Non-bilayer Lipid Phases Research Articles

Cationic Proteins Rich in Lysine Residue Trigger Formation of Non-bilayer Lipid Phases in Model and Biological Membranes: Biophysical Methods of Study.

Cationic membrane-active toxins are the most abundant group of proteins in the venom of snakes and insects. Cationic proteins such as cobra venom cytotoxin and bee venom melittin are known for their pharmacological reactions including anticancer and antimicrobial effects which arise from the toxin-induced alteration in the dynamics and structure of plasma membranes and membranes of organelles. It has been established that these cationic toxins trigger the formation of non-bilayer lipid phase transitions in artificial and native mitochondrial membranes. Remarkably, the toxin-induced formation of non-bilayer lipid phase increases at certain conditions mitochondrial ATP synthase activity. This observation opens an intriguing avenue for using cationic toxins in the development of novel drugs for the treatment of cellular energy deficiency caused by aging and diseases. This observation also warrants a thorough investigation of the molecular mechanism(s) of lipid phase polymorphisms triggered by cationic proteins. This article presents a review on the application of powerful biophysical methods such as resonance spectroscopy (31P-, 1H-, 2H-nuclear magnetic resonance, and electron paramagnetic resonance), luminescence, and differential scanning microcalorimetry in studies of non-bilayer lipid phase transitions triggered by cationic proteins in artificial and biological membranes. A phenomenon of the triggered by cationic proteins the non-bilayer lipid phase transitions occurring within 10-2-10-11s is discussed in the context of potential pharmacological applications of cationic proteins. Next to the ATP dimer is an inverted micelle made of cardiolipin that serves as a vehicle for the transport of H+ ions from the intra-crista space to the matrix. It is proposed that such inverted micelles are triggered by the high density of H+ ions and the cationic proteins rich in lysine residue which compete with the conserved lysine residues of the ATP synthase rotor for binding to cardiolipin in the inner mitochondrial membrane and perturb the bilayer lipid packing of cristae. Phospholipids with a blue polar head represent cardiolipin and those with a red polar head represent other phospholipids found in the crista membrane.

Non-bilayer Lipid Phases Modulate the Structure and Functions of Mitochondrial Membranes: Pharmacological Relevance

Non-bilayer Lipid Phases Modulate the Structure and Functions of Mitochondrial Membranes: Pharmacological Relevance

Structural Entities Associated with Different Lipid Phases of Plant Thylakoid Membranes-Selective Susceptibilities to Different Lipases and Proteases.

It is well established that plant thylakoid membranes (TMs), in addition to a bilayer, contain two isotropic lipid phases and an inverted hexagonal (HII) phase. To elucidate the origin of non-bilayer lipid phases, we recorded the 31P-NMR spectra of isolated spinach plastoglobuli and TMs and tested their susceptibilities to lipases and proteases; the structural and functional characteristics of TMs were monitored using biophysical techniques and CN-PAGE. Phospholipase-A1 gradually destroyed all 31P-NMR-detectable lipid phases of isolated TMs, but the weak signal of isolated plastoglobuli was not affected. Parallel with the destabilization of their lamellar phase, TMs lost their impermeability; other effects, mainly on Photosystem-II, lagged behind the destruction of the original phases. Wheat-germ lipase selectively eliminated the isotropic phases but exerted little or no effect on the structural and functional parameters of TMs—indicating that the isotropic phases are located outside the protein-rich regions and might be involved in membrane fusion. Trypsin and Proteinase K selectively suppressed the HII phase—suggesting that a large fraction of TM lipids encapsulate stroma-side proteins or polypeptides. We conclude that—in line with the Dynamic Exchange Model—the non-bilayer lipid phases of TMs are found in subdomains separated from but interconnected with the bilayer accommodating the main components of the photosynthetic machinery.

Short-Chained Alcohols Make Membrane Surfaces Conducive for Melittin Action: Implication for the Physiological Role of Alcohols in Cells.

