The effect of protein stability on protein-monoglyceride interactions.

We have previously shown that proteins such as beta-lactoglobulin and lysozyme insert into monoglyceride monolayers and are able to induce an L(beta) to coagel phase transition in monoglyceride bilayers. These studies gave a first indication that protein stability could be an important factor for these interactions. This study therefore aims at further investigating the potential role of protein stability on protein-monoglyceride interactions. To this end we studied the interaction of stable and destabilized alpha-lactalbumin with monostearoylglycerol. Our results show that protein stability is important for the insertion of proteins into a monostearoylglycerol monolayer, such that the lower the stability of the protein the better the protein inserts. In marked contrast to beta-lactoglobulin and lysozyme we found that destabilized alpha-lactalbumin does not induce the L(beta) to coagel phase transition in monoglyceride bilayers. We propose that this is due to an increased surface coverage by the protein which could result from the unfolding of the protein upon binding to the interface.

Thermotropic phase behavior of monoglyceride-dicetylphosphate dispersions and interactions with proteins: a (2)H and (31)P NMR study.

The phase behavior of a 1-[(2)H(35)]-stearoyl-rac-glycerol ([(2)H(35)]-MSG)/dicetylphosphate (DCP) mixture and its interaction with beta-lactoglobulin and lysozyme were studied by (2)H and (31)P nuclear magnetic resonance (NMR). The behavior of the lipids was monitored by using deuterium-labeled [(2)H(35)]-MSG as a selective probe for (2)H NMR and DCP for (31)P NMR. Both (2)H and (31)P NMR spectra exhibit characteristic features representative of different phases. In the lamellar phases, (31)P NMR spectra of DCP are different from the spectra of natural phospholipids, which is attributable to differences in the intramolecular motions and the orientation of the shielding tensor of DCP compared with phospholipids. The presence of the negatively charged amphiphile DCP has a large effect on the phase behavior of [(2)H(35)]-MSG. At low temperature, the presence of DCP inhibits crystallization of the gel phase into the coagel. Upon increasing the temperature, the gel phase of [(2)H(35)]-MSG transforms in the liquid-crystalline lamellar phase. In the presence of DCP, the gel phase directly transforms into an isotropic phase. The negatively charged beta-lactoglobulin and the positively charged lysozyme completely neutralize the destabilizing effect of DCP on the monoglyceride liquid-crystalline phase and they even stabilize this phase. Without DCP the proteins do not seem to interact with the monoglyceride. These results suggest that interaction is facilitated by electrostatic interactions between the negatively charged DCP and positively charged residues in the proteins. In addition, the nonbilayer-forming DCP creates insertion sites for proteins in the bilayer.

Effect of nonbilayer lipids on membrane binding and insertion of the catalytic domain of leader peptidase.

Biological membranes contain a substantial amount of "nonbilayer lipids", which have a tendency to form nonlamellar phases. In this study the hypothesis was tested that the presence of nonbilayer lipids in a membrane, due to their overall small headgroup, results in a lower packing density in the headgroup region, which might facilitate the interfacial insertion of proteins. Using the catalytic domain of leader peptidase (delta2-75) from Escherichia coli as a model protein, we studied the lipid class dependence of its insertion and binding. In both lipid monolayers and vesicles, the membrane binding of (catalytically active) delta2-75 was much higher for the nonbilayer lipid DOPE compared to the bilayer lipid DOPC. For the nonbilayer lipids DOG and MGDG a similar effect was observed as for DOPE, strongly suggesting that no specific interactions are involved but that the small headgroups create hydrophobic interfacial insertion sites. On the basis of the results of the monolayer experiments, calculations were performed to estimate the space between the lipid headgroups accessible to the protein. We estimate a maximal size of the insertion sites of 15 +/- 7 A2/lipid molecule for DOPE, relative to DOPC. The size of the insertion sites decreases with an increase in headgroup size. These results show that nonbilayer lipids stimulate the membrane insertion of delta2-75 and support the idea that such lipids create insertion sites by reducing the packing density at the membrane-water interface. It is suggested that PE in the bacterial membrane facilitates membrane insertion of the catalytic domain of leader peptidase, allowing the protein to reach the cleavage site in preproteins.

The N-terminal portion of the preToc75 transit peptide interacts with membrane lipids and inhibits binding and import of precursor proteins into isolated chloroplasts.

