Fluorescent proteins have been extensively engineered and applied as optical indicators for chloride in a variety of biological contexts. Surprisingly, given the biodiversity of fluorescent proteins, a naturally occurring chloride sensor has not been reported to date. Here, we present the identification and spectroscopic characterization of the yellow fluorescent protein from the jellyfish Phialidium sp. (phiYFP), a rare example of a naturally occurring, excitation ratiometric, and turn-on fluorescent protein sensor for chloride. Our results show that chloride binding tunes the pKa of the chromophore Y66 and shifts the equilibrium from the fluorescent phenolate form to the weakly fluorescent phenol form. The latter likely undergoes excited state proton transfer to generate a turn-on fluorescence response that is pH dependent. Moreover, anion selectivity and mutagenesis in the chloride binding pocket provide additional evidence for the proposed chloride sensing mechanism. Given these properties, we anticipate that phiYFP, with further engineering, could be a new tool for imaging cellular chloride dynamics.

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Living cells rely on a finely tuned symphony of inorganic ion gradients composed of both cations and anions. This delicate balance is maintained by biological receptors all acting in concert to selectively recognize and position ions for homeostasis. These dynamic processes can be intercepted and visualized with optical microscopy at the organismal, tissue, cellular and subcellular levels using fluorescent protein-based biosensors. Since the first report of such tool for calcium (Ca2+) in 1997, outstanding biological questions and innovations in protein engineering along with associated fields have driven the development of new biosensors for Ca2+ and beyond. In this Review, we summarize a workflow that can be used to generate fluorescent protein-based biosensors to study monoatomic ions in biology. To showcase the scope of this approach, we highlight recent advances reported for Ca2+ biosensors and in detail discuss representative case studies of biosensors reported in the last four years for potassium (K+), magnesium (Mg2+), copper (Cu2+/+), lanthanide (Ln3+) and chloride (Cl−) ions.