Thermotropic phase behavior and headgroup interactions of the nonbilayer lipids phosphatidylethanolamine and monogalactosyldiacylglycerol in the dry state

Abstract

Background: Although biological membranes are organized as lipid bilayers, they contain a substantial fraction of lipids that have a strong tendency to adopt a nonlamellar, most often inverted hexagonal (HII) phase. The polymorphic phase behavior of such nonbilayer lipids has been studied previously with a variety of methods in the fully hydrated state or at different degrees of dehydration. Here, we present a study of the thermotropic phase behavior of the nonbilayer lipids egg phosphatidylethanolamine (EPE) and monogalactosyldiacylglycerol (MGDG) with a focus on interactions between the lipid molecules in the interfacial and headgroup regions.

Results: Liposomes were investigated in the dry state by Fourier-transform Infrared (FTIR) spectroscopy and Differential Scanning Calorimetry (DSC). Dry EPE showed a gel to liquid-crystalline phase transition below 0°C and a liquid-crystalline to HII transition at 100°C. MGDG, on the other hand, was in the liquid-crystalline phase down to -30°C and showed a nonbilayer transition at about 85°C. Mixtures (1:1 by mass) with two different phosphatidylcholines (PC) formed bilayers with no evidence for nonbilayer transitions up to 120°C. FTIR spectroscopy revealed complex interactions between the nonbilayer lipids and PC. Strong H-bonding interactions occurred between the sugar headgroup of MGDG and the phosphate, carbonyl and choline groups of PC. Similarly, the ethanolamine moiety of EPE was H-bonded to the carbonyl and choline groups of PC and probably interacted through charge pairing with the phosphate group.

Conclusions: This study provides a comprehensive characterization of dry membranes containing the two most important nonbilayer lipids (PE and MGDG) in living cells. These data will be of particular relevance for the analysis of interactions between membranes and low molecular weight solutes or soluble proteins that are presumably involved in cellular protection during anhydrobiosis.

Figure 1

Lipid melting curves determined as the temperature dependent increase in the position of the CH₂ symmetric stretching vibration band (νCH₂s) of the fatty acyl chains in FTIR spectra. Samples contained either the pure lipids or binary lipid mixtures at a 1:1 mass ratio as indicated in the panels. Phase transition temperatures (T_m) were determined as the midpoints of the melting curves and are shown in Table 1.

Figure 2

DSC heating thermograms of dry samples. Panel (A) shows data for pure EPE (a) and for dry liposomes containing 50% EPE/50% EPC (b), pure EPC (c), pure DMPC (d) and 50% EPE/50% DMPC (e). Panel (B) shows data for dry pure MGDG (a) and for dry liposomes containing 50% MGDG/50% EPC (b) and 50% MGDG/50% DMPC (c). Thermograms are from the second heating scan from -30°C to 120°C. Phase transition temperatures (T_m and T_hex) and transition enthalpies (ΔH) are shown in Table 1.

Figure 3

Infrared spectra in the carbonyl stretching region. Spectra were taken from dry EPE and from dry liposomes containing pure EPC, pure DMPC, 50% EPE/50% EPC and 50% EPE/50% DMPC (A) and from dry MGDG and from dry liposomes containing pure EPC, pure DMPC, 50% MGDG/50% EPC and 50% MGDG/50% DMPC (B). Spectra were measured at 90°C, except for DMPC at 100°C.

Figure 4

Ratio between the fitted peak areas of the two component bands of the carbonyl stretching peaks shown in Fig. 3. The ratio A C=O_H-bonded/A C=O_free, indicating the relative amount of H-bonding to the lipid C=O groups, is shown for the pure lipids and all binary mixtures. The values represent the means ± SE from at least 3 different samples.

Figure 5

νP=Oas peak positions determined from dry samples containing the indicated lipid compositions (compare Fig. 3). The values represent the means ± SE from at least 3 different samples.

Figure 6

νN⁺(CH₃)₃as peak positions determined from dry samples containing the indicated lipid compositions (compare Fig. 3). The values represent the means ± SE from at least 3 different samples.

Figure 7

Infrared spectra in the OH stretching vibration (νOH) region of the galactose headgroups of MGDG in pure MGDG and the indicated binary mixtures.

Figure 8

νOH of the galactose headgroups of MGDG in pure MGDG and in the indicated binary mixtures (compare Fig. 7) as a function of temperature. The peak positions determined from the two binary mixtures were almost identical at all temperatures and therefore only the symbols for one lipid composition are visible.