Toc75 is an outer envelope membrane protein of chloroplasts. It is unusual among the outer membrane proteins in that its precursor form has a bipartite transit peptide. The N-terminal portion of the Toc75 transit peptide is sufficient to target the protein to the stromal space of chloroplasts. We prepared a 45 amino-acid peptide containing the stromal targeting domain of the Toc75 transit peptide in Escherichia coli, using the intein-mediated system, and purified it by reverse-phase HPLC. Its identity was confirmed by N-terminal amino-acid sequencing and matrix assisted laser desorption ionization mass spectrometry. In monolayer experiments, the peptide inserted into the chloroplastic membrane lipids sulfoquinovosyl diacylglycerol and phosphatidylglycerol and into a nonchloroplastic lipid phosphatidylethanolamine. However, it did not insert into other chloroplastic lipids, such as mono- and digalactosyl diacylglycerol, and phosphatidylcholine. Furthermore, the peptide significantly inhibited binding of radiolabeled precursors of Toc75 and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase to intact chloroplasts as effectively as did a bacterially produced precursor of the small subunit of 1,5-bisphosphate carboxylase/oxygenase. The peptide also inhibited import of radiolabeled precursors into isolated chloroplasts, however, to a lesser extent than did nonlabeled precursor of the small subunit of 1,5-bisphosphate carboxylase/oxygenase.

Fructans insert between the headgroups of phospholipids.

Fructans are polysaccharides consisting of one glucose unit and two or more fructose units. It was hypothesized that fructans play a role in drought tolerance in plants by interacting directly with the membrane. In this paper we investigated this hypothesis by studying fructan-membrane interactions in hydrated mono- and bilayer systems. It was found that fructans inserted between the headgroups of different kinds of phospholipids with some preference for phosphatidylethanolamine. Insertion occurred even under conditions of very tight lipid packing. The presence of a surface associated layer of fructan was observed in both model systems. This layer was able to reduce the ability of a surface-active protein to interact with the lipids. Fructans showed a much stronger effect on the different lipid systems than other (poly)saccharides, which appears to be related to their hydrophobic properties. Fructans were able to stabilize the liquid-crystalline lamellar phase, which is consistent with a drought protecting role in plants.

The specificity of monoglyceride-protein interactions and mechanism of the protein induced L(beta) to coagel phase transition.

This study aims at gaining insight into the specificity and molecular mechanism of monoglyceride-protein interactions. We used beta-lactoglobulin (beta-LG) and lysozyme as model proteins and both monostearoylglycerol and monopalmitoylglycerol as defined gel phase monoglycerides. The monoglycerides were used in different combinations with the two negatively charged amphiphiles dicetylphosphate and distearylphosphate. The interactions were characterized using the monolayer technique, isothermal titration calorimetry, (2)H-nuclear magnetic resonance (NMR) using deuterium labelled monoglycerides and freeze fracture electron microscopy (EM). Our results show that lysozyme inserts efficiently into all monolayers tested, including pure monoglyceride layers. The insertion of beta-LG depends on the lipid composition of the monolayer and is promoted when the acylchains of the negatively charged amphiphile are shorter than that of the monoglyceride. The binding parameters found for the interaction of beta-LG and lysozyme with monoglyceride bilayers were generally similar. Moreover, in all cases a large exothermic binding enthalpy was observed which was found to depend on the nature of the monoglycerides but not of the proteins. (2)H-NMR and freeze fracture EM showed that this large enthalpy results from a protein mediated catalysis of the monoglyceride L(beta) to coagel phase transition. The mechanism of this phase transition consists of two steps, an initial protein mediated vesicle aggregation step which is followed by stacking and probably fusion of the bilayers.

Membrane activity of the peptide antibiotic clavanin and the importance of its glycine residues.

