In the present paper we used both FTIR spectroscopy and DSC to determine the phase behavior and the molecular interactions of bilayer and nonbilayer lipids. This combination was necessary, as none of the methods alone would have yielded a complete picture of these dry lipid systems. The position of the νCH2
s peak in FTIR spectra is sensitive to the mobility of the fatty acyl chains and can thus be used to discriminate between lipids in the gel state and lipids in either the liquid-crystalline or nonlamellar state [43
]. Consequently, only transitions between gel phase and either of the other two phases were detected, but not transitions from liquid-crystalline to nonlamellar. In addition to the gel phase, fully saturated lipids such as DMPC can also form a solid (crystalline) phase [59
]. However, we found no evidence of an additional low-temperature phase transition in either the FTIR or DSC data. In DSC thermograms gel to liquid-crystalline and liquid-crystalline to nonlamellar transitions can be easily distinguished due to the large difference in transition enthalpy [7
]. However, with DSC interactions between lipid molecules can not be detected, making a combination of these two methods especially useful for the characterization of interactions between lipids and between lipids and interacting solutes or proteins. It should, however, be noted that neither method allows absolute phase assignments, but only records transitions between phases.
Pure, dry EPE showed one phase transition in the FTIR, corresponding to a gel to liquid-crystalline transition and an additional low enthalpy transition to HII
was detected by DSC. This sequence of transitions has also been reported for different PEs in the hydrated state [20
]. The Tm
for pure EPE was significantly lower than that determined by DSC for pure dry POPE (73°C, [60
]). In addition, the same Tm
was found for dry POPE and POPC, while we found a large difference in the Tm
of dry EPE and EPC. These differences have to be attributed to the different fatty acid composition of the lipids, as EPE and EPC are not composed of identical fatty acids (compare Methods section).
Due to the high degree of unsaturation of the fatty acyl chains of the MGDG from spinach leaves used in this study and the resultant high mobility of the chains, νCH2
s was located at high wavenumbers (above 2854 cm-1
) even at -30°C and there was no indication for a phase transition in the FTIR data. There was only one low enthalpy phase transition apparent in the DSC thermograms, indicating that dry MGDG was in the liquid-crystalline phase at low temperature and in a nonlamellar phase at high temperature. For fully hydrated MGDG previous DSC measurements have shown a transition from HII
to lamellar phase at -30°C during cooling [26
] and a lamellar to HII
transition at -44°C during heating [16
In all mixed bilayer systems (EPE/EPC, EPE/DMPC, MGDG/EPC, MGDG/DMPC) only gel to liquid-crystalline phase transitions were detected by FTIR and DSC, while the DSC thermograms indicated that the transition from liquid-crystalline to nonbilayer was completely abolished. As expected, Tm
was significantly higher in membranes containing DMPC than in those containing EPC. Only in the case of MGDG/DMPC membranes, there was an indication for a partial demixing of the two lipids during the phase transition. This was only apparent in the DSC thermograms, where two peaks were resolved, but not in the FTIR melting curves that only showed one transition. We have shown previously that the resolution of more than one lipid melting event is possible with our FTIR method [53
]. This suggests that the more unsaturated MGDG started to melt at a lower temperature than DMPC, but that the two melting events were too close together to be resolved by FTIR. In the liquid-crystalline state, the two lipids were completely mixed again, thereby preventing a further transition to HII
, that was observed in pure MGDG. Interestingly, EPE/EPC membranes showed a phase transition that was almost identical to that of EPE (and not halfway between EPE and EPC), indicating the importance of headgroup interactions for the phase behavior of this lipid mixture.
In the fully hydrated state it has been shown previously for MGDG/DMPC [64
], EPE/EPC, DOPE/DOPC [22
] and DMPC/DMPE [11
] that stable bilayers were formed, where the two lipids were well mixed and did not form separate domains. On the other hand, it has been suggested that during dehydration, when the formation of nonlamellar structures is favoured, phase separation of lipids with different phase preferences can take place, leading to freezing and dehydration induced membrane damage [32
]. The results presented here suggest that for the investigated lipid mixtures such phase separation and HII
formation did not occur during drying.
FTIR has provided evidence for complex interactions between lipid headgroups in the dry state that was observed at the C=O (all lipids), P=O (EPE, EPC, DMPC), choline (EPC, DMPC) and sugar OH (MGDG) levels. These interactions are made possible by the strong tilt of the headgroups in PC [66
] and PE [67
] towards the membrane surface in the hydrated state, which is increased during dehydration, as shown for PC [68
]. For MGDG, some results also suggest a headgroup orientation almost parallel to the surface of the lipid bilayer [28
], while other studies indicated that β-anomeric linkages of monosacharides, as in MGDG, lead to an orientation of the sugar away from the surface of fully hydrated membranes [69
]. The strong involvement of the MGDG headgroup in H-bonding interactions that we found in the present study indicates that at least in the dry state the headgroup is bent towards the membrane surface, similar to the DGDG headgroup [70
]. A comparison of the interaction patterns of the different functional groups across all relevant lipid combinations yielded results consistent with this assumption.
The ethanolamine in PE has been shown previously to form a network of strong H-bonds and electrostatic interactions with P=O groups [9
]. These interactions result in a relatively low frequency of the νP=Oas vibration compared to PC. In the dry state νP=Oas of DMPC is situated at 1262 cm-1
, while in the fully hydrated state it is shifted to around 1220 cm-1
]. For hydrated DMPE the frequency of the P=O vibration was found at 1217 cm-1
], while for dry PE values between 1231 cm-1
and 1234 cm-1
] were reported, in good agreement with the value of 1230 cm-1
measured here for EPE.
The νP=Oas of mixed membranes containing EPE/EPC or EPE/DMPC had intermediate positions, in agreement with recent data on hydrated EPE/EPC mixtures [11
]. The spectra also indicate that the interactions of ethanolamine groups were limited to the P=O groups and that access to the C=O groups was strongly restricted. This was reflected in the position of the C=O peak, which was highest in pure EPE and in the A C=OH-bonded
ratio, which also showed the lowest value for pure EPE. In the mixed membranes, this ratio increased, indicating increased H-bonding of the ethanolamine groups to the C=O groups with a concomitant decrease in the interactions with the P=O groups. A similar increase was also observed in H-bonding to the choline groups, in agreement with a general redistribution of the interaction pattern between pure EPE and EPE/EPC mixtures. H-bonding to the C=O groups was strikingly lower in membranes containing DMPC than in those containing EPC, indicating that the tight packing of the membranes in the presence of a fully saturated lipid restricted access of other groups to the C=O moieties.
The OH groups in the galactose headgroup of MGDG H-bond effectively to the C=O, P=O and choline groups. In pure MGDG the fraction of C=O groups involved in H-bonding was more than twice that of free C=O and also the mixtures with PCs showed high A C=OH-bonded
ratios. Similarly, these mixtures showed reduced νP=Oas and increased νN+
as values in comparison to pure EPC and DMPC, indicating H-bonding with the galactose OH groups. Additional evidence for these H-bonding interactions was found in the massive shift of the νOH vibration by more than 100 wavenumbers in the mixed membranes compared to pure MGDG. A similar pattern of H-bonding has previously been shown in dry samples of mixed EPC/DGDG liposomes [39
While the H-bonding with the C=O groups in the mixed membranes was always weaker in the presence of DMPC than in the presence of EPC, the degree of lipid unsaturation was less important for interactions with the P=O groups. The phosphate group is situated further away from the hydrophobic core of the membrane than the carbonyl esters and therefore the packing of the acyl chains and the area per lipid molecule have less influence on the degree of interaction.