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

Abstract

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.

Front Cover: Identification of mNeonGreen as a pH‐Dependent, Turn‐On Fluorescent Protein Sensor for Chloride (ChemBioChem 14/2019)

“…[6,15] Interestingly,E 2 GFP and its derivatives operate via as tatic quenching mechanism with no shifts in the chromophore equilibriuma nd have completely differentc hloride binding pockets from avYFP-H148Qi nw hich the chloride ion directly interacts with the chromophore( Figure 1B). [19][20][21] The residues in the putative chloride binding pocket of phiYFP are identicala nd in as imilara rrangement to that observed for avYFP-H148Q ( Figure 1C). (phiYFP) as an aturally occurring, turn-on fluorescent protein sensorf or chloride.…”

“…[5,6,8,21] We speculate that this unique sensingm echanism could be linked to the fact that mNeonGreen has more ionizable residues in the chloride binding pocket that could be positively charged in the presence of chloride at pH 4.5, thus stabilizing both the phenolate form of the chromophore and the coordination complex. [5,6,8,21] We speculate that this unique sensingm echanism could be linked to the fact that mNeonGreen has more ionizable residues in the chloride binding pocket that could be positively charged in the presence of chloride at pH 4.5, thus stabilizing both the phenolate form of the chromophore and the coordination complex.…”

“…(phiYFP) as an aturally occurring, turn-on fluorescent protein sensorf or chloride. [21] The discoveryo fp hiYFP inspired us to ask if af luorescent protein with ad ifferent chloride binding pocket and potentially different properties could have evolved in Nature.Through a Protein Data Bank (PDB) search, [23] we uncovered the crystal structure of the tetrameric yellow fluorescent protein lanYFP from the cephalochordate Branchiostomal anceolatum with two foldingm utations, V171A and N174T,b ound to chloride ( Figure 1D). [22] Like avYFP-basedc hloride sensors, phiYFPi sp H-dependent, and chloride binding shifts the chromophore Y66 equilibrium from the fluorescent phenolate form to the weakly fluorescent phenol form.…”

Identification of mNeonGreen as a pH‐Dependent, Turn‐On Fluorescent Protein Sensor for Chloride

“…To expand on this body of work, we recently identified and spectroscopically characterized the yellow fluorescent protein from the jellyfish Phialidium sp. (phiYFP) as a naturally occurring, turn‐on fluorescent protein sensor for chloride . The residues in the putative chloride binding pocket of phiYFP are identical and in a similar arrangement to that observed for avYFP‐H148Q (Figure C) .…”

“…Like avYFP‐based chloride sensors, phiYFP is pH‐dependent, and chloride binding shifts the chromophore Y66 equilibrium from the fluorescent phenolate form to the weakly fluorescent phenol form. But in the case of phiYFP, the phenol form likely undergoes an excited state proton transfer to regenerate the fluorescent phenolate form, giving rise to the turn‐on fluorescence response …”