Alcohols are a part of cellular metabolism, but their physiological roles are not well understood. We investigated the effects of short-chain alcohols on Daphnia pulex and model membranes mimicking the lipid composition of eukaryotic inner mitochondrial membranes. We also studied the synergistic effects of alcohols with the bee venom membrane-active peptide, melittin, which is structurally similar to endogenous membrane-active peptides. The alcohols, from ethanol to octanol, gradually decreased the heart rate and the mitochondrial ATP synthesis of daphnia; in contrast, in combination with melittin, which exerted no sizeable effect, they gradually increased both the heart rate and the ATP synthesis. Lipid packing and the order parameter of oriented films, monitored by EPR spectroscopy of the spin-labeled probe 5-doxylstrearic acid, revealed gradual alcohol-assisted bilayer to non-bilayer transitions in the presence of melittin; further, while the alcohols decreased, in combination with melittin they increased the order parameter of the film, which is attributed to the alcohol-facilitated association of melittin with the membrane. A 1H-NMR spectroscopy of the liposomes confirmed the enhanced induction of a non-bilayer lipid phase that formed around the melittin, without the permeabilization of the liposomal membrane. Our data suggest that short-chain alcohols, in combination with endogenous peptides, regulate protein functions via modulating the lipid polymorphism of membranes.

Modulation of non-bilayer lipid phases and the structure and functions of thylakoid membranes: effects on the water-soluble enzyme violaxanthin de-epoxidase

The role of non-bilayer lipids and non-lamellar lipid phases in biological membranes is an enigmatic problem of membrane biology. Non-bilayer lipids are present in large amounts in all membranes; in energy-converting membranes they constitute about half of their total lipid content—yet their functional state is a bilayer. In vitro experiments revealed that the functioning of the water-soluble violaxanthin de-epoxidase (VDE) enzyme of plant thylakoids requires the presence of a non-bilayer lipid phase. 31P-NMR spectroscopy has provided evidence on lipid polymorphism in functional thylakoid membranes. Here we reveal reversible pH- and temperature-dependent changes of the lipid-phase behaviour, particularly the flexibility of isotropic non-lamellar phases, of isolated spinach thylakoids. These reorganizations are accompanied by changes in the permeability and thermodynamic parameters of the membranes and appear to control the activity of VDE and the photoprotective mechanism of non-photochemical quenching of chlorophyll-a fluorescence. The data demonstrate, for the first time in native membranes, the modulation of the activity of a water-soluble enzyme by a non-bilayer lipid phase.

Lipid Dependence of Xanthophyll Cycling in Higher Plants and Algae.

The xanthophyll cycles of higher plants and algae represent an important photoprotection mechanism. Two main xanthophyll cycles are known, the violaxanthin cycle of higher plants, green and brown algae and the diadinoxanthin cycle of Bacillariophyceae, Xanthophyceae, Haptophyceae, and Dinophyceae. The forward reaction of the xanthophyll cycles consists of the enzymatic de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin or diadinoxanthin to diatoxanthin during periods of high light illumination. It is catalyzed by the enzymes violaxanthin or diadinoxanthin de-epoxidase. During low light or darkness the back reaction of the cycle, which is catalyzed by the enzymes zeaxanthin or diatoxanthin epoxidase, restores the epoxidized xanthophylls by a re-introduction of the epoxy groups. The de-epoxidation reaction takes place in the lipid phase of the thylakoid membrane and thus, depends on the nature, three dimensional structure and function of the thylakoid lipids. As the xanthophyll cycle pigments are usually associated with the photosynthetic light-harvesting proteins, structural re-arrangements of the proteins and changes in the protein-lipid interactions play an additional role for the operation of the xanthophyll cycles. In the present review we give an introduction to the lipid and fatty acid composition of thylakoid membranes of higher plants and algae. We introduce the readers to the reaction sequences, enzymes and function of the different xanthophyll cycles. The main focus of the review lies on the lipid dependence of xanthophyll cycling. We summarize the current knowledge about the role of lipids in the solubilization of xanthophyll cycle pigments. We address the importance of the three-dimensional lipid structures for the enzymatic xanthophyll conversion, with a special focus on non-bilayer lipid phases which are formed by the main thylakoid membrane lipid monogalactosyldiacylglycerol. We additionally describe how lipids and light-harvesting complexes interact in the thylakoid membrane and how these interactions can affect the structure of the thylakoids. In a dedicated chapter we offer a short overview of current membrane models, including the concept of membrane domains. We then use these concepts to present a model of the operative xanthophyll cycle as a transient thylakoid membrane domain which is formed during high light illumination of plants or algal cells.

