Cholesterol is a lipid that plays a key role mainly in the plasma membrane of animal cells, determining their structural and mechanical properties. While Langmuir monolayers have been a traditional model for studying these membranes, they may not fully capture microscopic phenomena. We studied mixed Langmuir monolayers of DPPC and cholesterol in various proportions, using a Langmuir trough for surface compression isotherms and micro-Brewster Angle Microscopy (MicroBAM) for imaging. Furthermore, atomistic Molecular Dynamics (MD) simulations were used to examine molecular distributions, geometric conformations, and emergence of a gas phase in the monolayers. Both experimental and simulation results were compared to obtain a comprehensive understanding. The results reveal strong attraction between DPPC tails and cholesterol, increasing monolayer stiffness and density. These interactions align the lipid tails more perpendicularly to the water surface, effectively minimizing their projected area and preventing tail entanglement. Consequently, the reduced flexibility triggers a phase separation in the monolayer at smaller areas per lipid, with low-density voids and denser regions, as observed in both MicroBAM and simulations. These outcomes emphasize the critical role of cholesterol in biological membranes. Finally, the consistency of MD simulations with experiments validate them as a valuable tool for investigating lipid monolayer behavior.
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