Semirational Protein Engineering Yields Archaerhodopsin-3-Based Fluorescent Genetically Encoded Voltage Indicators
Dmitrii M Nikolaev1,2, Ekaterina M Metelkina1, Nikita A Domskii1
1Institute of Chemistry, Saint Petersburg State University, 26 Universitetskii pr, St. Petersburg 198504, Russia.
Chem & Bio Engineering
|October 29, 2025
Summary
Researchers engineered brighter archaerhodopsin-based genetically encoded voltage indicators (GEVIs). This semirational approach yielded novel GEVIs with improved optical properties and voltage sensitivity for cellular imaging.
Area of Science:
- Biophysics
- Molecular Biology
- Protein Engineering
Background:
- Genetically encoded voltage indicators (GEVIs) are crucial tools for monitoring cellular electrical activity.
- Archaerhodopsin-based GEVIs offer unique spectral properties but often require optimization for brightness and sensitivity.
Purpose of the Study:
- To engineer novel, brighter variants of archaerhodopsin-based GEVIs.
- To enhance fluorescence quantum yield and red-shift absorption bands for improved optical imaging.
Main Methods:
- Semirational engineering combining directed evolution data, computational modeling, and evolutionary information.
- Protein library synthesis in *Escherichia coli*, followed by purification and characterization.
- Evaluation of spectral properties (absorption, quantum yield, extinction coefficient) and pKa.
- Expression in HEK293T cells to confirm voltage-dependent fluorescence.
Main Results:
- Development of a focused protein sequence library based on diverse data sources.
- Characterization of novel GEVIs demonstrating enhanced brightness and fluorescence quantum yield.
- Confirmation of red-shifted absorption bands and voltage sensitivity in cellular expression systems.
Conclusions:
- A series of novel, brighter archaerhodopsin-based GEVIs with enhanced optical properties were successfully developed.
- The semirational engineering approach is versatile and applicable to broader protein engineering challenges.
- These improved GEVIs hold potential for advanced neuroscience and cellular imaging applications.
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