Binary mixtures of C20BAS and POPC membranes were studied by solid-state 2H NMR spectroscopy and small angle x-ray scattering (SAXS) over a wide range of concentrations and at different temperatures. Three specifically deuterated C20BAS derivatives-[1′,1′, 20′,20′-2H4]C20BAS, [2′,2′,19′,19′-2H4]C 20BAS, and [10′,11′-2H2]C 20BAS - combined with protiated 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), as well as membranes containing POPC-d31 and fully protiated bolalipid, were used in NMR experiments to obtain structural information for the mixtures. The 2H NMR spectra of [10′,11′-2H2]C20BAS/POPC membrane dispersions reveal that the bolalipid is predominantly in the transmembrane conformation at high bolalipid concentrations (100, 90, and 70 mol %). At ≤50 mol % C20BAS, 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°C and 10 mol % C20BAS, where it accounts for ∼50% of the bolalipid population. The large fraction of C20BAS transmembrane conformers, regardless of the C20BAS/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 C20BAS-and POPC-rich domains. These observations are consistent with the presence of microphase-separated domains in the mixed membrane samples that arise from POPC-C20BAS hydrophobic mismatch.
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