| Popis: |
Chloride is a ubiquitous ion essential for regulating biological processes. Long thought to be just a counterion in biological systems, chloride is emerging as a vital anion that plays a major role in a range of diseases. The ability to visualize intracellular chloride has been enabled by fluorescent protein-based sensors. Although the yellow fluorescent protein from Aequorea victoria, and variants thereof, quench in the presence of chloride and other anions, they have provided valuable insights in biological studies and have established the benchmark for applying protein-based sensors for chloride. However, a single domain fluorescent protein sensor that generates a turn-on response to provide a spatial and temporal map of endogenous chloride has yet to be reported. Here, we highlight how mutagenesis of non-coordinating residues in the binding pocket of mNeonGreen has unlocked this new sensing capability. Our protein engineering efforts coupled with in vitro spectroscopy have led us to discover the ChlorON series as turn-on fluorescent sensors for chloride that operate at physiological pH. Fluorescence microscopy experiments with the ChlorON sensors reveal that cells have baseline levels of chloride, which could not have been directly observed with turn-off sensors. These advancements set the stage for visualizing endogenous chloride dynamics in real-time and uncovering new roles for chloride in biology.Abstract Figure |
