Phase Separation in Binary Mixtures of Bipolar and Monopolar Lipid Dispersions Revealed by Solid-State 2H NMR Spectroscopy and Small Angle X-ray Scattering

Abstract

Binary mixtures of C20BAS and POPC membranes have been studied by 2H NMR spectroscopy and small angle X-ray scattering (SAXS) over a wide range of concentrations and at different temperatures. Experiments tested the possibility of formation of phase-separated lipid domains predicted by the mean field theory [1]. Membranes composed of three specifically deuterated C20BAS derivatives [1,1,20,20-2H4]C20BAS, [2,2,19,19-2H4] C20BAS, and [10,11-2H2] C20BAS with protiated POPC and with membranes containing POPC-d31 and fully protiated bolalipid were used in NMR experiments to obtain structural information for the mixture. The 2H NMR spectra of 10,11-2H2-C20BAS:POPC membrane dispersion reveal that the bolalipid is predominantly in the transmembrane conformation at high bolalipid concentrations. At 50 mole percent C20BAS and lower, components appear in the spectra with smaller quadrupolar couplings, most likely indicating the presence of U-shaped conformers. The proportion of U-shaped bolalipids becomes more prominent as the amount of POPC in the membrane increases. However, the transmembrane component is still the dominant bolalipid conformation in the membrane even at 45 °C and 10 mole percent C20BAS, where it accounts for roughly 50% of the bolalipid population. The large fraction of C20BAS transmembrane conformers regardless of the C20BAS:POPC ratio together with POPC-bolalipid hydrophobic mismatch can be explained by co-existence of bolalipid-rich domains separate from the POPC-rich domains. In SAXS experiments only a single distinct lamellar repeat distance was observed, corresponding roughly to the average of bolalipid-rich and POPC-rich domains. These observations are consistent with the presence of microphase-separated domains in the mixed membrane samples. [1] G.S. Longo et al. (2007) Biophys. J. 93, 2609.