The peptide antibiotic clavanin A (VFQFLGKIIHHVGNFVHGFSHVF-NH(2)) is rich in histidine and glycine residues. In this study the antimicrobial activity and membrane activity of wild-type clavanin A and seven Gly --> Ala mutants thereof were investigated. Clavanin A effectively killed the test microorganism Micrococcus flavus and permeabilized its cytoplasmic membrane in the micromolar concentration range, suggesting that the membrane is the target for this molecule. Consistent with this suggestion, it was observed that clavanin A efficiently inserted into different phospholipid monolayers mainly via hydrophobic interactions. Bilayer permeabilization was observed for both low and high molecular mass fluorophores enclosed in unilamellar vesicles and occurred at the same concentration as the antimicrobial activity. It is therefore suggested that the loss of barrier function does not involve specific receptors in the target membrane. Circular dichroism spectroscopy indicated that under membrane mimicking conditions a random coil --> helical transition was induced for all clavanin derivatives tested. Observed differences in peptide-membrane interaction and biological activity between the various clavanin derivatives demonstrated the functional importance of Gly at the positions 6 and 13. These two glycines may act as flexible hinges that facilitate the hydrophobic N-terminal end of clavanin to deeply insert into the bilayer. On the contrary, no such role is evident for Gly 18, as its substitution by Ala actually stimulated membrane interaction and biological activity. This study suggests that the combined hydrophobicity, overall state of charge, and conformational flexibility of the peptide determine the (membrane) activity of clavanin A and its Gly --> Ala mutants.

Spontaneous insertion of gene 9 minor coat protein of bacteriophage M13 in model membranes.

Gene 9 minor coat protein from bacteriophage M13 is known to be located in the inner membrane after phage infection of Escherichia coli. The way of insertion of this small protein (32 amino acids) into membranes is still unknown. Here we show that the protein is able to insert in monolayers. The limiting surface pressure of 35 mN/m for 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol lipid systems indicates that this spontaneous insertion can also occur in vivo. By carboxyfluorescein leakage experiments of vesicles it is demonstrated that protein monomers, or at least small aggregates, are more effective in releasing carboxyfluorescein than highly aggregated protein. The final orientation of the protein in the bilayer after insertion was addressed by proteinase K digestion, thereby making use of the unique C-terminal location of the antigenic binding site. After insertion the C-terminus is still available for the enzymatic digestion, while the N-terminus is not. This leads to the overall conclusion that the protein is able to insert spontaneously into membranes without the need of any machinery or transmembrane gradient, with the positively charged C-terminus remaining on the outside. The orientation after insertion of gene 9 protein is in agreement with the 'positive inside rule'.

Lipid organization and dynamics of the monostearoylglycerol-water system. A 2H NMR study.

Deuterium labeled monostearoylglycerols with fully ([2H(35)]-MSG) and selectively ([11-(2)H(2)]-MSG) deuterated chains have been synthesized and used as a probe for 2H NMR. At low temperature monoglyceride-water systems form the coagel or crystalline phase, which transforms with increasing temperature subsequently into the gel, liquid crystalline and cubic phase. The 2H NMR spectra exhibit characteristic features representative of these phases. The gel phase is metastable and gradually transforms into the coagel at temperatures below 40 degrees C. The undercooled cubic phase transforms into the liquid crystalline phase during days. In the liquid crystalline phase, the chain order profile indicates an increase of the chain flexibility towards the methyl group. In the liquid crystalline phase, bilayers spontaneously align in a magnetic field with their normal perpendicular to the field. The results demonstrate that 2H NMR can serve as a convenient tool to study both structure and dynamics of different monoglyceride-water phases.

The S-layer protein of Lactobacillus acidophilus ATCC 4356: identification and characterisation of domains responsible for S-protein assembly and cell wall binding.

Lactobacillus acidophilus, like many other bacteria, harbors a surface layer consisting of a protein (S(A)-protein) of 43 kDa. S(A)-protein could be readily extracted and crystallized in vitro into large crystalline patches on lipid monolayers with a net negative charge but not on lipids with a net neutral charge. Reconstruction of the S-layer from crystals grown on dioleoylphosphatidylserine indicated an oblique lattice with unit cell dimensions (a=118 A; b=53 A, and gamma=102 degrees ) resembling those determined for the S-layer of Lactobacillus helveticus ATCC 12046. Sequence comparison of S(A)-protein with S-proteins from L. helveticus, Lactobacillus crispatus and the S-proteins encoded by the silent S-protein genes from L. acidophilus and L. crispatus suggested the presence of two domains, one comprising the N-terminal two-thirds (SAN), and another made up of the C-terminal one-third (SAC) of S(A)-protein. The sequence of the N-terminal domains is variable, while that of the C-terminal domain is highly conserved in the S-proteins of these organisms and contains a tandem repeat. Proteolytic digestion of S(A)-protein showed that SAN was protease-resistant, suggesting a compact structure. SAC was rapidly degraded by proteases and therefore probably has a more accessible structure. DNA sequences encoding SAN or Green Fluorescent Protein fused to SAC (GFP-SAC) were efficiently expressed in Escherichia coli. Purified SAN could crystallize into mono and multi-layered crystals with the same lattice parameters as those found for authentic S(A)-protein. A calculated S(A)-protein minus SAN density-difference map revealed the probable location, in projection, of the SAC domain, which is missing from the truncated SAN peptide. The GFP-SAC fusion product was shown to bind to the surface of L. acidophilus, L. helveticus and L. crispatus cells from which the S-layer had been removed, but not to non-stripped cells or to Lactobacillus casei.

