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Creators/Authors contains: "Dodani, Sheel C."

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  1. Free, publicly-accessible full text available April 15, 2023
  2. Free, publicly-accessible full text available February 16, 2023
  3. 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 Gloeobacter violaceus into GR1, a red-shifted, turn-on fluorescent sensor for chloride in detergent micelles and in live Escherichia coli . 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 binding of chloride tunes the p K a 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 E. coli , expressing a GR1 fusion with a cyan fluorescent protein, show that GR1 does not pump ions nor sense membrane potential but instead provides a reversible, ratiometric readout of changes in extracellular chloride at the membrane. This discovery sets the stage to use natural and laboratory-guidedmore »evolution to build a family of rhodopsin-based fluorescent chloride sensors with improved properties for cellular applications and learn how proteins can evolve and adapt to bind anions in water.« less
  4. Natural and laboratory-guided evolution has created a rich diversity of fluorescent protein (FP)-based sensors for chloride (Cl − ). To date, such sensors have been limited to the Aequorea victoria green fluorescent protein (avGFP) family, and fusions with other FPs have unlocked ratiometric imaging applications. Recently, we identified the yellow fluorescent protein from jellyfish Phialidium sp. (phiYFP) as a fluorescent turn-on, self-ratiometric Cl − sensor. To elucidate its working mechanism as a rare example of a single FP with this capability, we tracked the excited-state dynamics of phiYFP using femtosecond transient absorption (fs-TA) spectroscopy and target analysis. The photoexcited neutral chromophore undergoes bifurcated pathways with the twisting-motion-induced nonradiative decay and barrierless excited-state proton transfer. The latter pathway yields a weakly fluorescent anionic intermediate , followed by the formation of a red-shifted fluorescent state that enables the ratiometric response on the tens of picoseconds timescale. The redshift results from the optimized π–π stacking between chromophore Y66 and nearby Y203, an ultrafast molecular event. The anion binding leads to an increase of the chromophore p K a and ESPT population, and the hindrance of conversion. The interplay between these two effects determines the turn-on fluorescence response to halides such as Cl −more »but turn-off response to other anions such as nitrate as governed by different binding affinities. These deep mechanistic insights lay the foundation for guiding the targeted engineering of phiYFP and its derivatives for ratiometric imaging of cellular chloride with high selectivity.« less