Permeability of bilayers composed of mixtures of saturated phospholipids

Liposomes composed of an equimolar binary mixture of phospholipids were formed from a series of saturated phosphatidylcholines (PC) and phosphatidylethanolamines (PE). Mixtures were chosen such that the two phospholipids differed either in terms of head group alone, chain length alone, or both head group and chain length. Cation effluxes, both with and without ionophores (nigericin and valinomycin) were measured over a range of temperatures that encompassed the regions of phase separation for these different lipid mixtures. There was a good correlation between the temperatures at which permeability maxima and phase separation occur. For phospholipid mixtures with the same acyl chain but different head groups (PC vs. PE), the PC component ‘controls’ permeability. For mixtures of PCs differing in chain length, the short chain lipid dominates the permeability pattern particularly if the chain lengths are sufficiently different. Lipids differing in both head group and chain length give rise to more complex permeability patterns. The results of the present study are interpreted in terms of a model in which one of the lipid components of the mixture may specifically congregate at defects between co-existing phases and thus ‘regulate’ permeability.     

Structure of bacterial lipopolysaccharides

Bacterial lipopolysaccharides are the major components of the outer surface of Gram-negative bacteria They are often of interest in medicine for their immunomodulatory properties. In small amounts they can be beneficial, but in larger amounts they may cause endotoxic shock. Although they share a common architecture, their structural details exert a strong influence on their activity. These molecules comprise: a lipid moiety, called lipid A, which is considered to be the endotoxic component, a glycosidic part consisting of a core of approximately 10 monosaccharides and, in "smooth-type" lipopolysaccharides, a third region, named O-chain, consisting of repetitive subunits of one to eight monosaccharides responsible for much of the immunospecificity of the bacterial cell.     

Packing characteristics of two-component bilayers composed of ester- and ether-linked phospholipids.

The miscibility properties of ether- and ester-linked phospholipids in two-component, fully hydrated bilayers have been studied by differential scanning calorimetry (DSC) and Raman spectroscopy. Mixtures of 1,2-di-O-hexadecyl-rac-glycero-3-phosphocholine (DHPC) with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DHPE) and of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with 1,2-di-O-hexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) have been investigated. The phase diagram for the DPPC/DHPE mixtures indicates that these two phospholipids are miscible in all proportions in the nonrippled bilayer gel phase. In contrast, the DHPC/DPPE mixtures display two regions of gel phase immiscibility between 10 and 30 mol% DPPE. Raman spectroscopic measurements of DHPC/DPPE mixtures in the C-H stretching mode region suggest that this immiscibility arises from the formation of DHPC-rich interdigitated gel phase domains with strong lateral chain packing interactions at temperatures below 27 degrees C. However, in the absence of interdigitation, our findings, and those of others, lead to the conclusion that the miscibility properties of mixtures of ether- and ester-linked phospholipids are determined by the nature of the phospholipid headgroups and are independent of the character of the hydrocarbon chain linkages. Thus it seems unlikely that the ether linkage has any significant effect on the miscibility properties of phospholipids in biological membranes.     

Structure of the liposome composed of lipid-heme and phospholipids

The amphiphilic heme derivative, 5,10,15,20-tetra(α,α,α,α-o-(2′,2′-dimethyl-20′-(2′-trimethylammonioethyl) phosphonatoxyicosanamido)pheny)phorphinatoiron(II) (lipid-heme), formed a stable liposome (Φ ≈ 400 Å) with phospholipids. Differential scanning calorimetry showed that incorporation of the lipid-heme in the liposome bilayer (lipid/lipid-heme > 25) causes no disordering of the bilayer structure. Ligation of a bulky ligand to the lipid-heme liposome indicated that the lipid-heme situates facing predominantly outwards in the liposome. The closed vesicle structure and the stability of the lipid-heme liposome were also confirmed by the encapsulating capability of the fluorescence compound.     

Macro-ripple phase formation in bilayers composed of galactosylceramide and phosphatidylcholine.

