Probing the Orientation and Membrane Permeation of Rhodamine Voltage Reporters through Molecular Dynamics and Free Energy Calculations

The transmembrane potential of plasma membranes and membrane-bound organelles plays a fundamental role in cellular functions such as signal transduction, ATP synthesis, and homeostasis. Rhodamine Voltage Reporters (RhoVRs), which operate based on Photoinduced electron Transfer (PeT) mechanisms, are non-invasive, small-molecule voltage sensors that can detect rapid voltage changes, with some of them specifically targeting the inner mitochondrial membrane. In this work, we conducted extensive molecular dynamics simulations and free energy calculations to investigate the orientation of three RhoVRs. Our results indicate that the relative positioning of their polarized headgroups dictates RhoVRs' alignment with the membrane normal, thereby, significantly affecting their voltage sensitivity. Free energy calculations of the three RhoVRs in different membrane systems identified an extra energy barrier for RhoVR1 compared to SPIRIT RhoVR1, explaining their distinct cellular localization profiles. A divide-and-conquer approach employed to speed up the free energy calculation provides additional insight into the physicochemical properties governing the membrane permeation of RhoVRs. The connection between chemical composition and membrane orientation as well as permeation behaviors of RhoVRs revealed by these calculations provides general guidance to the design of PeT-based fluorescent dyes with enhanced voltage sensitivity and desired cellular localization.