Phase separation in binary mixtures of bipolar and monopolar lipid dispersions revealed by 2H NMR spectroscopy, small angle x-ray scattering, and molecular theory

Abstract

Binary mixtures of C(20)BAS and POPC membranes were studied by solid-state (2)H NMR spectroscopy and small angle x-ray scattering (SAXS) over a wide range of concentrations and at different temperatures. Three specifically deuterated C(20)BAS derivatives--[1',1',20',20'-(2)H(4)]C(20)BAS, [2',2',19',19'-(2)H(4)]C(20)BAS, and [10',11'-(2)H(2)]C(20)BAS--combined with protiated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), as well as membranes containing POPC-d(31) and fully protiated bolalipid, were used in NMR experiments to obtain structural information for the mixtures. The (2)H NMR spectra of [10',11'-(2)H(2)]C(20)BAS/POPC membrane dispersions reveal that the bolalipid is predominantly in the transmembrane conformation at high bolalipid concentrations (100, 90, and 70 mol %). At < or =50 mol % C(20)BAS, smaller quadrupolar couplings appear in the spectra, indicating the presence of U-shaped conformers. The proportion of U-shaped bolalipids increases as the amount of POPC in the membrane increases; however, the transmembrane component remains the dominant bolalipid conformation in the membrane even at 45 degrees C and 10 mol % C(20)BAS, where it accounts for approximately 50% of the bolalipid population. The large fraction of C(20)BAS transmembrane conformers, regardless of the C(20)BAS/POPC ratio, together with the findings from molecular mean-field theory calculations, suggests the coexistence of phase-separated bolalipid-rich domains and POPC-rich domains. A single lamellar repeat distance was observed in SAXS experiments corresponding to the average repeat spacing expected for C(20)BAS- and POPC-rich domains. These observations are consistent with the presence of microphase-separated domains in the mixed membrane samples that arise from POPC-C(20)BAS hydrophobic mismatch.

Figure 1

(A) Structures of POPC, C₂₀BAS, and deuterated C₂₀BAS derivatives 1–3. (B) Schematic representation of the predicted phase separation for a mixed C₂₀BAS/POPC lipid membrane. The bolalipid-rich domain consists of transmembrane bolalipids (A_T) and a small molar ratio of POPC (M). The monopolar lipid-rich domain consists of POPC with a small molar ratio of U-shaped bolalipids (A_U). (C) Predicted miscibility diagram showing the regime of lipid mixtures where phase separation between fluid phase C₂₀BAS and monopolar lipids (adapted from (42)) is expected (gray shaded area). X_A is the molar fraction of C₂₀BAS, and n_L is the number of carbons in the hydrophobic chain of the monopolar lipid. (D) Diagram of various membrane lipid configurations in different regions of the miscibility diagram. I = POPC-rich phase with U-shaped C₂₀BAS conformations; II = mixture of C₂₀BAS and POPC that generates significant hydrophobic mismatch leading to a separation into type I and III domains; III = C₂₀BAS-rich phase with modest POPC content (predominantly transmembrane C₂₀BAS with some U-conformers, especially at higher temperatures).

Figure 2

Powder-type (black line) and de-Paked (gray line) ²H NMR spectra obtained from [10′,11′-²H₂]C₂₀BAS/POPC (X_A/X_M) mixed membranes at various concentrations. Spectra were acquired at 25°C (left) and 45°C (right). Data for 1:0 [10′,11′-²H₂]C₂₀BAS/POPC at 25°C (21) are used with permission from the American Chemical Society.

Figure 3

(A) Powder-type (black line) and de-Paked (gray line) ²H NMR spectra recorded for C₂₀BAS/POPC-d₃₁ (X_A/X_M) membranes at X_A = 0.9, 0.7, 0.5, 0.3, 0.1, and 0 at 25°C. (B) Order parameter of [C6′] (□), [C9′] (×), and [C12′] (Δ) methylene units in POPC-d₃₁, as a function of C₂₀BAS mole fraction in POPC at 25°C.

Figure 4

Powder (top) and de-Paked (bottom) spectra of C₂₀BAS/POPC-d₃₁ (1:1) at 25°C, 10°C, 4°C, and −60°C.

Figure 5

Free energy per molecule, f_N, as a function of the area per lipid headgroup, a_h, for (A) pure C₂₀ BAS membranes at 17°C, the melting transition of C₂₀BAS; (B) C₂₀BAS-rich membranes (X_A = 0.9) in the gel phase at 4°C; and (C) POPC-rich membranes (X_A = 0.1) in the L_α phase at 4°C. (D) Orientational order parameters for POPC as a function of alkyl chain segment number at X_A = 0.9 in the gel phase (□) and X_A = 0.1 (•) in the L_α phase.

Figure 6

(A) SAXS patterns of C₂₀BAS/POPC vesicles for X_A = 0, 0.1, 0.3, 0.4, 0.5, 0.7, and 0.9, at 30°C. Y axis = intensity in arbitrary units. (B) D spacings of C₂₀BAS/POPC membranes for X_A = 0, 0.1, 0.3, 0.5, 0.7, 0.9, and 1.0 at 30°C.