Effect of Divalent Cations on Polarity and Hydration at the Lipid/Water Interface Probed by 4-Aminophthalimide-Based Dyes

The ion binding to the lipid/water interface can substantially influence the structural, functional, and dynamic properties of the cell membrane. Despite extensive research on ion-lipid interactions, the specific effects of ion binding on the polarity and hydration at the lipid/water interface remain poorly understood. This study explores the influence of three biologically relevant divalent cations─Mg²⁺, Ca²⁺, and Zn²⁺─on the depth-dependent interfacial polarity and hydration of zwitterionic DPPC lipid in its gel phase at room temperature. To measure these depth-dependent properties, we use a series of solvatochromic fluorescent probes synthesized based on 4-aminophthalimide with varying alkyl chain lengths (4AP-Cn; n = 5, 7, and 9). Employing steady-state fluorescence experiments and all-atom molecular dynamics (MD) simulations, we quantify changes in interfacial polarity and hydration induced by the cations binding to the lipid/water interface. Our results reveal that Zn²⁺ induces a significant blue shift in the fluorescence spectra of all 4AP-Cn dyes, indicating a marked decrease in local polarity (E_T^N ≤ 0.05) at the lipid/water interface compared to Mg²⁺ and Ca²⁺, which results in a higher polarity (E_T^N ≥ 0.2). The depth-dependent fluorescence spectra of dyes at the interface in the presence of Mg²⁺ and Ca²⁺ remain similar to those in the absence of cations, with only a minor red shift observed for Mg²⁺, implying a slight hydration effect. MD simulations show that cations primarily bind to the headgroup and glycerol regions of lipid. Simulations also reveal that Zn²⁺ causes substantial dehydration at the lipid/water interface, as detected by the 4AP-Cn dyes, while Mg²⁺ and Ca²⁺ have less pronounced effects, with only slight hydration induced by Mg²⁺. This study highlights the distinct positional effects of cations probed by 4AP-Cn probes at the lipid-water interface, underscoring the potential of 4AP-Cn dyes for monitoring depth-dependent changes in membrane properties induced by external agents or environmental conditions.