De Novo Designed Proteins for Ultrafast Detection of Membrane Potential Changes

Abstract

Neuronal networks communicate through changes in membrane potentials. Optically recording these changes on the sub-microsecond timescale provides a better understanding of interneuronal communication. While organic probes that have been developed detect signals with fast temporal resolution, these probes lack cell and membrane specificity. Genetically-encoded voltage indicators (GEVIs) remedy this problem; however, these protein-based optical voltage indicators are dimmer and slower than their organic probe counterparts. Here, we present our progress on the design, development, and characterization of novel GEVIs based on artificial 4-α-helical bundle proteins, called maquettes. We demonstrated that membrane maquettes are able to bind 3 hemes upon assembly in vesicles, each with different redox midpoint potentials. To sense changes in membrane potential, we are modifying our first prototype maquettes to develop two classes of maquette GEVIs. The first class of maquette GEVIs covalently binds a fluorescent cofactor, and we are using directed evolution to improve its fluorescent signal and quantum yield. The second class of maquette GEVIs is a fusion construct of a maquette and a fluorescent protein, whose fluorescence can be modulated by a chain of voltage-sensitive hemes embedded within the maquette. Energy transfer has been demonstrated between the fluorescent protein and the membrane maquette. These maquette GEVI prototypes have been expressed in E. coli and purified for in vitro characterization. Parallel maquettes are being developed for expression in hippocampal neuronsto validate the in vitro biophysical approach in mammalian cells.