Curvature, Order, and Dynamics of Lipid Hexagonal Phases Studied by Deuterium NMR Spectroscopy

Solid-state deuterium (²H) NMR spectroscopy enables one to study both equilibrium and dynamical properties of membrane constituents at the molecular level and can yield significant insights regarding the organization of non-bilayer lipid aggregates. We have investigated a representative unsaturated phosphatidylethanolamine, viz., l-perdeuteriopalmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine, PLPE-d₃₁, in the lamellar, or L_α, phase and the reversed hexagonal, or H₁₁, phase. Phosphorus-31 (³¹P) NMR studies of PLPE-d₃₁ in the H₁₁ phase revealed that the chemical shift anisotropy of the phosphoethanolamine head groups, Δσ, was scaled by the expected geometrical factor of-1/2 relative to the lamellar state. However, we found the occurrence of a further reduction in the ²H NMR quadrupolar splittings, Δv_Q, of the ²H-labeled palmitoyl acyl chain segments. These observations point toward the role of interfacial curvature with regard to properties of reverse hexagonal phase lipids, and indicate that the pivotal position or neutral surface of approximately constant area may lie near the glycerol or polar head group region. Variations in the acyl chain packing due to curvature of the aqueous interface yield significant differences in the segmental order profiles as determined by ²H NMR spectroscopy. The latter reflect the local orientational order of the acyl chains and can be used together with simple statistical theories to extract positional or structural information. Average projected acyl chain lengths and mean interfacial or cross-sectional areas for PLPE-d₃₁ in the different phases have been calculated. In addition, we describe a new means of estimating the radius of curvature of H₁₁ phase lipid aggregates utilizing ²H NMR spectroscopy, which is based on the difference between the lamellar and hexagonal phase order profiles. Here the radius of curvature, R_c, is defined as the distance from the center of the water core to the lipid/water interface, near the carbonyl segments of the acyl chains, giving R_c = 25.4–28.1 Å for PLPE-d₃₁ in the H₁₁ phase at 60 °C. This value is in good agreement with previous X-ray diffraction studies of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Alternatively, the data yield for the radius of the central water core that R_w = 17.8–20.5 Å at 60 °C. The differences in geometry also lead to higher quadrupolar echo relaxation rates (R_2e) for the lipid acyl segments closest to the aqueous interface in the H₁₁ versus the L_α phase. We propose that this enhancement is due to an additional relaxation mechanism found in the hexagonal phases, namely, translational diffusion of lipids about the cylinder axes. For comparison, the normal hexagonal (H₁ and lamellar (L_α) phases of a lyotropic system comprising perdeuterated potassium laurate were also studied. This research indicates clearly that the packing and dynamical properties of the acyl chains of phospholipids depend on the curvature of the aqueous interface and, thus, the aggregate geometry. The latter is related to the average shape of lipids in their respective phases and to the curvature free energy, which in the planar state may influence protein-mediated functions of membranes.