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

Chloride is a vital ion for all forms of life. Protein-based fluorescent biosensors can enable researchers to visualize chloride in cells but remain underdeveloped. Here, we demonstrate how a single point mutation in an engineered microbial rhodopsin results 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 diverse biological contexts.

[1]  Jun O. Liu,et al.  ATP-releasing SWELL1 channel in spinal microglia contributes to neuropathic pain , 2023, Science advances.

[2]  C. Akerman,et al.  Intracellular chloride regulation mediates local sleep pressure in the cortex , 2022, Nature Neuroscience.

[3]  F. Morcos,et al.  Coupling a Live Cell Directed Evolution Assay with Coevolutionary Landscapes to Engineer an Improved Fluorescent Rhodopsin Chloride Sensor. , 2022, ACS synthetic biology.

[4]  L. Brown.  Light-driven proton transfers and proton transport by microbial rhodopsins - A biophysical perspective. , 2022, Biochimica et biophysica acta. Biomembranes.

[5]  A. Trevelyan,et al.  Genetically encoded sensors for Chloride concentration , 2021, Journal of Neuroscience Methods.

[6]  O. Béjà,et al.  Microbial Rhodopsins: The Last Two Decades. , 2021, Annual review of microbiology.

[7]  M. Shoulders,et al.  Directed evolution in mammalian cells , 2021, Nature Methods.

[8]  A. Flood,et al.  Polarity-Tolerant Chloride Binding in Foldamer Capsules by Programmed Solvent-Exclusion. , 2021, Journal of the American Chemical Society.

[9]  Sheel C. Dodani,et al.  The design and evolution of fluorescent protein-based sensors for monoatomic ions in biology. , 2021, Protein engineering, design & selection : PEDS.

[10]  Yamuna Krishnan,et al.  Proton-activated chloride channel PAC regulates endosomal acidification and transferrin receptor-mediated endocytosis , 2021, Cell reports.

[11]  I. Shin,et al.  A mitochondrial Cl--selective fluorescent probe for biological applications. , 2020, Analytical chemistry.

[12]  A. Cohen,et al.  Multiplexed Optical Sensors in Arrayed Islands of Cells for multimodal recordings of cellular physiology , 2020, Nature Communications.

[13]  De‐Xian Wang,et al.  Artificial Chloride-Selective Channel: Shape and Function Mimic of the ClC Channel Selective Pore. , 2020, Journal of the American Chemical Society.

[14]  Yamuna Krishnan,et al.  What biologists want from their chloride reporters – a conversation between chemists and biologists , 2020, Journal of Cell Science.

[15]  S. Muallem,et al.  Cl- as a bona fide signalling ion , 2019, American journal of physiology. Cell physiology.

[16]  J. Marvin,et al.  Quantitative in vivo imaging of neuronal glucose concentrations with a genetically encoded fluorescence lifetime sensor , 2019, Journal of neuroscience research.

[17]  G. Duflos,et al.  Use of Ratiometric Probes with a Spectrofluorometer for Bacterial Viability Measurement. , 2018, Journal of microbiology and biotechnology.

[18]  Jasmine N. Tutol,et al.  Discovery and Characterization of a Naturally Occurring, Turn-On Yellow Fluorescent Protein Sensor for Chloride. , 2018, Biochemistry.

[19]  Joel M. Kralj,et al.  Voltage-gated calcium flux mediates Escherichia coli mechanosensation , 2017, Proceedings of the National Academy of Sciences.

[20]  O. Yizhar,et al.  Optogenetic control of mitochondrial metabolism and Ca2+ signaling by mitochondria-targeted opsins , 2017, Proceedings of the National Academy of Sciences.

[21]  K. Tye,et al.  A light- and calcium-gated transcription factor for imaging and manipulating activated neurons , 2017, Nature Biotechnology.

[22]  Takeharu Nagai,et al.  Dependence of fluorescent protein brightness on protein concentration in solution and enhancement of it , 2016, Scientific Reports.

[23]  K. Deisseroth.  Optogenetics: 10 years of microbial opsins in neuroscience , 2015, Nature Neuroscience.

[24]  G. Whitesides,et al.  Interactions between Hofmeister anions and the binding pocket of a protein. , 2015, Journal of the American Chemical Society.

[25]  Stanford Schor,et al.  Directed evolution of Gloeobacter violaceus rhodopsin spectral properties. , 2015, Journal of molecular biology.

[26]  Philip A. Gale,et al.  Synthetic ion transporters can induce apoptosis by facilitating chloride anion transport into cells. , 2014, Nature chemistry.

[27]  Frances H. Arnold,et al.  Directed evolution of a far-red fluorescent rhodopsin , 2014, Proceedings of the National Academy of Sciences.

[28]  Christopher A. Werley,et al.  Screening Fluorescent Voltage Indicators with Spontaneously Spiking HEK Cells , 2013, PloS one.

[29]  Manfred T Reetz,et al.  Reducing codon redundancy and screening effort of combinatorial protein libraries created by saturation mutagenesis. , 2013, ACS synthetic biology.

[30]  Kenrick Berend,et al.  Chloride: the queen of electrolytes? , 2012, European journal of internal medicine.

[31]  D. Maclaurin,et al.  Optical recording of action potentials in mammalian neurons using a microbial rhodopsin , 2011, Nature Methods.

[32]  Adam E. Cohen,et al.  Electrical Spiking in Escherichia coli Probed with a Fluorescent Voltage-Indicating Protein , 2011, Science.

[33]  Y. Shichida,et al.  Multiple functions of Schiff base counterion in rhodopsins , 2010, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[34]  Michael A. Henninger,et al.  High-Performance Genetically Targetable Optical Neural Silencing via Light-Driven Proton Pumps , 2009, Nature.

[35]  Sreekanth H. Chalasani,et al.  Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators , 2009, Nature Methods.

[36]  L. Brown,et al.  The photocycle and proton translocation pathway in a cyanobacterial ion-pumping rhodopsin. , 2009, Biophysical journal.

[37]  Fabio Beltram,et al.  Spectroscopic and structural study of proton and halide ion cooperative binding to gfp. , 2007, Biophysical journal.

[38]  J. Watson,et al.  A novel main-chain anion-binding site in proteins: the nest. A particular combination of phi,psi values in successive residues gives rise to anion-binding sites that occur commonly and are found often at functionally important regions. , 2002, Journal of molecular biology.

[39]  Yizhak Marcus,et al.  A simple empirical model describing the thermodynamics of hydration of ions of widely varying charges, sizes, and shapes , 1994 .

[40]  J. Wieth,et al.  Organotin-mediated exchange diffusion of anions in human red cells , 1979, The Journal of general physiology.