Single Lipid Bilayers Constructed on Polymer Cushion Studied by Sum Frequency Generation Vibrational Spectroscopy

Abstract

Planar solid supported single lipid bilayers on mica, glass, or other inorganic surfaces have been widely used as models for cell membranes. To more closely mimic the cell membrane environment, soft hydrophilic polymer cushions were introduced between the hard inorganic substrate and the lipid bilayer to completely avoid the possible substrate-lipid interactions. In this article, sum frequency generation (SFG) vibrational spectroscopy was used to examine and compare single lipid bilayers assembled on the CaF(2) prism surface and on poly (L-lactic acid) (PLLA) cushion. By using asymmetric lipid bilayers composed of a hydrogenated 1,2-dipalmitoyl-sn-glycerol-3-phosphoglycerol (DPPG) leaflet and a deuterated 1,2-dipalmitoyl-(d62)-sn-glycerol-3-phosphoglycerol (d-DPPG) leaflet, it was shown that the DPPG lipid bilayers deposited on the CaF(2) and PLLA surfaces have similar structures. SFG has also been applied to investigate molecular interactions between an antimicrobial peptide Cecropin P(1) (CP1) and the lipid bilayers on the above two different surfaces. Similar results were again obtained. This research demonstrated that the hydrophilic PLLA cushion can serve as an excellent substrate to support single lipid bilayers. We believe that it can be an important cell membrane model for future studies on transmembrane proteins, for which the possible inorganic substrate-bilayer interactions may affect the protein structure or function.

Fig. 1

Molecular structure of A) 1,2-dipalmitoyl-sn-glycerol-3-phosphoglycerol (DPPG); B) 1,2-dipalmitoyl-(d62)-sn-glycerol-3-phosphoglycerol (d-DPPG); C) poly (L-lactic acid) (PLLA).

Fig. 2

Schematic of the SFG experimental geometry

Fig. 3

SFG ssp spectra collectd from the d-DPPG (A, B) leaflet in the C-D stretching frequency region and the DPPG (C, D) leaflet in the C-H stretching frequency region. Spectra A and C were collected from the lipid bilayer deposited on the CaF₂ prism, while spectra B and D were detected from the lipid bilayer deposited on the PLLA cushion.

Fig. 4

SFG signal intensities at 2875 cm⁻¹ (for C-H signals) and at 2070 cm⁻¹ (for C-D signals) monitored as a functional of time. The signals were offset. The signals are stable for at least one hour, indicating that the bilayers are stable during the time period of the SFG experiments.

Fig. 5

Schematic showing the methyl tilt angle and the alkyl chain orientation. The upper leaflet is d-DPPG, and the lower leaflet is DPPG.

Fig. 6

SFG ssp and ppp spectra collected from CP1 associated with CaF₂ supported lipid bilayer (left) and from CP1 associated with PLLA cushion supported lipid bilayer (right). The CP1 concentrations (from top to bottom) are 12, 15, and 20 μM.

Fig. 7

SFG ssp (left) and ppp (right) spectra collected from the d-DPPG leaflet in the lipid bilayer in contact with CP1 solutions of different concentrations: Top: CaF₂ supported lipid bilayer; Bottom: PLLA cushion supported lipid bilayer.