Lipid-polymorphism of plant thylakoid membranes. Enhanced non-bilayer lipid phases associated with increased membrane permeability.

Earlier experiments, using 31 P-NMR and time-resolved merocyanine fluorescence spectroscopy, have shown that isolated intact, fully functional plant thylakoid membranes, in addition to the bilayer phase, contain three non-bilayer (or non-lamellar) lipid phases. It has also been shown that the lipid polymorphism of thylakoid membranes can be characterized by remarkable plasticity, i.e. by significant variations in 31 P-NMR signatures. However, changes in the lipid-phase behaviour of thylakoids could not be assigned to changes in the overall membrane organization and the photosynthetic activity, as tested by circular dichroism and 77 K fluorescence emission spectroscopy and the magnitude of the variable fluorescence of photosystem II, which all showed only marginal variations. In this work, we investigated in more detail the temporal stability of the different lipid phases by recording 31 P-NMR spectra on isolated thylakoid membranes that were suspended in sorbitol- or NaCl-based media. We observed, at 5°C during 8h in the dark, substantial gradual enhancement of the isotropic lipid phases and diminishment of the bilayer phase in the sorbitol-based medium. These changes compared well with the gradually increasing membrane permeability, as testified by the gradual acceleration of the decay of flash-induced electrochromic absorption changes and characteristic changes in the kinetics of fast chlorophyll a-fluorescence transients; all variations were much less pronounced in the NaCl-based medium. These observations suggest that non-bilayer lipids and non-lamellar lipid phases play significant roles in the structural dynamics and functional plasticity of thylakoid membranes.

Lipid polymorphism in chloroplast thylakoid membranes \u2013 as revealed by 31P-NMR and time-resolved merocyanine fluorescence spectroscopy

Chloroplast thylakoid membranes contain virtually all components of the energy-converting photosynthetic machinery. Their energized state, driving ATP synthesis, is enabled by the bilayer organization of the membrane. However, their most abundant lipid species is a non-bilayer-forming lipid, monogalactosyl-diacylglycerol; the role of lipid polymorphism in these membranes is poorly understood. Earlier 31P-NMR experiments revealed the coexistence of a bilayer and a non-bilayer, isotropic lipid phase in spinach thylakoids. Packing of lipid molecules, tested by fluorescence spectroscopy of the lipophilic dye, merocyanine-540 (MC540), also displayed heterogeneity. Now, our 31P-NMR experiments on spinach thylakoids uncover the presence of a bilayer and three non-bilayer lipid phases; time-resolved fluorescence spectroscopy of MC540 also reveals the presence of multiple lipidic environments. It is also shown by 31P-NMR that: (i) some lipid phases are sensitive to the osmolarity and ionic strength of the medium, (ii) a lipid phase can be modulated by catalytic hydrogenation of fatty acids and (iii) a marked increase of one of the non-bilayer phases upon lowering the pH of the medium is observed. These data provide additional experimental evidence for the polymorphism of lipid phases in thylakoids and suggest that non-bilayer phases play an active role in the structural dynamics of thylakoid membranes.

Role of MGDG and Non-bilayer Lipid Phases in the Structure and Dynamics of Chloroplast Thylakoid Membranes.

In this chapter we focus our attention on the enigmatic structural and functional roles of the major, non-bilayer lipid monogalactosyl-diacylglycerol (MGDG) in the thylakoid membrane. We give an overview on the state of the art on the role of MGDG and non-bilayer lipid phases in the xanthophyll cycles in different organisms. We also discuss data on the roles of MGDG and other lipid molecules found in crystal structures of different photosynthetic protein complexes and in lipid-protein assemblies, as well as in the self-assembly of the multilamellar membrane system. Comparison and critical evaluation of different membrane models--that take into account and capitalize on the special properties of non-bilayer lipids and/or non-bilayer lipid phases, and thus to smaller or larger extents deviate from the 'standard' Singer-Nicolson model--will conclude this review. With this chapter the authors hope to further stimulate the discussion about, what we think, is perhaps the most exciting question of membrane biophysics: the why and wherefore of non-bilayer lipids and lipid phases in, or in association with, bilayer biological membranes.

