Semirational Protein Engineering Yields Archaerhodopsin-3-Based Fluorescent Genetically Encoded Voltage Indicators with Enhanced Brightness and Red Shifted Absorption Bands

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 pKa 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.