Changes in molecular composition and packing during lipidmembrane reconstitution from phospholipid–surfactant micelles

Abstract

Total internal reflection (TIR) Raman spectroscopy was used to investigate changes in the properties of phospholipid bilayers at the quartz–water interface during the process of membrane reconstitution from lipid–detergent micelles. The non-ionic detergent β-D-dodecyl maltoside (β-DDM) was used in 9 : 1 ratio to deuterated and non-deuterated dipalmitoylphosphatidyl choline (DPPC and d75-DPPC) to form detergent–lipid micelles. Exposure of the quartz surface to decreasing concentrations of such detergent–lipid solutions resulted in the formation of a d75-DPPC bilayer indistinguishable from that of a bilayer formed by fusion of small unilamellar vesicles of the pure lipid, indicating that the use of detergent micelles does not alter the packing or conformation of lipids, or leave a residue of surfactant in the bilayer. Polarised measurements of d75DPPC spectra in the C–H and C–D regions at each step of the process showed that the lipid surface density increases as the detergent is removed by rinsing and replaced by more lipid. The absence of shifts in the frequencies demonstrates that DPPC is in a gel-like conformation even at high relative surfactant fractions. The TIR geometry also allowed us to record high resolution spectra in the low frequency C–D bending region of d75-DPPC, which showed that the lipid molecules have an upright orientation with respect to the support surface.