Normal vibrations of ternary DOPC/DPPC/cholesterol lipid bilayers by low-frequency Raman spectroscopy

Abstract

A lipid bilayer containing a ternary mixture of low- and high-melting lipids and cholesterol (Chol) can give rise to domain formation, referred to as lipid rafts. Low-frequency Raman spectroscopy at reduced temperatures allows detection of normal membrane mechanical vibrations. In this work, Raman spectra were obtained in the spectral range between 5 and 90 cm^-1 for bilayers prepared from dioleoyl-glycero-phosphocholine (DOPC), dipalmitoyl-glycero-phosphocholine (DPPC) and Chol. A narrow peak detected between 13 and 16 cm^-1 was attributed to the vibrational eigenmode of a lipid monolayer (a leaflet). For the equimolar DOPC/DPPC ratio, the Chol concentration dependence for the peak position, width and amplitude may be divided into three distinct ranges: below 9 mol%, the intermediate range between 9 mol% and 38 mol%, and above 38 mol%. In the intermediate range the peak position attains its minimum, and the peak width drops approximately by a factor of two as compared with the Chol-free bilayers. Meanwhile, this range is known for raft formation in a fluid state. The obtained results may be interpreted as evidence that bilayer structures in the raft-containing fluid state may be frozen at low temperatures. The drop of peak width indicates that at the spatial scale of the experiment (∼2.5 nm) the intermolecular bilayer structure with raft formation becomes more homogeneous and more cohesive.

Fig. 1. Phase diagram of the DOPC/DPPC/Chol ternary system at 18 °C retrieved from literature data. The vertical line corresponds to the compositions studied in this work. The grey triangle with vertexes A, B and C corresponds to the compositions where the Ld, Lo and g phases are present; see text for meaning of the A′ and B′ points.

Fig. 2. Low-frequency Raman spectra of cholesterol-free DOPC/DPPC bilayers obtained at 100 K and 170 K. In the inset the experimental spectrum at T = 100 K (circles) is approximated by four Lorentzians shown by dashed, dotted and two solid lines, with their sum presented by the solid line drawn through the circles.

Fig. 3. The peak position (A) and the peak width (B) versus Chol concentration at 100 K (black squares) and 170 K (red triangles). The solid straight-line segments are drawn to guide the eye, and the dotted lines are predictions of eqn (3) (see text).

Fig. 4. The peak amplitude versus Chol concentration at 100 K. Three solid straight-line segments are drawn to guide the eye, and the dashed lines are predictions of eqn (3) (see text).

Fig. 5. Left: The circles show experimental Raman spectra for the DOPC/DPPC/Chol bilayers taken at 100 K for the compositions A, B, and C of the triangle vertexes shown in Fig. 1. Data are shifted upwards for convenience. The solid lines drawn through the experimental points are fits by a sum of Loretzians (cf.Fig. 2). The insert shows the Lorentzians describing the peak between 13 and 16 cm⁻¹, for the A, B and C compositions. Right: the peak weights a, b and c found from eqn (4), as a function of Chol concentration.