Semirational Protein Engineering Yields Archaerhodopsin-3-Based Fluorescent Genetically Encoded Voltage Indicators...

A semirational engineering of novel bright variants of archaerhodopsin-based genetically encoded voltage indicators (GEVIs) is performed. The corresponding focused protein sequence library was generated using data derived from prior directed evolution experiments, computational modeling, evolutionary information, and a physics-based theoretical framework. The proposed variants were synthesized in Escherichia coli, extracted, and purified. Their absorption spectra, fluorescence quantum yields, extinction coefficients, and pK _a of the retinal protonated Schiff base were evaluated. The brightest variants were also expressed in HEK293T cells, and the voltage-dependence of the fluorescence signal was confirmed. As a result, a series of novel GEVIs with enhanced brightness, fluorescence quantum yield, and red-shifted absorption bands have been developed. The approach proposed in this study is general and can be applied to a wide range of protein engineering problems.