been reported, data are usually limited to their spectral properties. Photophysical properties are often reported for a single pH value. In this study, the photophysical properties of pHluorins and superfolder GFP-including a novel pH sensor pHluorin4-were compared. Purified pHluorin4 effectively reported ASL pH differences in cystic fibrosis (CF) and non-CF epithelia. These findings provide a comprehensive summary of the photophysical and spectral properties of popular pHluorins, including the new member pHluorin4, which can be used to optimize their application in different systems.