The importance of membrane defects-lessons from simulations.

The defects and pores within lipid membranes are scientifically interesting and have a number of biological applications. Although lipid bilayers are extremely thin hydrophobic barriers, just ∼3 nm thick, they include diverse chemistry and have complex structures. Bilayers are soft and dynamic, and as a result, they can bend and deform in response to different stimuli by means of structural changes in their component lipids. Though defects occur within these structures, their transience and small size have made it difficult to characterize them. However, with recent advances in computer power and computational modeling techniques, researchers can now use simulations as a powerful tool to probe the mechanism and energies of defect and pore formation in a number of situations. In this Account, we present results from our detailed molecular dynamics computer simulations of hydrophilic pores and related defects in lipid bilayers at an atomistic level. Electroporation can be used to increase the permeability of cellular membranes, with potential therapeutic applications. Atomistic simulations of electroporation have illustrated the molecular details of this process, including the importance of water dipole interactions at the water-membrane interface. Characterization of the lipid-protein interactions provides an important tool for understanding transmembrane protein structure and thermodynamic stability. Atomistic simulations give a detailed picture of the free energies of model peptides and side chains in lipid membranes; the energetic cost of defect formation strongly influences the energies of interactions between lipids and polar and charged residues. Many antimicrobial peptides form hydrophilic pores in lipid membranes, killing bacteria or cancer cells. On the basis of simulation data, at least some of these peptides form defects and pores near the center of the bilayer, with a common disordered structure where hydrated headgroups form an approximately toroidal shape. The localization and trafficking of lipids supports general membrane structure and a number of important signaling cascades, such as those involving ceramide, diacylglycerol, and cholesterol. Atomistic simulations have determined the rates and free energies of lipid flip-flop. During the flip-flop of most phosphatidylcholine lipids, a hydrophilic pore forms when the headgroup moves near the center of the bilayer. Simulations have provided novel insight into many features of defects and pores in lipid membranes. Simulation data from very different systems and models show how water penetration and defect formation can determine the free energies of many membrane processes. Bilayers can deform and allow transient defects and pores when exposed to a diverse range of stimuli. Future work will explore many aspects of membrane defects with increased resolution and scope, including the study of more complex lipid mixtures, membrane domains, and large-scale membrane remodeling. Such studies will examine processes including vesicle budding and fusion, non-bilayer lipid phases, and interactions between lipid bilayers and other biomolecules. Simulations provide information that complements experimental studies, allowing microscopic insight into experimental observations and suggesting novel hypotheses and experiments. These studies should enable a deeper understanding of the role of lipid bilayers in cellular biology and support the development of future lipid-based biotechnology.

Phosphatidic acid mediates the targeting of tBid to induce lysosomal membrane permeabilization and apoptosis

Upon apoptotic stimuli, lysosomal proteases, including cathepsins and chymotrypsin, are released into cytosol due to lysosomal membrane permeabilization (LMP), where they trigger apoptosis via the lysosomal-mitochondrial pathway of apoptosis. Herein, the mechanism of LMP was investigated. We found that caspase 8-cleaved Bid (tBid) could result in LMP directly. Although Bax or Bak might modestly enhance tBid-triggered LMP, they are not necessary for LMP. To study this further, large unilamellar vesicles (LUVs), model membranes mimicking the lipid constitution of lysosomes, were used to reconstitute the membrane permeabilization process in vitro. We found that phosphatidic acid (PA), one of the major acidic phospholipids found in lysosome membrane, is essential for tBid-induced LMP. PA facilitates the insertion of tBid deeply into lipid bilayers, where it undergoes homo-oligomerization and triggers the formation of highly curved nonbilayer lipid phases. These events induce LMP via pore formation mechanisms because encapsulated fluorescein-conjugated dextran (FD)-20 was released more significantly than FD-70 or FD-250 from LUVs due to its smaller molecular size. On the basis of these data, we proposed tBid-PA interactions in the lysosomal membranes form lipidic pores and result in LMP. We further noted that chymotrypsin-cleaved Bid is more potent than tBid at binding to PA, inserting into the lipid bilayer, and promoting LMP. This amplification mechanism likely contributes to the culmination of apoptotic signaling.

