Single-protein diffusion in micrometer-size vesicles is governed by viscosity, surface charge, and membrane interactions
- 1University of Groningen
Abstract
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 um) 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.
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BibTeX
@misc{coenradij2026singleprotein,
title = {{Single-protein diffusion in micrometer-size vesicles is governed by viscosity, surface charge, and membrane interactions}},
author = {J. Coenradij and D. Linnik and K. Postmus and Z. Sensan and M. Accorsi and C. M. Punter and R. Dimova and B. Poolman},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.21.760703},
url = {https://www.biorxiv.org/content/10.64898/2026.10.21.760703v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Single-protein diffusion in micrometer-size vesicles is governed by viscosity, surface charge, and membrane interactions
AU - J. Coenradij
AU - D. Linnik
AU - K. Postmus
AU - Z. Sensan
AU - M. Accorsi
AU - C. M. Punter
AU - R. Dimova
AU - B. Poolman
PY - 2026
DA - 2026/10/21
PB - bioRxiv
DO - 10.64898/2026.10.21.760703
UR - https://www.biorxiv.org/content/10.64898/2026.10.21.760703v1
AB - 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 um) 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.
ER -