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

## TL;DR

This paper studies how pH affects the light properties of pH-sensitive fluorescent proteins, introducing a new sensor useful for measuring pH in airway surfaces.

## Contribution

The study introduces pHluorin4, a novel pH sensor, and provides detailed photophysical and spectral data for pHluorins.

## Key findings

- pHluorin4 effectively reports pH differences in airway surface liquid of cystic fibrosis and non-CF epithelia.
- Photophysical properties of pHluorins and superfolder GFP were compared across varying pH levels.
- The findings offer a comprehensive resource for optimizing pHluorin applications in biological systems.

## Abstract

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.