Understanding the voltage-sensitivity of Arch3-based microbial rhodopsins

The visualization of membrane potentials in living cells allows new insights into the function of neurons and, in particular, the brain. Although a lot of effort has been made during the last years, this task still remains very challenging facing problems such as the low signal intensity. The recently reported voltage-dependent fluorescence in archaerhodopsin-3 (Arch3) offered a new very encouraging basis to develop new optogenetic sensors. Both voltage-sensitivity and fluorescence quantum yields, however, are very low which makes the tuning of this property necessary for application in living rodents. Current approaches focused on this aim used high-throughput techniques without taking the mechanistic understanding on an atomic level into account. In our study, we predicted the voltage response of known Arch3 derivatives such as Arch2, Arch3, Archon1 and its mutants using the atomistic molecular dynamics (MD) based computational electrophysiology approach at the nanosecond to microsecond time scale. The simulations indicated a significant rearrangement of the hydrogen bonding network including the retinal Schiff base and water molecules upon voltage. Based on these findings, so far unrecognized point mutants interrupting the hydrogen bonding network showed an alternative voltage-sensing mechanism. Instead of the well-known arginine R92 adjacent to the counter ions, an intracellular aspartic acid D125 in helix D changed its orientation as response to changes in the applied voltage. The side-chain orientation of D125 was in turn modulated by the voltage-dependent dynamics of the intracellular part of helix D. The mutation D125N which interrupted the aforementioned reorientation in the MD simulations was validated experimentally and showed a higher voltage-sensitive fluorescence. This knowledge on the molecular level will be used in the future to further deepen the understanding of the voltage-sensing mechanism and to rationally predict new generation of fluorescence voltage sensors with improved optogenetic properties.