Dynamin is membrane-active: lipid insertion is induced by phosphoinositides and phosphatidic acid.

Dynamin is a large GTPase involved in the regulation of membrane constriction and fission during receptor-mediated endocytosis. Dynamin contains a pleckstrin-homology domain which is essential for endocytosis and which binds to anionic phospholipids. Here, we show for the first time that dynamin is a membrane-active molecule capable of penetrating into the acyl chain region of membrane lipids. Lipid penetration is strongly stimulated by phosphatidic acid (PA), phosphatidylinositol 4-phosphate, and phosphatidylinositol 4, 5-bisphosphate. Though binding is more efficient in the presence of the phosphoinositides, a much larger part of the dynamin molecule penetrates into PA-containing mixed-lipid systems. Thus, local lipid metabolism will dramatically influence dynamin-lipid interactions, and dynamin-lipid interactions are likely to play an important role in dynamin-dependent endocytosis. Our data suggest that dynamin is directly involved in membrane destabilization, a prerequisite to membrane fission.

Visualization of highly ordered striated domains induced by transmembrane peptides in supported phosphatidylcholine bilayers.

We used atomic force microscopy (AFM) to study the lateral organization of transmembrane TmAW(2)(LA)(n)W(2)Etn peptides (WALP peptides) incorporated in phospholipid bilayers. These well-studied model peptides consist of a hydrophobic alanine-leucine stretch of variable length, flanked on each side by two tryptophans. They were incorporated in saturated phosphatidylcholine (PC) vesicles, which were deposited on a solid substrate via the vesicle fusion method, yielding hydrated gel-state supported bilayers. At low concentrations (1 mol %) WALP peptides induced primarily line-type depressions in the bilayer. In addition, striated lateral domains were observed, which increased in amount and size (from 25 nm up to 10 microm) upon increasing peptide concentration. At high peptide concentration (10 mol %), the bilayer consisted mainly of striated domains. The striated domains consist of line-type depressions and elevations with a repeat distance of 8 nm, which form an extremely ordered, predominantly hexagonal pattern. Overall, this pattern was independent of the length of the peptides (19-27 amino acids) and the length of the lipid acyl chains (16-18 carbon atoms). The striated domains could be pushed down reversibly by the AFM tip and are thermodynamically stable. This is the first direct visualization of alpha-helical transmembrane peptide-lipid domains in a bilayer. We propose that these striated domains consist of arrays of WALP peptides and fluidlike PC molecules, which appear as low lines. The presence of the peptides perturbs the bilayer organization, resulting in a decrease in the tilt of the lipids between the peptide arrays. These lipids therefore appear as high lines.

Anionic phospholipids are involved in membrane association of FtsY and stimulate its GTPase activity.

FtsY, the Escherichia coli homologue of the eukaryotic signal recognition particle (SRP) receptor alpha-subunit, is located in both the cytoplasm and inner membrane. It has been proposed that FtsY has a direct targeting function, but the mechanism of its association with the membrane is unclear. FtsY is composed of two hydrophilic domains: a highly charged N-terminal domain (the A-domain) and a C-terminal GTP-binding domain (the NG-domain). FtsY does not contain any hydrophobic sequence that might explain its affinity for the inner membrane, and a membrane-anchoring protein has not been detected. In this study, we provide evidence that FtsY interacts directly with E.coli phospholipids, with a preference for anionic phospholipids. The interaction involves at least two lipid-binding sites, one of which is present in the NG-domain. Lipid association induced a conformational change in FtsY and greatly enhanced its GTPase activity. We propose that lipid binding of FtsY is important for the regulation of SRP-mediated protein targeting.

Blistering of langmuir-blodgett bilayers containing anionic phospholipids as observed by atomic force microscopy.

