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Electron spin echo envelope modulation (ESEEM) spectroscopy was used to investigate binary mixtures of single-chain micelle-forming lipids and diacyl bilayer-forming lipids dispersed in D 2 O at 77 K. Mixtures of dipalmitoylphosphatidylcholine (DPPC) and lyso-palmitoylphosphatidylcholine (Lyso-PPC) over the entire composition range (0–100 mol%) and phosphatidylcholine spin-labeled at selected carbon atom position along the sn-2 chain (n-PCSL) were considered. On increasing the content of the lysolipids incorporated in DPPC, the lipid bilayers are first transformed in interdigitated lamellae and then converted in micelles of Lyso-PPC. In the interdigitated phase, the profile of translamellae water accessibility is rather uniform as all the hydrocarbon segments are equally exposed to the solvent. In Lyso-PPC micelle, water penetrates at any depth of the hydrocarbon region with a tendency to increase toward the chain termini. The extent of water penetration is higher in the interdigitated DPPC/Lyso-PPC dispersions than in Lyso-PPC micelles. The profiles of water permeation revealed directly by D 2 O-ESEEM are also confirmed by more indirect evaluation of the polarity profiles based on the 14 N-hyperfine splitting in the conventional electron paramagnetic resonance spectra of n-PCSL in frozen DPPC/Lyso-PPC mixtures at 77 K. The ESEEM data reveal that H-bonding formation between the –NO group of the spin-label and the D 2 O molecules is favored in the intergitated phase with respect to the micellar phase and, in any lipid dispersion, the fraction of nitroxides that are singly H-bonded to deuterons is higher than the fraction that are doubly H-bonded. The overall results highlight the differences in the accessibility and properties of the solvent in the hydrocarbon region of lipid bilayers, interdigitated bilayers and micelles.

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