A Single Point Mutation Converts a Proton-pumping Rhodopsin into a Turn-on Fluorescent Sensor for Chloride

Abstract

<p>The visualization of chloride in living cells with fluorescent sensors is linked to our ability to design hosts that can overcome the energetic penalty of desolvation to bind chloride in water. Fluorescent proteins can be used as biological supramolecular hosts to address this fundamental challenge. Here, we showcase the power of protein engineering to convert the fluorescent proton-pumping rhodopsin GR from <i>Gloeobacter violaceus</i> into GR1, a turn-on fluorescent sensor for chloride in detergent micelles and in live <i>Escherichia coli</i>. This non-natural function was unlocked by mutating D121, which serves as the counterion to the protonated retinylidene Schiff base chromophore. Substitution from aspartate to valine at this position (D121V) creates a binding site for chloride. The addition of chloride tunes the p<i>K</i><sub>a </sub>of the chromophore towards the protonated, fluorescent state to generate a pH-dependent response. Moreover, ion pumping assays combined with bulk fluorescence and single cell fluorescence microscopy experiments with <i>E. coli</i>, expressing a GR1 fusion with cyan fluorescent protein, show that GR1 does not pump ions nor sense membrane potential but instead provides a reversible, ratiometric readout of chloride. This discovery sets the stage to use natural and laboratory-guided evolution to build a family of rhodopsin fluorescent chloride sensors for cellular applications and learn how proteins can evolve and adapt to bind anions in water.</p>

Strategies of Detecting Bacteria Using Fluorescence-Based Dyes

“…For example, Dodani et al reported a chloride-sensitive fluorescent probe 39 comprising a counterion-mutated rhodopsin (GR1) fused to a cyan fluorescent protein (CFP) (Tutol et al, 2021). Rhodopsin is a transmembrane of chloride ion pump composed of a retinylidene Schiff base chromophore (SBC) bonded to seven α-helices (Figure 20A).…”

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

“…23,[41][42][43][44] These efforts are part of our larger program aimed at decoding the sequence level determinants of biological anion recognition. [45][46][47] In our rst report in 2019, we used a global similarity analysis with reference to avYFP-H148Q to identify a homologous sequence in the jellysh Phialidium sp. that encodes for a dimeric yellow uorescent protein called phiYFP.…”