Asymmetric bilayers of different phospholipid compositions have been prepared by the Langmuir-Blodgett (L-B) method, and imaged by atomic force microscopy (AFM). Such bilayers can function as a model for biological membranes. The first leaflet consisted of zwitterionic phospholipids phosphatidylcholine (PC) or phosphatidylethanolamine (PE). The second leaflet consisted of the anionic phospholipid phosphatidylglycerol (PG), in either the condensed or liquid phase or, for comparison, of PC. Different bilayers showed different morphology. In all bilayers defects in the form of holes were present. In some bilayers with a first leaflet consisting of PC, polygonal line-shaped defects were observed, whereas when the first leaflet consisted of PE, mainly round defects were seen. Not only the shape, but also the amount of defects varied, depending on the condition and the composition of the second leaflet. In most of the PG-containing systems the defects were surrounded by elevations, which reversibly disappeared in the presence of divalent cations. This is the first time that such elevations have been observed on phospholipid bilayers. We propose that they are induced by phospholipid exchange between the two leaflets around the defects, leading to the presence of negatively charged phospholipids in the first leaflet. Because the substrate is also negatively charged, the bilayer around the edges is repelled and lifted up. Since it was found that the elevations are indeed detached from the substrate, we refer to this effect as bilayer blistering.

Domain structure and lipid interaction of recombinant yeast Tim44.

Tim44 is an essential component of the machinery that mediates the translocation of nuclear-encoded proteins across the mitochondrial inner membrane. It functions as a membrane anchor for the ATP-driven protein import motor whose other subunits are the mitochondrial 70-kDa heat-shock protein (mhsp70) and its nucleotide exchange factor, mGrpE. To understand how this motor is anchored to the inner membrane, we have overexpressed Tim44 in Escherichia coli and studied the properties of the pure protein and its interaction with model lipid membranes. Limited proteolysis and analytical ultracentrifugation indicate that Tim44 is an elongated monomer with a stably folded C-terminal domain. The protein binds strongly to liposomes composed of phosphatidylcholine and cardiolipin but only weakly to liposomes containing phosphatidylcholine alone. Studies with phospholipid monolayers suggest that Tim44 binds to phospholipids of the mitochondrial inner membrane both by electrostatic interactions and by penetrating the polar head group region.

Interaction mode specific reorganization of gel phase monoglyceride bilayers by beta-lactoglobulin.

The interaction between beta-lactoglobulin and sonicated aqueous dispersions of the gel phase forming monoglyceride monostearoylglycerol were studied using isothermal titration calorimetry, direct binding experiments, differential scanning calorimetry, leakage of a fluorescent dye and solid-state (31)P- and (2)H-NMR. In the absence of a charged amphiphile, monostearoylglycerol forms a precipitate. Under these conditions, no interaction with beta-lactoglobulin was observed. In the presence of the negatively charged amphiphile dicetylphosphate, the gel phase monostearoylglycerol formed stable and closed, probably unilamellar, vesicles with an average diameter of 465 nm. beta-Lactoglobulin interacts with these bilayer structures at pH 4, where the protein is positively charged, as well as at pH 7 where the protein is negatively charged. Under both conditions of pH, the binding affinity of beta-lactoglobulin is in the micromolar range as observed with ITC and the direct binding assay. At pH 4, two binding modes were found, one of which is determined with ITC while the direct binding assay determines the net result of both. The first binding mode is observed with ITC and is characterized by a large binding enthalpy, a decreased enthalpy of the MSG L(beta) to L(alpha) phase transition and leakage of a fluorescent dye. These characteristics are explained by a beta-lactoglobulin induced partial L(beta) to coagel phase transition that results from a specific electrostatic interaction between the protein and the charged amphiphile. This explanation is confirmed by solid-state (2)H-NMR using 1-monostearoylglycerol with a fully deuterated acyl chain. Upon interaction with beta-lactoglobulin, the isotropic signal in the (2)H-NMR spectrum of the monostearoylglycerol-dicetylphosphate mixture partially transforms into a broad anisotropic signal which could be assigned to coagel formation. The second binding mode probably results from an aspecific electrostatic attraction between the negatively charged bilayer and the positively charged protein and causes the precipitation of the dispersion. At pH 7, only the first binding mode is observed.

Affinity of the periplasmic chaperone Skp of Escherichia coli for phospholipids, lipopolysaccharides and non-native outer membrane proteins. Role of Skp in the biogenesis of outer membrane protein.

