Specific interactions of tryptophan with phosphatidylcholine and digalactosyldiacylglycerol in pure and mixed bilayers in the dry and hydrated state
Introduction
Living cells contain various amphiphilic compounds (e.g. phenolics and flavonoids; Rice-Evans et al., 1997), which are mainly found in the aqueous phase under fully hydrated conditions. During dehydration-induced by drying or freezing, amphiphilic substances partition from the aqueous into the lipid phase (Golovina et al., 1998, Buitink et al., 2000). The degree of partitioning into membranes depends on the water content of the cells and the hydrophobicity and chemical structure of the amphiphiles. This insertion into membranes is reversible on subsequent rehydration (Hoekstra and Golovina, 2002). The interaction between biological membranes and amphiphiles can have both positive and negative consequences for membrane stability and consequently for cell survival during desiccation (Hoekstra et al., 2001). Many amphiphiles are potent antioxidants (Larson, 1988, Rice-Evans et al., 1997) and some, such as arbutin (4-hydroxyphenyl-β-glucopyranoside) can inhibit phospholipase A2 activity under conditions of low hydration (Oliver et al., 2002). In addition, the insertion of an amphiphile into a membrane can increase the fluidity of the lipid phase, and thereby counteract the dehydration-induced increase of the gel to liquid-crystalline phase transition temperature of the membrane lipids (Jain et al., 1985, Casal et al., 1987). On the other hand, the perturbation of membrane structure could result in a destabilization of the host membrane under stress conditions (Hoekstra and Golovina, 2002).
The effect of some amphiphiles on membrane stability during drying or freezing can be profoundly affected by the lipid composition of the membrane. Arbutin, for instance, destabilizes membranes containing only bilayer lipids during drying or freezing, while membranes containing both bilayer and non-bilayer lipids are stabilized under the same conditions (Hincha et al., 1999, Oliver et al., 2001). This is due to a stabilization of the lamellar phase in the presence of non-bilayer lipids (Oliver et al., 2001).
The aromatic amino acid tryptophan (Trp) has an amphiphilic character due to the hydrophobic indole ring structure and the hydrophilic amino acid part. Therefore, although Trp is usually only present in very low concentrations in cells, it is an interesting model substance to investigate the effects of relatively hydrophilic amphiphiles on membrane stability and structure. We have previously shown that Trp destabilizes both biological and model membranes during freezing (Popova et al., 2002). The degree of destabilization, especially at low Trp concentrations, depends on the lipid composition of the membranes. Membranes made up entirely of phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylethanolamine are very strongly affected, while much less destabilization is observed for galactolipid-containing liposomes. It was obvious from this study that the phase preference of the lipids (lamellar or hexagonal II) was not decisive for the effect of Trp, in contrast to arbutin. We hypothesized that Trp interacts differently with phospholipids and glycolipids (Popova et al., 2002).
In the present study, we have investigated the interactions of Trp with the phospholipid egg phosphatidylcholine (EPC) and the chloroplast galactolipid digalactosyldiacylglycerol (DGDG). As a comparison, we have used the hydrophilic amino acid glycine (Gly). Since Gly has only an H atom as a side chain, effects of this amino acid can be expected to result from interactions of the lipids with the hydrophilic amine or carbonic acid groups. We have chosen to use two bilayer lipids in these experiments, to avoid complications arising from different phase preferences of the membrane lipids. DGDG is a typical plant chloroplast lipid, that makes up approximately 25% of the thylakoid lipids (Webb and Green, 1991). It contains predominantly C18 fatty acids with three double bonds (Quinn and Williams, 1983, Klaus et al., 2002). The physiological role of DGDG has been investigated with knock-out mutants in the biosynthetic pathway, which showed that this lipid is essential for plant growth, thylakoid function, and protein import into chloroplasts (see Dörmann and Benning, 2002 for a recent review). It has recently been shown that under conditions of phosphate starvation, plants reduce the amount of phospholipids in favor of DGDG. Under these conditions, DGDG is also found in extraplastidial membranes and can account for up to 70% of the total plasma membrane lipids (Andersson et al., 2003). Effects on function and stability of the plasma membrane under such conditions have not been reported to date.
It has, however, been shown that up to 50% DGDG in EPC membranes has no destabilizing influence on model membranes during freezing or hyperosmotic stress (Hincha, 2003). In addition, we have shown recently (Popova and Hincha, 2003) that DGDG and EPC show strong interactions between their headgroups. This facilitates lipid mixing in mixed EPC/DGDG membranes and an extremely low gel to liquid-crystalline phase transition temperature (Tm below −20 °C) in both mixed EPC/DGDG and pure DGDG membranes in the dry state. For pure DGDG in the fully hydrated state, a Tm of −50 °C has been reported previously (Shipley et al., 1973).
Section snippets
Materials
Egg phosphatidylcholine (EPC), 1,2-dimyristoylphosphatidylcholine (DMPC), 1,2-dimyristoyl(D54)phosphatidylcholine (D54DMPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), and 1-palmitoyl(D31)-2-oleoylphosphatidylcholine (D31POPC) were obtained from Avanti Polar Lipids (Alabaster, AL). The chloroplast glycolipid digalactosyldiacylglycerol (DGDG) was purchased from Lipid Products (Redhill, Surrey, UK), Gly from Sigma, and Trp from Fluka. Diphenylhexatriene (DPH), trimethylammonium-DPH
Results
We had found in an earlier investigation (Popova et al., 2002) that Trp affects the stability of liposomes during freezing to −20 °C differently, depending on whether they contain glycolipids or not. We used several different fluorescence spectroscopical methods to investigate the interactions of Trp with fully hydrated liposomes of different lipid compositions (data not shown). The probes we used report on the dynamics of the lipids from the hydrophobic interior (DPH) to the lipid–water
Interactions of Trp with dry liposomes
The position of the symmetric CH2 stretching vibration of dry DGDG liposomes was substantially higher than that of dry EPC liposomes in both the gel and liquid crystalline phase (Fig. 2). Dry liposomes containing 50% EPC and 50% DGDG showed an intermediate behavior. In a recent paper, we have hypothesized that this is the result of a higher degree of motional freedom of fatty acyl chains in liposomes containing DGDG, not only due to the high degree of unsaturation of the fatty acids (mainly
Conclusions
In conclusion, this study shows that in dry membranes both Trp and Gly interact with the membrane lipids. However, while some of those interactions are common to both amino acids, some are unique to Trp, such as the effect on Tm in pure EPC membranes and the upshift in the CO peak in pure DGDG membranes. After rehydration, most of the effects of Gly that were observed in dry membranes were abolished. However, effects of Trp on acyl chain mobility were still present, as were the effects of Trp
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Permanent address: Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.