Orientation and Membrane Partition Free Energy of PeT-Based Voltage-Sensitive Dyes from Molecular Simulations

Voltage measurement via small-molecule fluorescent indicators is a valuable approach in deciphering complex dynamics in electrically excitable cells. However, our understanding of various physicochemical properties governing the performance of fluorescent voltage sensors based on the photoinduced electron transfer (PeT) mechanism remains incomplete. Here, through extensive molecular dynamics and free energy calculations, we systematically examine the orientation and membrane partition of three PeT-based voltage-sensing VoltageFluor (VF) dyes in different lipid environment. We show that the symmetry of the molecular scaffold and the net charge of the hydrophilic headgroup of a given VF dye dominate its orientation and membrane partition, respectively. Our work provides a mechanistic understanding of the physical properties contributing to the voltage sensitivity, signal-to-noise ratio, as well as membrane distribution of VF dyes and sheds light onto rational design principles of PeT-based fluorescent probes in general.