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

AI Article Synopsis

Article Abstract

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 GR from 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 GR 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 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 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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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9184236PMC
http://dx.doi.org/10.1021/acssynbio.2c00033DOI Listing

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