Efficient Light Harvesting by Photosystem II Requires an Optimized Protein Packing Density in Grana Thylakoids

A recently developed technique for dilution of the naturally high protein packing density in isolated grana membranes was applied to study the dependence of the light harvesting efficiency of photosystem (PS) II on macromolecular crowding. Slight dilution of the protein packing from 80% area fraction to the value found in intact grana thylakoids (70%) leads to an improved functionality of PSII (increased antenna size, enhanced connectivity between reaction centers). Further dilution induces a functional disconnection of light-harvesting complex (LHC) II from PSII. It is concluded that efficient light harvesting by PSII requires an optimal protein packing density in grana membranes that is close to 70%. We hypothesize that the decreased efficiency in overcrowded isolated grana thylakoids is caused by excited state quenching in LHCII, which has previously been correlated with neoxanthin distortion. Resonance Raman spectroscopy confirms this increase in neoxanthin distortion in overcrowded grana as compared with intact thylakoids. Furthermore, analysis of the changes in the antenna size in highly diluted membranes indicates a lipid-induced dissociation of up to two trimeric LHCII from PSII, leaving one trimer connected. This observation supports a hierarchy of LHCII-binding sites on PSII.

Anionic liposomal delivery system for DNA transfection

The present study investigates the use of novel anionic lipoplexes composed of physiological components for plasmid DNA delivery into mammalian cells in vitro. Liposomes were prepared from mixtures of endogenously occurring anionic and zwitterionic lipids, 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), respectively, at a molar ratio of 17:83 (DOPG:DOPE). Anionic lipoplexes were formed by complexation between anionic liposomes and plasmid DNA molecules encoding green fluorescence protein (GFP) using Ca2+ ions. Transfection and toxicity were evaluated in CHO-K1 cells using flow cytometry and propidium iodide staining, respectively. Controls included Ca2+-DNA complexes (without lipids), anionic liposomes (no Ca2+), and a cationic liposomal formulation. Efficient delivery of plasmid DNA and subsequent GFP expression was achieved using anionic lipoplexes. Transfection efficiency increased with Ca2+ concentration up to 14 mM Ca2+, where transfection efficiency was 7-fold higher than in untreated cells, with minimum toxicity. Further increase in Ca2+ decreased transfection. Transfection efficiency of anionic lipoplexes was similar to that of cationic liposomes (lipofectAmine), whereas their toxicity was significantly lower. Ca2+-DNA complexes exhibited minimal and irregular transfection with relatively high cytotoxicity. A model was developed to explain the basis of anionic lipoplex uptake and transfection efficacy. Effective transfection is explained on the formation of nonbilayer hexagonal lipid phases. Efficient and relatively safe DNA transfection using anionic lipoplexes makes them an appealing alternative to be explored for gene delivery.

Synergistic perturbation of phosphatidylcholine/sphingomyelin bilayers by diacylglycerol and cholesterol

The perturbations induced by second messenger diacylglycerols (DAGs) into bovine brain phosphatidylcholine (BBPC) bilayers in the presence or absence of bovine brain sphingomyelin (SM) and/or cholesterol were studied by 2 H NMR. Addition of 15 mol% DAG to BBPC bilayers did not induce non-bilayer lipid phases in the temperature range 30–60 °C. Similar measurements performed in the presence of cholesterol revealed that cholesterol progressively destabilizes PC bilayers with respect to DAG-induced perturbations. Thus, at 40 mol% cholesterol, addition of 15 mol% DAG induced the formation of non-bilayer (isotropic and inverted hexagonal) phases at 60 °C. Whereas some lateral separation of the bilayers into domains of different cholesterol contents was observed in BBPC/cholesterol membranes, such a lateral heterogeneity was greatly facilitated by the addition of SM. Since both a tendency to form non-bilayer lipid phases and lateral heterogeneity of the membranes are associated with increased activation of a number of membrane-associated enzymes, our results suggest that SM- and cholesterol-enriched regions of biological membranes (rafts) provide an environment with increased sensitivity to the generation of lipid second messengers and modified transmembrane signal transduction properties.

