Influence of Charges on Lipid Membrane‐Incorporation Behaviors of Artificial Channels

Comprehensive Summary The influence of charges on cell membrane‐incorporation behaviors of channel proteins remains incompletely understood due to the structural complexity of these proteins. In this study, the influence was investigated by using asymmetric unimolecular artificial channels as simplified models. The study revealed a profound influence of terminal charge on interaction strength and kinetics. The negatively charged Ch⁻ exhibited significantly stronger interactions with lipid bilayers, demonstrated by an 83% membrane incorporation efficiency, compared to only 16% for the positively charged Ch⁺ . Fluorescence correlation spectroscopy (FCS) and confocal microscopy confirmed that Ch⁻ rapidly inserts and redistributes within the membrane, while Ch⁺ shows slow, sporadic incorporation, attributed to electrostatic repulsion from positively charged lipid headgroups. Furthermore, the terminal charge critically dictated the channel's orientation within the bilayer. Using a fluorescence quenching assay, it was revealed that Ch⁻ predominantly adopts an outward‐facing configuration (83%). In contrast, Ch⁺ favors an inward‐facing orientation (only 19% outward). This orientation is driven by the initial anchoring of the hydrophobic end for Ch⁺ , while for Ch⁻ , electrostatic attraction facilitates a charged‐end‐first insertion. Single‐particle tracking via total internal reflection fluorescence (TIRF) microscopy provided further kinetic insights: Ch⁻ aggregates adsorbed quickly and underwent dynamic redistribution, whereas Ch⁺ displayed rigid, immobilized binding once incorporated. This work unequivocally establishes molecular charge as a fundamental design parameter controlling the adsorption kinetics, binding stability, and ultimate orientation of artificial channels in lipid membranes. These findings provide essential principles for guiding the rational design of next‐generation membrane‐active therapeutic agents and biomimetic sensors.