Single-protein diffusion in micrometer-size vesicles is governed by viscosity, surface charge, and membrane interactions

Giant unilamellar vesicles (GUVs) are a powerful model system for studying protein dynamics in cell-sized compartments with a precisely tunable physicochemical environment. Here, we apply Single-Molecule Displacement Mapping (SMdM) to quantify how luminal viscosity, ionic strength, membrane lipid composition, and protein surface charge of soluble proteins govern intraluminal single-molecule diffusion. Increasing viscosity led to a predictable decrease in the diffusion coefficient. However, spatially resolved diffusion measurements reveal reduced protein mobility near the membrane, suggesting additional membrane-proximal interactions not captured by particle-based simulations. We observe long-range (∼1 µm) effects of membrane charge on protein mobility that extend well beyond classical Debye screening, which may reflect shape fluctuations of the GUV membrane. For the membrane-associated protein LactC2, we resolve two distinct diffusing populations within a single vesicle, corresponding to free and membrane-bound states. Our workflow provides a quantitative framework for probing single-protein mobility in vesicle-based synthetic cells. These results advance our understanding of how molecular crowding and transient membrane interactions shape protein diffusion within cell-sized compartments, with possible implications for metabolic organization.