Mechanism of the interaction of beta(2)-glycoprotein I with negatively charged phospholipid membranes.

In an attempt to understand the multifunctional involvement of beta(2)-glycoprotein I (beta(2)GPI) in autoimmune diseases, thrombosis, atherosclerosis, and inflammatory processes, substantial interest is focused on the interaction of beta(2)GPI with negatively charged ligands, in particular, with acidic phospholipids. In this study, unilamellar vesicles composed of cardiolipin were used as in vitro membrane system to test and further refine a model of interaction based on the crystal structure of beta(2)GPI. The data suggest that beta(2)GPI anchors to the membrane surface with its hydrophobic loop adjacent to the positively charged lysine rich region in domain V. Subsequently, beta(2)GPI penetrates the membrane interfacial headgroup region as indicated by a restriction of the lipid side chain mobility, but without formation of a nonbilayer lipid phase. A structural rearrangement of beta(2)GPI upon lipid binding was detected by microcalorimetry and may result in the exposure of cryptic epitopes located in the complement control protein domains. This lipid-dependent conformational change may induce oligomerization of beta(2)GPI and promote intermolecular associations. Thus, the aggregation tendency of beta(2)GPI may serve as the basis for the formation of a molecular link between cells but may also be an essential feature for binding of autoantibodies and hence determine the role of beta(2)GPI in autoimmune diseases.

Tunable pH-Sensitive Liposomes Composed of Mixtures of Cationic and Anionic Lipids

The pH-dependent fusion properties of large unilamellar vesicles (LUVs) composed of binary mixtures of anionic and cationic lipids have been investigated. It is shown that stable LUVs can be prepared from the ionizable anionic lipid cholesteryl hemisuccinate (CHEMS) and the permanently charged cationic lipid N,N-dioleoyl- N,N-dimethylammonium chloride (DODAC) at neutral pH values and that these LUVs undergo fusion as the pH is reduced. The critical pH at which fusion was observed (pH f) was dependent on the cationic lipid-to-anionic lipid ratio. LUVs prepared from DODAC/CHEMS mixtures at molar ratios of 0 to 0.85 resulted in vesicles with pH f values that ranged from pH 4.0 to 6.7, respectively. This behavior is consistent with a model in which fusion occurs at pH values such that the DODAC/CHEMS LUV surface charge is zero. Related behavior was observed for LUVs composed of the ionizable cationic lipid 3 α-[ N-( N′,N′-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Chol) and the acidic lipid dioleoylphosphatidic acid (DOPA). Freeze-fracture and 31P NMR evidence is presented which indicates that pH-dependent fusion results from a preference of mixtures of cationic and anionic lipid for “inverted” nonbilayer lipid phases under conditions where the surface charge is zero. It is concluded that tunable pH-sensitive LUVs composed of cationic and anionic lipids may be of utility for drug delivery applications. It is also suggested that the ability of cationic lipids to adopt inverted nonbilayer structures in combination with anionic lipids may be related to the ability of cationic lipids to facilitate the intracellular delivery of macromolecules.

Nucleotide chain length and the morphology of complexes with cationic amphiphiles: 31P-NMR observations

31P-NMR and UV spectroscopies were used to study the interactions between cationic amphiphile-containing lipid bilayers and either a phosphorothioate oligonucleotide (OligoS) ( n=21) or polyadenylic acid (PolyA) ( n≈18 000). Multilamellar vesicles (MLVs) were composed of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) or 1,2-dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE) in binary mixture with either of the cationic lipids, N-[1-(2,3-dioleoyloxy)propyl]- N′, N′, N′-trimethylammonium chloride (DOTAP) or cetyltrimethylammonium bromide (CTAB). A UV-difference assay showed that OligoS binding ceased above a 1:1 anion/cation ratio, while PolyA binding continued until a 2:1 ratio was reached, indicating a ‘flat’ conformation for bound OligoS, but not necessarily for PolyA. Cross-polarization 31P-NMR of the nucleotide chains bound to 100% DOTAP MLVs produced spectra virtually identical to those of dry powders of OligoS or PolyA, indicating effective immobilization of the surface-bound nucleotide chains. Hahn echo 31P-NMR showed that MLVs composed of binary mixtures of POPC with DOTAP or CTAB retained a lamellar bilayer architecture upon adding nucleotide chains. At less than stoichiometric anion/cation ratios little or no signal attributable to free nucleotide chains was visible. A narrow signal at the chemical shift expected for phosphorothiodiesters or phosphodiesters became visible at greater levels of added OligoS or PolyA, respectively, indicating the presence of mobile nucleotide chains. Salt addition caused complete desorption of the nucleotide chains. When POPC was replaced with DOPE, binding of OligoS or PolyA produced non-bilayer lipid phases in the presence of DOTAP, but not in the presence of CTAB.

