Designing Neuronal Optical Voltage-Sensing Probes using Artificial Proteins

Abstract

Communication between neurons in neuronal networks is associated with changes in neuronal membrane potentials. The ability to image these changes on sub-ms timescales is vital for better understanding of inter-neuronal connections. Inorganic probes have been developed for this purpose and proved to be fast enough to detect neuronal action potentials with sufficient time resolution. However, these probes non-selectively partition into membranes other than plasma membranes and they cannot be targeted to specific cells. Protein-based optical voltage-sensing probes can address this problem, but current genetically encoded voltage indicators (GEVIs) lack behind the inorganic probes in speed and amplitude. Here, we present the design and characterization of new sensors that are based on artificial proteins (maquettes). These artificial membrane maquettes contain four membrane-spanning α-helices that are linked into a single protein chain and form electron transfer chain across a lipid bilayer. To facilitate in vitro characterization, they have been expressed in high yields in inclusion bodies of E. coli, purified and refolded in charged and uncharged detergent micelles, as well as in lipid vesicles. Circular dichroism studies revealed 70% α-helicity in SDS and no melting in high temperatures and common denaturants, indicating their strong structural stability. The maquettes assemble in different membrane environments and bind 3 hemes upon assembly in vesicles (two hemes strongly with 150 nM affinity and third one more weakly with kd ∼ 1 µM). The redox midpoint potentials of the three hemes at pH=8.0 are −38, −150, and −203 mV, suggesting that they will be in mixed oxidation states when incorporated in resting plasma membranes. We are currently conjugating these maquettes with fluorescent proteins and nanoparticles to enable fast electron and energy transfers that will lead to strong fluorescent signals deltaF/F as a function of transmembrane voltage.