Asymmetric Distribution of Lipids in a Phase Segregated Phospholipid Bilayer Observed by Sum-Frequency Vibrational Spectroscopy

Abstract

The intrinsic symmetry constraints on sum-frequency vibrational spectroscopy (SFVS) have been exploited to measure the asymmetric distribution of lipid domains in the proximal and distal layers of a planar supported lipid bilayer (PSLB) in the gel−liquid-crystalline (l.c.) coexistence region of the phase diagram for several lipid systems. Four saturated phospholipids, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (DHPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and two equal-molar binary mixtures of DSPC with DMPC, and DSPC with a unsaturated lipid DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) were investigated. The destructive interference of the symmetric stretch (vs) transition moments from the lipid fatty acid methyl groups (CH3) was used to monitor changes in the symmetry of the bilayer structure. A maximum in the CH3 vs intensity is observed at the phase transition temperature (Tm) due to the break in the local symmetry of lipid bilayers caused by the dislocation of the gel and l.c. phase domains. The SFVS results were correlated to phase segregation in the membranes as measured by fluorescence microscopy. The SFVS measured Tm for DMPC, DPPC, DHPC, and DSPC were 23.4 ± 0.9 °C, 41.0 ± 0.4 °C, 52.4 ± 0.7 °C, and 57.5 ± 0.5 °C, respectively. These values correlate well with the literature values of 23.6 ± 1.5 °C, 41.3 ± 1.8 °C, 49 ± 3 °C, and 54.5 ± 1.5 °C for DMPC obtained by differential scanning calorimetry (DSC). In addition to providing a direct spectroscopic probe of the Tm of PSLBs, these studies also provide evidence for the delocalization of the gel and l.c. domain structures between the two layers of lipid bilayers.