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Identification of mNeonGreen as a pH‐Dependent, Turn‐On Fluorescent Protein Sensor for Chloride
Author(s) -
Tutol Jasmine N.,
Kam Hiu C.,
Dodani Sheel C.
Publication year - 2019
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201900147
Subject(s) - fluorescence , chromophore , chloride , chemistry , residue (chemistry) , green fluorescent protein , tyrosine , fluorescent protein , dansyl chloride , guanidinium chloride , biophysics , photochemistry , biochemistry , organic chemistry , biology , chromatography , mass spectrometry , physics , quantum mechanics , gene , enzyme , derivatization
Chloride‐sensitive fluorescent proteins generated from laboratory evolution have a characteristic tyrosine residue that interacts with a chloride ion and π‐stacks with the chromophore. However, the engineered yellow‐green fluorescent protein mNeonGreen lacks this interaction but still binds chloride, as seen in a recently reported crystal structure. Based on its unique coordination sphere, we were curious if chloride could influence the optical properties of mNeonGreen. Here, we present the structure‐guided identification and spectroscopic characterization of mNeonGreen as a turn‐on fluorescent protein sensor for chloride. Our results show that chloride binding lowers the chromophore p K a and shifts the equilibrium away from the weakly fluorescent phenol form to the highly fluorescent phenolate form, resulting in a pH‐dependent, turn‐on fluorescence response. Moreover, through mutagenesis, we link this sensing mechanism to a non‐coordinating residue in the chloride binding pocket. This discovery sets the stage to further engineer mNeonGreen as a new fluorescent protein‐based tool for imaging cellular chloride.