The Skp protein of Escherichia coli has been proposed to be a periplasmic molecular chaperone involved in the biogenesis of outer membrane proteins. In this study, evidence is obtained that Skp exists in two different states characterized by their different sensitivity to proteases. The conversion between these states can be modulated in vitro by phospholipids, lipopolysaccharides and bivalent cations. Skp is able to associate with and insert into phospholipid membranes in vitro, indicating that it may associate with phospholipids in the inner and/or outer membrane in vivo. In addition, it interacts specifically with outer membrane proteins that are in their non-native state. We propose that Skp is required in vivo for the efficient targeting of unfolded outer membrane proteins to the membrane.

An investigation into the lipid interactions of peptides corresponding to the C-terminal anchoring domains of Escherichia coli penicillin-binding proteins 4, 5 and 6.

The Escherichia coli low molecular mass penicillin-binding proteins PBP4, PBP5 and PBP6 are DD-peptidases involved in murein biosynthesis. It has been suggested that these proteins may be anchored to the periplasmic face of the inner membrane via their C termini. Here, peptide homologues (P4, P5 and P6) of the PBP4, PBP5 and PBP5 C-terminal regions have been used to investigate potential protein-lipid interactions involved in this anchoring mechanism. Surface pressure changes observed for the interactions of P5 and P6 with a range of monolayers indicated that the peptides are membrane interactive and that the interactions proceeded via predominantly hydrophobic forces with only minor requirements for anionic lipid. In contrast, P4 interactions with monolayers appeared to proceed via predominantly electrostatic forces with a major requirement for anionic lipid. The lipid interactions of all three peptides were generally enhanced by low pH and for P5 and P6 were in the range of 10-15 mN m-1 whereas for P4 interactions they were in the range of 3-7 mN m-1. CD analysis implied the presence of alpha-helical structure in P5 and P6 and molecular area determinations implied that P4 may also possess helical architecture in the presence of dioleoylphosphatidylglycerol monolayers. Overall, our results support the view that C-terminal amphiphilic alpha-helices are involved in the membrane anchoring of PBP5 and PBP6 and suggest that a similar mechanism could contribute to PBP4-membrane anchoring. Furthermore, we have speculated that the presence of cationic residues in the hydrophilic face of these alpha-helices may help facilitate membrane interaction.

Pore formation by nisin involves translocation of its C-terminal part across the membrane.

Nisin is an amphiphilic peptide with a strong antimicrobial activity against various Gram-positive bacteria. Its activity results from permeabilization of bacterial membranes, causing efflux of cytoplasmic compounds. To get information on the molecular mechanism of membrane permeabilization, a mutant of nisin Z containing the C-terminal extension Asp-(His)6 was produced. The biological and anionic lipid-dependent membrane activity of this peptide was very similar to that of nisin Z. Analysis of the pH dependence of model membrane interactions with the elongated peptide indicated the importance of electrostatic interactions of the C-terminus with the target membrane for membrane permeabilization. Most importantly, the membrane topology of the C-terminus of the molecule could be determined by trypsin digestion experiments, in which trypsin was encapsulated in the lumen of large unilamellar vesicles. The results show that the C-terminal part of the peptide translocates across model membranes. The pH and anionic lipid dependence of translocation closely paralleled the results of membrane permeabilization studies. Binding of nickel ions to the histidines blocked translocation of the C-terminus and concomitantly resulted in a 4-fold reduced capacity to induce K+ leakage. The results demonstrate for the first time that pore formation of nisin involves translocation of the C-terminal region of the molecule across the membrane.

Fructans interact strongly with model membranes.

Bacterial fructans with a high degree of polymerisation cause a very large increase in surface pressure of lipid monolayers at the air-water interface with a broad range of lipids, including phosphatidylethanolamine and several types of phosphatidylcholines. The surface active effect of fructans contrasts strongly with the maximal effects observed for trehalose, sucrose and glucose under comparable conditions (20 and 0.6 mN/m for fructans and the other sugars, respectively). The results demonstrate a profound and specific membrane interaction of the fructans which is probably very different from the effect of the smaller carbohydrates. The fructan concentrations used in this study are within the physiological range observed in fructan-accumulating plants. The suggested water-stress protective effect of fructans may be induced by membrane-fructan interaction which prevent lipid condensation and phase transitions to take place.