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

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 ( v s ) transition moments from the lipid fatty acid methyl groups (CH 3 ) was used to monitor changes in the symmetry of the bilayer structure. A maximum in the CH 3 v s intensity is observed at the phase transition temperature ( T m ) 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 T m 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 T m 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.