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

Abstract

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 the photoinduced electron transfer (PeT) mechanism, 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 physicochemical properties governing the orientation as well as membrane permeation barriers of three RhoVRs. Our results indicate that the positioning of the most polarized functional group relative to the hydrophobic molecular wire dictates the alignment of RhoVRs with the membrane normal, thereby, significantly affecting their voltage sensitivity. Free-energy calculations in different membrane systems identify significantly higher barriers against the permeation of RhoVR 1 compared to SPIRIT RhoVR 1, explaining their distinct subcellular localization profiles. Subsequent free-energy calculations of the distinguishing components from the two different RhoVRs provide additional insight into the physicochemical properties governing their membrane permeation. The connection between chemical composition and membrane orientation, as well as permeation behaviors of RhoVRs revealed by our calculations provides general guiding principles for the rational design of PeT-based fluorescent dyes with enhanced voltage sensitivity and desired subcellular distribution.