Chloride is a ubiquitous ion essential for regulating biological processes. Long thought to be just a counterion in biological systems, chloride is emerging as a vital anion that plays a major role in a range of diseases. The ability to visualize intracellular chloride has been enabled by fluorescent protein-based sensors. Although the yellow fluorescent protein from Aequorea victoria, and variants thereof, quench in the presence of chloride and other anions, they have provided valuable insights in biological studies and have established the benchmark for applying protein-based sensors for chloride. However, a single domain fluorescent protein sensor that generates a turn-on response to provide a spatial and temporal map of endogenous chloride has yet to be reported. Here, we highlight how mutagenesis of non-coordinating residues in the binding pocket of mNeonGreen has unlocked this new sensing capability. Our protein engineering efforts coupled with in vitro spectroscopy have led us to discover the ChlorON series as turn-on fluorescent sensors for chloride that operate at physiological pH. Fluorescence microscopy experiments with the ChlorON sensors reveal that cells have baseline levels of chloride, which could not have been directly observed with turn-off sensors. These advancements set the stage for visualizing endogenous chloride dynamics in real-time and uncovering new roles for chloride in biology.
Our understanding of chloride in biology has been accelerated through the application of fluorescent protein-based sensors in living cells. These sensors can be generated and diversified to have a range of properties using laboratory-guided evolution. 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 (D121V) was introduced into wtGR fused to 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 a sequence can be used to quantify the 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.
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...
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.
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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