Antoaneta V. Popova

Intermolecular Interactions in Dry and Rehydrated Pure and Mixed Bilayers of Phosphatidylcholine and Digalactosyldiacylglycerol: A Fourier Transform Infrared Spectroscopy Study

Glycolipids are an important part of almost all biological membranes. Their effects on membrane structure and their interactions with phospholipids, however, have not been extensively studied so far. We have investigated the phase behavior and intermolecular interactions in dry and rehydrated bilayers made from the phospholipid egg phosphatidylcholine (EPC) and the plant chloroplast glycolipid digalactosyldiacylglycerol (DGDG), or from a mixture (1:1) of these lipids, using Fourier transform infrared spectroscopy. We show that there are extensive interactions between EPC and DGDG in mixed membranes, and also between DGDG molecules in pure DGDG membranes, involving sugar OH groups and C[double bond]O, P[double bond]O, and choline moieties in dry membranes. These interactions persist to a certain degree even after rehydration. We present evidence that these interactions influence the mixing behavior in phosphatidylcholine/DGDG membranes and also the phase behavior of both EPC/DGDG and pure DGDG membranes in the dry state.

Interaction of two intrinsically disordered plant stress proteins (COR15A and COR15B) with lipid membranes in the dry state.

COR15A and COR15B form a tandem repeat of highly homologous genes in Arabidopsis thaliana. Both genes are highly cold induced and the encoded proteins belong to the Pfam LEA_4 group (group 3) of the late embryogenesis abundant (LEA) proteins. Both proteins were predicted to be intrinsically disordered in solution. Only COR15A has previously been characterized and it was shown to be localized in the soluble stroma fraction of chloroplasts. Ectopic expression of COR15A in Arabidopsis resulted in increased freezing tolerance of both chloroplasts after freezing and thawing of intact leaves and of isolated protoplasts frozen and thawed in vitro. In the present study we have generated recombinant mature COR15A and COR15B for a comparative study of their structure and possible function as membrane protectants. CD spectroscopy showed that both proteins are predominantly unstructured in solution and mainly alpha-helical after drying. Both proteins showed similar effects on the thermotropic phase behavior of dry liposomes. A decrease in the gel to liquid-crystalline phase transition temperature depended on both the unsaturation of the fatty acyl chains and lipid headgroup structure. FTIR spectroscopy indicated no strong interactions between the proteins and the lipid phosphate and carbonyl groups, but significant interactions with the galactose headgroup of the chloroplast lipid monogalactosyldiacylglycerol. These findings were rationalized by modeling the secondary structure of COR15A and COR15B. Helical wheel projection indicated the presence of amphipathic alpha-helices in both proteins. The helices lacked a clear separation of positive and negative charges on the hydrophilic face, but contained several hydroxylated amino acids.

Structural transitions in the intrinsically disordered plant dehydration stress protein LEA7 upon drying are modulated by the presence of membranes

Dehydration stress-related late embryogenesis abundant (LEA) proteins have been found in plants, invertebrates and bacteria. Most LEA proteins are unstructured in solution, but some fold into amphipathic α-helices during drying. The Pfam LEA_4 (Group 3) protein LEA7 from the higher plant Arabidopsis thaliana was predicted to be 87% α-helical, while CD spectroscopy showed it to be largely unstructured in solution and only 35% α-helical in the dry state. However, the dry protein contained 15% β-sheets. FTIR spectroscopy revealed the β-sheets to be largely due to aggregation. β-Sheet content was reduced and α-helix content increased when LEA7 was dried in the presence of liposomes with secondary structure apparently influenced by lipid composition. Secondary structure was also affected by the presence of membranes in the fully hydrated state. A temperature-induced increase in the flexibility of the dry protein was also only observed in the presence of membranes. Functional interactions of LEA7 with membranes in the dry state were indicated by its influence on the thermotropic phase transitions of the lipids and interactions with the lipid headgroup phosphates.

Influence of drying on the secondary structure of intrinsically disordered and globular proteins

Circular dichroism (CD) spectroscopy of five Arabidopsis late embryogenesis abundant (LEA) proteins constituting the plant specific families LEA_5 and LEA_6 showed that they are intrinsically disordered in solution and partially fold during drying. Structural predictions were comparable to these results for hydrated LEA_6, but not for LEA_5 proteins. FTIR spectroscopy showed that verbascose, but not sucrose, strongly affected the structure of the dry proteins. The four investigated globular proteins were only mildly affected by drying in the absence, but strongly in the presence of sugars. These data highlight the larger structural flexibility of disordered compared to globular proteins and the impact of sugars on the structure of both disordered and globular proteins during drying.

Low temperature tolerance of tobacco plants transformed to accumulate proline, fructans, or glycine betaine. Variable chlorophyll fluorescence evidence

Tobacco (Nicotiana tabacum L.) has been transformed to accumulate different compatible solutes (proline, fructans, or glycine betaine) in order to improve its tolerance to abiotic stress. Photosynthetic activity of wild Type (wt) and transformed tobacco plants before and after freezing stress was studied by measuring chlorophyll (Chl) fluorescence. The JIP test of Chl fluorescence induction was used to analyze in details the functional activity of photosystem 2. No significant differences were found among wild Type and transgenic plants after 12 h of freezing. Both plant Types maintained the same values of the measured parameters [FV/FM, PI(CSM), ABS/RC, TR0/RC, ET/RC] after recovery of stress. The studied Chl fluorescence parameters decreased only for the wild Type plants, stressed for 24 h at −2 °C. The strong inhibition of photosynthetic reactions in the wt plant after 24 h of freezing could not be restored. The evaluated parameters of transgenic plants did not change significantly after 24 h at −2 °C and successfully survived freezing stress.

