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

Peng, Weicheng; Maydew, Caden; Kam, Hiu; Lynd, Jacob K.; Tutol, Jasmine N.; Phelps, Shelby M; Abeyrathna, Sameera; Meloni, Gabriele; Dodani, Sheel C.

doi:10.1039/d2sc03903f

Cited by 12 publications

(11 citation statements)

References 81 publications

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“…Moreover, as expected, the intracellular pH remained constant, as indicated by the BCECF fluorescence (Δ F < 10%, Figure S22). ,, …”

Section: Resultsmentioning

confidence: 99%

Engineering the ChlorON Series: Turn-On Fluorescent Protein Sensors for Imaging Labile Chloride in Living Cells

Tutol,

Ong,

Phelps

et al. 2023

ACS Cent. Sci.

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Beyond its role as the "queen of electrolytes", chloride can also serve as an allosteric regulator or even a signaling ion. To illuminate this essential anion across such a spectrum of biological processes, researchers have relied on fluorescence imaging with genetically encoded sensors. In large part, these have been derived from the green fluorescent protein found in the jellyfish Aequorea victoria. However, a standalone sensor with a turn-on intensiometric response at physiological pH has yet to be reported. Here, we address this technology gap by building on our discovery of the anion-sensitive fluorescent protein mNeonGreen (mNG). The targeted engineering of two non-coordinating residues, namely K143 and R195, in the chloride binding pocket of mNG coupled with an anion walking screening and selection strategy resulted in the ChlorON sensors: ChlorON-1 (K143W/R195L), ChlorON-2 (K143R/R195I), and ChlorON-3 (K143R/R195L). In vitro spectroscopy revealed that all three sensors display a robust turn-on fluorescence response to chloride (20-to 45-fold) across a wide range of affinities (K d ≈ 30−285 mM). We further showcase how this unique sensing mechanism can be exploited to directly image labile chloride transport with spatial and temporal resolution in a cell model overexpressing the cystic fibrosis transmembrane conductance regulator. Building from this initial demonstration, we anticipate that the ChlorON technology will have broad utility, accelerating the path forward for fundamental and translational aspects of chloride biology.

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“…Moreover, as expected, the intracellular pH remained constant, as indicated by the BCECF fluorescence (Δ F < 10%, Figure S22). ,, …”

Section: Resultsmentioning

confidence: 99%

Engineering the ChlorON Series: Turn-On Fluorescent Protein Sensors for Imaging Labile Chloride in Living Cells

Tutol,

Ong,

Phelps

et al. 2023

ACS Cent. Sci.

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“…Moreover, protein engineering can be used to rapidly diversify the encoding sequence space and enrich for hosts with desired properties (e.g., selectivity, affinity) [19–21] . To decode these supramolecular principles, we are actively investigating, engineering, and applying protein‐based hosts for anions [22–28] . Along these lines, we have a growing program focused on anion sensors derived from the green fluorescent protein (GFP) family.…”

Section: Introductionmentioning

confidence: 99%

Rational Design of the β‐Bulge Gate in a Green Fluorescent Protein Accelerates the Kinetics of Sulfate Sensing**

Ong

Pathiranage

et al. 2023

Angew Chem Int Ed

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Detection of anions in complex aqueous media is a fundamental challenge with practical utility that can be addressed by supramolecular chemistry. Biomolecular hosts such as proteins can be used and adapted as an alternative to synthetic hosts. Here, we report how the mutagenesis of the β-bulge residues (D137 and W138) in mNeonGreen, a bright, monomeric fluorescent protein, unlocks and tunes the anion preference at physiological pH for sulfate, resulting in the turn-off sensor SulfOFF-1. This unprecedented sensing arises from an enhancement in the kinetics of binding, largely driven by position 138. In line with these data, molecular dynamics (MD) simulations capture how the coordinated entry and gating of sulfate into the β-barrel is eliminated upon mutagenesis to facilitate binding and fluorescence quenching.

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“…25 As part of our efforts in this area, we have expanded beyond the GFP family and are actively investigating other classes of chromophore-containing proteins such as fluorescent microbial rhodopsins. [26][27][28][29][30][31] The rhodopsin family is a diverse group of transmembrane, photoactive proteins with a covalently bound Schiff base chromophore (SBC) formed from the condensation of a conserved lysine residue with retinal (Fig. 1).…”

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confidence: 99%

Unlocking chloride sensing in the red at physiological pH with a fluorescent rhodopsin-based host

Phelps

Tutol

Advani

et al. 2023

Preprint

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Chloride is a vital ion for all forms of life. Nature has evolved elegant supramolecular machines to facilitate chloride transport across cell membranes. Protein-based fluorescent biosensors can enable researchers to intercept and monitor chloride in living cells but remain underdeveloped. Here, we demonstrate how this can be achieved through the introduction of a single point mutation in an engineered, microbial rhodopsin from the archaebacterium Halorubrum sodomense resulting in ChloRED-1-CFP. This membrane-bound host is a far-red emitting, ratiometric sensor that provides a reversible readout of chloride in live bacteria at physiological pH, setting the stage to investigate the roles of chloride in a wide range of biological contexts.

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

Abstract: 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...

Cited by 12 publications

References 81 publications

Engineering the ChlorON Series: Turn-On Fluorescent Protein Sensors for Imaging Labile Chloride in Living Cells

Engineering the ChlorON Series: Turn-On Fluorescent Protein Sensors for Imaging Labile Chloride in Living Cells

Rational Design of the β‐Bulge Gate in a Green Fluorescent Protein Accelerates the Kinetics of Sulfate Sensing**

Unlocking chloride sensing in the red at physiological pH with a fluorescent rhodopsin-based host

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