Characterization of DMPC Bilayer at Silica-D2O Interface ...



Supporting Information

Facile Lipid Flip-Flop in a Phospholipid Bilayer Induced by Gramicidin A Measured by Sum-Frequency Vibrational Spectroscopy

Jin Liu, Timothy C. Anglin, and John C. Conboy*

Department of Chemistry, University of Utah, 315 South 1400 East, RM 2020, Salt Lake City, Utah 84112

1. Materials

D2O (99.9%) was purchased from Cambridge Isotope Laboratories. GC grade CHCl3 was obtained from Mallinckrodt. Gramicidin A (gA) was obtained from Fluka with a purity greater than 90%. 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-D70-sn-glycero-3-phosphocholine (DSPC-d70) and 1,2-distearoyl-D70-sn-glycero-3-phosphocholine-1,1,2,2-D4-N,N,N-trimethyl-D9 (DSPC-d83) were purchased from Avanti Polar Lipids and used without any further purification. The water used in these studies was obtained from a NanopureTM Infinity Ultrapure water system with a minimum resistivity of 18.2 Mohm-cm. An IR/UV grade fused silica hemicylindrical prism (Almaz Optics) was used as the substrate for the planar supported lipid bilayers (PSLBs). The prism was mounted onto a Teflon flow cell with a volume of 1 mL. The prism and cell were cleaned by submerging them in a solution of 70% - 18M sulfuric acid / 30% - 30% H2O2 for at least 4 hours and then rinsed with copious amounts Nanopure water. The prism was then plasma (Harrick Scientific) cleaned for 3 minutes just prior to use.

2. Determination of the Gauche Content in a DSPC/DSPC-d70 Bilayer

Figure 2 shows a SFVS spectrum of DSPC/DSPC-d70 bilayer. This spectra and all the spectra and spectral kinetic data presented in the article were obtained with the ssp polarization combinations (s-polarized sum-frequency, s-polarized visible and p-polarized IR). The gauche defect content was determined by fitting the SFVS spectrum of a DSPC/DSPC-d70 bilayer to obtain the peak areas for the CH2 vs and CH3 vs resonances. The calculated ratio for the CH2 vs/CH3 vs in a DSPC/DSPC-d70 bilayer, such as that shown in Figure 1, is 0.38.

Figure 1. SFVS spectrum of a DSPC/DSPC-d70 bilayer (black) with the corresponding peaks obtained from a nonlinear least squares fit to the spectra (gray).

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3. Infrared Spectroscopy of 2mol % gA in a DSPC Bilayer

Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was used to determine if gA was successfully incorporated into a DSPC bilayer by the LB/LS deposition technique. A germanium ATR crystal (Pike Technologies) was plasma cleaned for three minutes and annealed at 300ºC for thirty minutes in order to prepare a clean surface and generate a stable oxide layer.(1) The clean crystal was then mounted into an ATR flow cell (Pike Technologies). A Perkin Elmer Spectrum-One FTIR with a liquid nitrogen cooled MCT detector was used to obtain the spectra. The instrument was purged with dry nitrogen gas to minimize interference from CO2 and H2O. A background spectrum was obtained by averaging 256 single-beam scans with instrument settings of 1cm/s scan speed and 1.00 cm-1 resolution. The ATR crystal was then removed from the flow cell and a bilayer of DSPC with 2 mol% gA was deposited on the crystal surface by the LB/LS technique. The crystal was maintained in an aqueous environment after sample deposition and assembled into the ATR flow cell underwater to ensure sample integrity. The flow cell was then purged with D2O in order to ensure that no spectral features from H2O would be observed. The flow cell was returned to the spectrometer and purged with dry nitrogen again before obtaining a sample spectrum under the same conditions mentioned before. The sample single-beam spectrum was divided by the background single-beam spectrum in order to obtain a transmittance spectrum for the deposited bilayer sample. This spectrum was baseline corrected and converted into absorbance units, and the peaks of interest fit using Grams/AI 7.01 (Thermo Galactic).

References

1. Hayashi, S., S. Tanimoto, M. Fujii, and K. Yamamoto. 1990. Surface Oxide Layers of Si and Ge Nanocrystals. Superlattices and Microstructures 8:13-18.

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