The intrinsically disordered protein LEA7 from Arabidopsis thaliana protects the isolated enzyme lactate dehydrogenase and enzymes in a soluble leaf proteome during freezing and drying.

The accumulation of Late Embryogenesis Abundant (LEA) proteins in plants is associated with tolerance against stresses such as freezing and desiccation. Two main functions have been attributed to LEA proteins: membrane stabilization and enzyme protection. We have hypothesized previously that LEA7 from Arabidopsis thaliana may stabilize membranes because it interacts with liposomes in the dry state. Here we show that LEA7, contrary to this expectation, did not stabilize liposomes during drying and rehydration. Instead, it partially preserved the activity of the enzyme lactate dehydrogenase (LDH) during drying and freezing. Fourier-transform infrared (FTIR) spectroscopy showed no evidence of aggregation of LDH in the dry or rehydrated state under conditions that lead to complete loss of activity. To approximate the complex influence of intracellular conditions on the protective effects of a LEA protein in a convenient in-vitro assay, we measured the activity of two Arabidopsis enzymes (glucose-6-P dehydrogenase and ADP-glucose pyrophosphorylase) in total soluble leaf protein extract (Arabidopsis soluble proteome, ASP) after drying and rehydration or freezing and thawing. LEA7 partially preserved the activity of both enzymes under these conditions, suggesting its role as an enzyme protectant in vivo. Further FTIR analyses indicated the partial reversibility of protein aggregation in the dry ASP during rehydration. Similarly, aggregation in the dry ASP was strongly reduced by LEA7. In addition, mixtures of LEA7 with sucrose or verbascose reduced aggregation more than the single additives, presumably through the effects of the protein on the H-bonding network of the sugar glasses.

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

The stability of cellular membranes during dehydration can be strongly influenced by the partitioning of amphiphilic solutes from the aqueous phase into the membranes. The effects of partitioning on membrane stability depend in a complex manner on the structural properties of the amphiphiles and on membrane lipid composition. Here, we have investigated the effects of the amphiphilic aromatic amino acids Trp and Phe on membrane stability during freezing. Both amino acids were cryotoxic to isolated chloroplast thylakoid membranes and to large unilamellar liposomes, but Trp had a much stronger effect than Phe. In liposomes, both amino acids induced solute leakage and membrane fusion during freezing. The presence of the chloroplast galactolipids monogalactosyldiacylglycerol or digalactosyldiacylglycerol in egg phosphatidylcholine (EPC) membranes reduced leakage from liposomes during freezing in the presence of up to 5 mM Trp, as compared to membranes composed of pure EPC. The presence of the nonbilayer-forming lipid phosphatidylethanolamine increased leakage. Membrane fusion followed a similar trend, but was dramatically reduced when the anthracycline antibiotic daunomycin was incorporated into the membranes. Daunomycin has been shown to stabilize the bilayer phase of membranes in the presence of nonbilayer lipids and was therefore expected to reduce fusion. Surprisingly, this had only a small influence on leakage. Collectively, these data indicate that Trp and Phe induce solute leakage from liposomes during freezing by a mechanism that is largely independent of fusion events.

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

BackgroundAlthough 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.ResultsLiposomes 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.ConclusionsThis 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.

Chapter 6 Effects of Sugars on the Stability and Structure of Lipid Membranes During Drying

Abstract Most plants, microbes, and animals that can survive complete dehydration (anhydrobiotes) accumulate high concentrations of sugars in their cells during desiccation. Since cellular membranes are a primary target of desiccation damage in cells, liposomes have been widely used as comparatively simple model systems to study the effects of sugars on the properties of membranes during drying. In addition, there is a pronounced technical interest in the stabilization of liposomes with encapsulated medical drugs in the dry state for pharmaceutical purposes. Most sugars induce a depression of the membrane phase transition temperature of the dry lipid and form a glass (vitrify) during drying. In this paper, we critically review the current state of knowledge about the physical mechanisms that may lead to membrane stabilization in dry systems, with a special emphasis on possible interactions between membrane lipids and sugars in the dry state. For this purpose we compare the effects of soluble disaccharides, oligosaccharides, and polysaccharides, and also the effects of lipid-bound sugars on the stability and physical behavior of liposomes.

Effects of flavonol glycosides on liposome stability during freezing and drying

Flavonoids are a large and diverse group of plant secondary metabolites that are mainly present as glycosides. They are often accumulated in response to abiotic stresses such as UV radiation, drought, cold and freezing. The most extensively studied function of flavonoids is their antioxidant activity although their importance as antioxidants in plants has been questioned. We therefore aim to study effects of flavonols on cellular stress tolerance that are independent of their antioxidant function. Here we investigate the effects of the glycosylated flavonols kaempferol-3-O-glucoside, kaempferol-7-O-glucoside, quercetin-3-O-glucoside and quercetin-3-O-rhamnoside on liposome stability after freezing and drying. Insertion of flavonols in lipid bilayers destabilized egg phosphatidylcholine (EPC) liposomes and to a lesser extent vesicles made from equal proportions of EPC and egg phosphatidylethanolamine (EPE) during a freeze-thaw cycle, while liposomes containing the unsaturated non-bilayer lipid 18:2 PE were either unaffected or slightly stabilized. In general, the kaempferol derivatives were more destabilizing for liposomes during freezing than the quercetin derivatives. Fourier-transform infrared spectroscopy revealed that all flavonols were localized in the interfacial region of the lipid bilayers, forming H-bonds with the lipid phosphate and carbonyl groups. The phase transition temperature of dry 16:0/18:1 PC (POPC) and POPC/EPE liposomes was decreased by 75°C and 55°C, respectively. Changes in the vibration bands attributed to the phenolic ring structures of the flavonols in the presence of liposomes provided further evidence of interactions of these molecules in particular with the interfacial region of the bilayers.