Specific interactions of tryptophan with phosphatidylcholine and digalactosyldiacylglycerol in pure and mixed bilayers in the dry and hydrated state

https://doi.org/10.1016/j.chemphyslip.2004.06.003Get rights and content

Abstract

Amphiphilic solutes play an important role in the desiccation tolerance of plant cells, because they can reversibly partition into cellular membranes during dehydration. Their effects on membrane stability depend on their chemical structure, but also on the lipid composition of the host membrane. We have shown recently that tryptophan destabilizes liposomes during freezing. The degree of destabilization depends on the presence of glycolipids in the membranes, but not on the phase preference (bilayer or non-bilayer) of the lipids in mixtures with the bilayer lipid phosphatidylcholine. Here, we have investigated the influence of tryptophan on the phase behavior and intermolecular interactions in dry and hydrated bilayers made from the phospholipid egg phosphatidylcholine and the plant chloroplast glycolipid digalactosyldiacylglycerol, or from a mixture (1:1) of these lipids, using Fourier-transform infrared spectroscopy. To distinguish effects of the hydrophobic ring structure of tryptophan from those of the amino acid moiety, we also performed experiments with the hydrophilic amino acid glycine. Our data show that there are specific interactions between tryptophan and either phospholipid or glycolipid in the dry state, as well as H-bonding interactions between the lipids and both solutes. In the rehydrated state, the H-bonding interactions between amino acids and lipids are mostly replaced by interactions between water and lipids, while the hydrophobic interactions between lipids and tryptophan mostly persist.

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 Cdouble bondO 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

References (50)

  • F.A. Hoekstra et al.

    Mechanisms of plant desiccation tolerance

    Trends Plant Sci.

    (2001)
  • F.A. Hoekstra et al.

    The role of amphiphiles

    Comp. Biochem. Physiol.

    (2002)
  • W. Hübner et al.

    Interactions at the lipid–water interface

    Chem. Phys. Lipids

    (1998)
  • M.K. Jain et al.

    Effect of tryptophan derivatives on the phase properties of bilayers

    Biochim. Biophys. Acta

    (1985)
  • R.A. Larson

    The antioxidants of higher plants

    Phytochemistry

    (1988)
  • R.N.A.H. Lewis et al.

    The structure and organization of phospholipid bilayers as revealed by infrared spectroscopy

    Chem. Phys. Lipids

    (1998)
  • R.C. MacDonald et al.

    Small-volume extrusion apparatus for preparation of large, unilamellar vesicles

    Biochim. Biophys. Acta

    (1991)
  • H.H. Mantsch et al.

    Phospholipid phase transitions in model and biological membranes as studied by infrared spectroscopy

    Chem. Phys. Lipids

    (1991)
  • A.E. Oliver et al.

    Arbutin inhibits PLA2 in partially hydrated model systems

    Biochim. Biophys. Acta

    (1996)
  • A.E. Oliver et al.

    Interactions of arbutin with dry and hydrated bilayers

    Biochim. Biophys. Acta

    (1998)
  • A.E. Oliver et al.

    The effect of arbutin on membrane integrity during drying is mediated by stabilization of the lamellar phase in the presence of nonbilayer-forming lipids

    Chem. Phys. Lipids

    (2001)
  • A.E. Oliver et al.

    Looking beyond sugars: the role of amphiphilic solutes in preventing adventitious reactions in anhydrobiotes at low water contents

    Comp. Biochem. Physiol.

    (2002)
  • C. Paré et al.

    Differential scanning calorimetry and 2H nuclear magnetic resonance and Fourier transform infrared spectroscopy studies of the effects of transmembrane α-helical peptides on the organization of phosphatidylcholine bilayers

    Biochim. Biophys. Acta

    (2001)
  • A.V. Popova et al.

    Differential destabilization of membranes by tryptophan and phenylalanine during freezing: the roles of lipid composition and membrane fusion

    Biochim. Biophys. Acta

    (2002)
  • A.V. Popova et al.

    Intermolecular interactions in dry and rehydrated pure and mixed bilayers of phosphatidylcholine and digalactosyldiacylglycerol: a Fourier-transform infrared spectroscopy study

    Biophys. J.

    (2003)
  • Cited by (0)

    1

    Permanent address: Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.

    View full text