Biological membranes play a vital role in cell functioning, providing structural integrity, controlling signal transduction, and controlling the transport of various chemical species. Owing to the complex nature of biomembranes, the self-assembly of lipids in aqueous media has been utilized to develop model systems mimicking the lipid bilayer structure, paving the way to elucidate the mechanisms underlying various biological processes, as well as to develop a number of biomedical and technical applications. The hydration properties of lipid bilayers are crucial for their activity in various cellular processes. Of particular interest is the local membrane dehydration, which occurs in membrane fusion events, including neurotransmission, fertilization, and viral entry. The lack of universal technique to evaluate the local hydration state of the membrane components hampers understanding of the molecular-level mechanisms of these processes. Here, we present a new approach to quantify the hydration state of lipid bilayers. It takes advantage of the change in the lateral diffusion of lipids that depends on the number of water molecules hydrating them. Using fluorescence recovery after photobleaching technique, we applied this approach to planar single and multicomponent supported lipid bilayers. The method enables the determination of the hydration level of a biomimetic membrane down to a few water molecules per lipid.
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