Discovery of a monomeric green fluorescent protein sensor for chloride by structure-guided bioinformatics

Chloride is an essential anion for all forms of life. Beyond electrolyte balance, an increasing body of evidence points to new roles for chloride in normal physiology and disease. Over the last two decades, this understanding has been advanced by chloride-sensitive fluorescent proteins for imaging a...

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Bibliographic Details
Published in:	Chemical science (Cambridge) Vol. 13; no. 43; pp. 12659 - 12672
Main Authors:	Peng, Weicheng, Maydew, Caden C, Kam, Hiu, Lynd, Jacob K, Tutol, Jasmine N, Phelps, Shelby M, Abeyrathna, Sameera, Meloni, Gabriele, Dodani, Sheel C
Format:	Journal Article
Language:	English
Published:	England Royal Society of Chemistry 09-11-2022 The Royal Society of Chemistry
Subjects:	Binding Bioinformatics Chemistry Chloride Chlorides Chromophores Electrolytic cells Fluorescence Oligomerization Proteins Sensors
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Summary:	Chloride is an essential anion for all forms of life. Beyond electrolyte balance, an increasing body of evidence points to new roles for chloride in normal physiology and disease. Over the last two decades, this understanding has been advanced by chloride-sensitive fluorescent proteins for imaging applications in living cells. To our surprise, these sensors have primarily been engineered from the green fluorescent protein (GFP) found in the jellyfish Aequorea victoria . However, the GFP family has a rich sequence space that could already encode for new sensors with desired properties, thereby minimizing protein engineering efforts and accelerating biological applications. To efficiently sample this space, we present and validate a stepwise bioinformatics strategy focused first on the chloride binding pocket and second on a monomeric oligomerization state. Using this, we identified GFPxm163 from GFPxm found in the jellyfish Aequorea macrodactyla . In vitro characterization shows that the binding of chloride as well as bromide, iodide, and nitrate rapidly tunes the ground state chromophore equilibrium from the phenolate to the phenol state generating a pH-dependent, turn-off fluorescence response. Furthermore, live-cell fluorescence microscopy reveals that GFPxm163 provides a reversible, yet indirect readout of chloride transport via iodide exchange. With this demonstration, we anticipate that the pairing of bioinformatics with protein engineering methods will provide an efficient methodology to discover and design new chloride-sensitive fluorescent proteins for cellular applications. We developed a workflow to identify and apply GFPxm163 as a new green fluorescent protein-based sensor for chloride.
Bibliography:	https://doi.org/10.1039/d2sc03903f Electronic supplementary information (ESI) available. See DOI ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	2041-6520 2041-6539
DOI:	10.1039/d2sc03903f