A single point mutation converts a proton-pumping rhodopsin into a red-shifted, turn-on fluorescent sensor for chloride

Tutol, Jasmine N.; Lee, Jessica; Chi, Hsichuan; Faizuddin, Farah; Abeyrathna, Sameera; Qin, Zhou; Morcos, Faruck; Meloni, Gabriele; Dodani, Sheel C.

doi:10.1039/d0sc06061e

Cited by 22 publications

(60 citation statements)

References 97 publications

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“…3-fold higher ratio change for GR2-CFP versus GR1-CFP. 21 However, the K d for chloride binding to GR2-CFP (53 ± 14 mM) is comparable to that of GR1-CFP (42 ± 1 mM, Figure S10). 21 Interestingly, even though we did not select for an expanded operational pH, the mutations along the proton-pumping pathway did result in measurable chloride sensing at pH 6 (Figure S14).…”

Section: Discussionmentioning

confidence: 87%

“…In comparison to GR1-CFP, GR2-CFP has a little to no background fluorescence from the rhodopsin (Figure 3, Figure 4A). 21 This in combination with the larger dynamic range, translates into a ca . 3-fold higher ratio change for GR2-CFP versus GR1-CFP.…”

Section: Discussionmentioning

confidence: 99%

“…25 As we previously reported, the non-natural D121V mutation in GR1-CFP eliminates proton-pumping activity and confers the fluorescence response to chloride at pH 5. 21,23 In GR2-CFP, the enrichment of positively charged lysine residues at positions 132 and 84 provides a ca . 2-fold higher fluorescence ratio change relative to GR1-CFP (Figure 3, Figure S7, Figure S8).…”

Section: Discussionmentioning

confidence: 99%

“…The pET-21a(+) based plasmid encoding GR1-CFP was generated in a prior study. 21 Three forward primers and one reverse primer were synthesized using the 22c-trick (Sigma-Aldrich) to generate each SSM library as previously described (Table S2). 21,59 E. cloni EXPRESS BL21 (DE3) cells (Lucigen) were transformed with each library and expressed in a 96-well deep well format.…”

Section: Methodsmentioning

confidence: 99%

“…Site-saturation mutagenesis at D121 resulted in the identification of GR1-CFP where mutation to valine not only resulted in a turn-on fluorescence response to 400 mM chloride (ΔF GR /F CFP ≈ 2.0 at pH 5) but also eliminated pumping activity in live E. coli . 21…”

Section: Introductionmentioning

confidence: 99%

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Coupling a Live Cell Directed Evolution Assay with Coevolutionary Landscapes to Engineer an Improved Fluorescent Rhodopsin Chloride Sensor

Chi

Qin

Tutol

et al. 2022

Preprint

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Our understanding of chloride in biology can be accelerated through the application of fluorescent protein-based sensors in living cells. Laboratory-guided evolution can be used to diversify and identify sensors with specific properties. Recently, we established that the fluorescent proton-pumping rhodopsin wtGR from Gloeobacter violaceus can be converted into a fluorescent sensor for chloride. To unlock this non-natural function, a single point mutation at the Schiff counterion position from D121V was introduced into wtGR fused with cyan fluorescent protein (CFP) resulting in GR1-CFP. Here, we have integrated coevolutionary analysis with directed evolution to understand how the rhodopsin sequence space can be explored and engineered to improve this starting point. We first show how evolutionary couplings are predictive of functional sites in the rhodopsin family and how a fitness metric based on sequence can be used to quantify known proton-pumping activities of GR-CFP variants. Then, we couple this ability to predict potential functional outcomes with a screening and selection assay in live Escherichia coli to reduce the mutational search space of five residues along the proton-pumping pathway in GR1-CFP. This iterative selection process results in GR2-CFP with four additional mutations, E132K, A84K, T125C, and V245I. Finally, bulk, and single fluorescence measurements in live E. coli reveal that GR2-CFP is a reversible, ratiometric fluorescent sensor for extracellular chloride with an improved dynamic range. We anticipate that our framework will be applicable to other systems, providing a more efficient methodology to engineer fluorescent-protein based sensors with desired properties.

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Section: Discussionmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Coupling a Live Cell Directed Evolution Assay with Coevolutionary Landscapes to Engineer an Improved Fluorescent Rhodopsin Chloride Sensor

Chi

Qin

Tutol

et al. 2022

Preprint

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

Ong

Pathiranage

et al. 2023

Angewandte Chemie

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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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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

Self Cite

View full text Add to dashboard Cite

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.

show abstract

A single point mutation converts a proton-pumping rhodopsin into a red-shifted, turn-on fluorescent sensor for chloride

Abstract: By utilizing laboratory-guided evolution, we have converted the fluorescent proton-pumping rhodopsin GR from Gloeobacter violaceus into GR1, a red-shifted, turn-on fluorescent sensor for chloride.

Cited by 22 publications

References 97 publications

Coupling a Live Cell Directed Evolution Assay with Coevolutionary Landscapes to Engineer an Improved Fluorescent Rhodopsin Chloride Sensor

Coupling a Live Cell Directed Evolution Assay with Coevolutionary Landscapes to Engineer an Improved Fluorescent Rhodopsin Chloride Sensor

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

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

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