Ceramides Modulate Protein Kinase C Activity and Perturb the Structure of Phosphatidylcholine/Phosphatidylserine Bilayers

We studied the effects of natural ceramide and a series of ceramide analogs with different acyl chain lengths on the activity of rat brain protein kinase C (PKC) and on the structure of bovine liver phosphatidylcholine (BLPC)/dipalmitoylphosphatidylcholine (DPPC)/dipalmitoylphosphatidylserine (DPPS) (3:1:1 molar ratio) bilayers using 2H-NMR and specific enzymatic assays in the absence or presence of 7.5 mol % diolein (DO). Only a slight activation of PKC was observed upon addition of the short-chain ceramide analogs (C 2-, C 6-, or C 8-ceramide); natural ceramide or C 16-ceramide had no effect. In the presence of 7.5 mol % DO, natural ceramide and C 16-ceramide analog slightly attenuated DO-enhanced PKC activity. 2H-NMR results demonstrated that natural ceramide and C 16-ceramide induced lateral phase separation of gel-like and liquid crystalline domains in the bilayers; however, this type of membrane perturbation has no direct effect on PKC activity. The addition of both short-chain ceramide analogs and DO had a synergistic effect in activating PKC, with maximum activity observed with 20 mol % C 6-ceramide and 15 mol % DO. Further increases in C 6-ceramide and/or DO concentrations led to decreased PKC activity. A detailed 2H-NMR investigation of the combined effects of C 6-ceramide and DO on lipid bilayer structure showed a synergistic effect of these two reagents to increase membrane tendency to adopt nonbilayer structures, resulting in the actual presence of such structures in samples exceeding 20 mol % ceramide and 15 mol % DO. Thus, the increased tendency to form nonbilayer lipid phases correlates with increased PKC activity, whereas the actual presence of such phases reduced the activity of the enzyme. Moreover, the results show that short-chain ceramide analogs, widely used to study cellular effects of ceramide, have biological effects that are not exhibited by natural ceramide.

Peptides and lipid bilayers: Dynamic interactions

The interactions of amphipathic a-helical peptides with lipid bilayers provide a model of the interactions of membrane proteins with their environment, as well as insights into the modes of action of biologically important peptides. Recent results have shown that antimicrobial peptides, such as magainin, may act by stabilising the formation of toroidal pores within lipid bilayers. In contrast, the influenza fusion peptide promotes nonbilayer lipid phases. Complex pore-forming bacterial toxins, such as colicins, unfold at the bilayer surface, yielding a dynamic structure containing both bilayer-inserted and bilayer-surface helices. Recent improvements in methods for computer simulations have enabled them to play an important role in helping elucidate the nature of peptide-bilayer interactions.

Phase changes in membrane lipids in sickle red cell shed-vesicles and sickle red cells.

Lipid phase transformations may occur in the membranes of sickle red cell shed-vesicles and sickle red cells. The presence of such phase changes could be important in sickle cell disease since membrane phase changes appear to contribute to the generation of antiphospholipid antibodies that are thrombophilic and occur in sickle cell disease. In the present study, we have evaluated sickle red cell shed-vesicles and sickle red cells for the presence of non-bilayer lipid phases using 31P-NMR spectroscopy. Results show that the spectra of both the shed-vesicles and the sickle red cells are compatible with the occurrence of non-bilayer phases in the membrane bilayers. The findings support the concept that these membranes could contribute to the generation of antiphospholipid antibodies in sickle cell disease.