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Channel Rhodopsins01:11

Channel Rhodopsins

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Most organisms use photoreceptors to sense and respond to light. Examples of photoreceptors include bacteriorhodopsins and bacteriophytochromes in some bacteria, phytochromes in plants, and rhodopsins in the photoreceptor cells of the vertebral retina. The light-sensitive property of these receptors is because of the bound chromophores, such as bilin in the phytochromes and retinal in the rhodopsins.
Rhodopsins belong to the family of cell surface proteins called G-protein coupled receptors,...
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Directed Evolution of a Genetically Encoded Indicator for Chloride.

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    Researchers engineered a new chloride indicator, ChlorOFF, from a non-sensing fluorescent protein using directed evolution. This advancement expands the toolkit for visualizing chloride in biological systems.

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    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Protein Engineering

    Background:

    • The green fluorescent protein (GFP) family is a versatile platform for developing biological sensors.
    • Chloride indicators based on GFP have been developed over the past 25 years.
    • Directed evolution has not yet been fully utilized for engineering GFP-based chloride sensors.

    Purpose of the Study:

    • To engineer a novel, functional chloride indicator using directed evolution.
    • To demonstrate the power of random mutagenesis and anion walking for protein engineering.
    • To create a new tool for visualizing chloride dynamics in living cells.

    Main Methods:

    • Utilized random mutagenesis and anion walking techniques.
    • Engineered a chloride-insensitive fluorescent protein (OFPxm) into a functional chloride indicator (ChlorOFF).
    • Analyzed the mutational landscape to identify key amino acid changes.

    Main Results:

    • Successfully engineered ChlorOFF, a functional chloride indicator, from OFPxm.
    • Identified an evolutionary convergent solution at one position within the anion binding pocket.
    • Discovered nine additional mutations across eight positions, with only one previously associated with chloride sensing.

    Conclusions:

    • Directed evolution is a powerful strategy for engineering novel fluorescent protein biosensors.
    • The identified mutations provide insights into the structural basis of chloride sensing in GFPs.
    • ChlorOFF represents a valuable new tool for studying chloride-related biological processes.