BPS2026 – Divalent cations and cholesterol modulate depth-dependent polarity and hydration at lipid/water interface

Lipid membranes are vital for cellular integrity and function, serving as dynamic platforms for biochemical processes. The binding of divalent cations and the presence of cholesterol within the lipid membrane significantly modulate the interfacial properties of lipid bilayers, yet their depth-specific effects on polarity and hydration at the sharp lipid/water interface remain poorly understood. This study investigates how biologically relevant divalent cations (Mg 2+ , Ca 2+ , and Zn 2+ ) and cholesterol affect the polarity and hydration of zwitterionic DPPC lipid bilayers in the gel phase at ambient temperature. We employed newly developed solvatochromic fluorescent probes based on 4-aminophthalamide (4AP-Cn; n = 5, 7, 9) with varying alkyl chain lengths to scan the depth-dependent changes, complemented by all-atom MD simulations. Steady-state fluorescence experiments revealed that Zn 2+ induces a pronounced blue shift in 4AP-Cn spectra, indicating a significant reduction in local polarity (≤0.05) and substantial dehydration at the lipid-water interface. Conversely, Mg 2+ and Ca 2+ exhibit milder effects, with Mg 2+ slightly increasing hydration (minor red shift) and Ca 2+ maintaining higher polarity (≈ 0.2). MD simulations show Zn 2+ strongly interacts with phosphate and glycerol regions of lipid headgroups, driving dehydration, while Mg 2+ and Ca 2+ have subtler impacts. Cholesterol, on the other hand, enhances bilayer rigidity and packing, reducing interfacial polarity and hydration, as evidenced by blue shifts in 4AP-Cn fluorescence spectra. MD results confirm that cholesterol alters water orientation and hydrogen-bonding near headgroups, promoting dehydration. The 4AP-Cn probes, with increasing penetration depth as alkyl chain length grows, effectively capturing sub-nanometer-scale changes in hydration and polarity. These findings highlight the distinct roles of divalent cations and cholesterol in modulating lipid-water interactions, demonstrating the ability of 4AP-Cn probes for depth-dependent biophysical studies and providing insights into membrane organization and potential applications in studying membrane-related pathologies.