Discovery and Characterization of a Naturally Occurring, Turn-On Yellow Fluorescent Protein Sensor for Chloride

摘要： Fluorescent proteins have been extensively engineered and applied as optical indicators for chloride in a variety of biological contexts. Surprisingly, given the biodiversity of ?uorescent proteins, a naturally occurring chloride sensor has not been reported to date. Here, we present the identi?cation and spectroscopic characterization of the yellow ?uorescent protein from the jelly?sh Phialidium sp. (phiYFP), a rare example of a naturally occurring, excitation ratiometric, and turn-on ?uorescent protein sensor for chloride. Our results show that chloride binding tunes the pKa of the chromophore Y66 and shifts the equilibrium from the ?uorescent phenolate form to the weakly ?uorescent phenol form. The latter likely undergoes excited-state proton transfer to generate a turn-on ?uorescence 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.