As determined by freeze fracture electron microscopy, increasing levels of bovine brain galactosylceramide (GalCer) altered the surface structure of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers by inducing a striking "macro-ripple" phase in the larger, multilamellar lipid vesicles at GalCer mole fractions between 0.4 and 0.8. The term "macro-ripple" phase was used to distinguish it from the P beta' ripple phase observed in saturated, symmetric-chain length phosphatidylcholines. Whereas the P beta' ripple phase displays two types of corrugations, one with a wavelength of 12-15 nm and the other with a wavelength of 25-35 nm, the macro-ripple phase occurring in GalCer/POPC dispersions was of one type with a wavelength of 100-110 nm. Also, in contrast to the extended linear arrays of adjacent ripples observed in the P beta' ripple phase, the macro-ripple phase of GalCer/POPC dispersions was interrupted frequently by packing defects resulting from double dislocations and various disclinations and, thus, appeared to be continuously twisting and turning. Control experiments verified that the macro-ripple phase was not an artifact of incomplete lipid mixing or demixing during preparation. Three different methods of lipid mixing were compared: a spray method of rapid solvent evaporation, a sublimation method of solvent removal, and solvent removal using a rotary evaporation apparatus. Control experiments also revealed that the macro-ripple phase was observed regardless of whether lipid specimens were prepared by either ultra-rapid or manual plunge freezing methods as well as either in the presence or absence of the cryo-protectant glycerol. The macro-ripple phase was always observed in mixtures that were fully annealed by incubation above the main thermal transition of both POPC and bovine brain GalCer before rapid freezing. If the GalCer mixed with POPC contained only nonhydroxy acyl chains or only 2-hydroxy acyl chains, then the occurrence of macro-ripple phase decreased dramatically.     

Sphingomyelin structure influences the lateral diffusion and raft formation in lipid bilayers

Liquid-disordered/liquid-ordered two-phase coexistence regions in hydrated bilayers have been investigated for sphingomyelins (SMs) of three different origins: egg, brain, and milk with the pulsed-field gradient NMR technique for lateral diffusion measurement. It is found that the three SMs have the same diffusional behavior in bilayers of SM alone, but in the multicomponent systems of dioleoylphosphatidylcholine/SM/cholesterol, the ability to form domains differs for the three SMs. The two-phase area is more extended for egg SM than for brain SM, and no two-phase coexistence is found for milk SM. The differences in behavior are correlated with the homogeneity of the SM hydrocarbon chain compositions, in which egg SM has the most homogeneous and milk SM has the most heterogeneous composition. The results indicate that a crucial element in the domain-forming process is the formation of highly packed bilayers of SM and cholesterol rather than specific interactions between SM and cholesterol.     

Lysylated phospholipids stabilize models of bacterial lipid bilayers and protect against antimicrobial peptides

Aminoacylated phosphatidylglycerols are common lipids in bacterial cytoplasmic membranes. Their presence in Staphylococcus aureus has been linked to increased resistance to a number of antibacterial agents, including antimicrobial peptides. Most commonly, the phosphatidylglycerol headgroup is esterified to lysine, which converts anionic phosphatidylglycerol into a cationic lipid with a considerably increased headgroup size. In the present work, we investigated the interactions of two well-studied antimicrobial peptides, cecropin A and mastoparan X, with lipid vesicles composed of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), containing varying fractions of an aminoacylated phosphatidylethanolamine, a stable analog of the corresponding phosphatidylglycerol-derivative. To differentiate between the effects of headgroup size and charge on peptide–lipid interactions, we synthesized two different derivatives. In one, the headgroup was modified by the addition of lysine, and in the other, by glutamine. The modification by glutamine results in a phospholipid with a headgroup size comparable to that of the lysylated version. However, whereas lysyl-phosphatidylethanolamine (Lys-PE) is cationic, glutaminyl-phosphatidylethanolamine (Gln-PE) is zwitterionic. We found that binding of mastoparan X and cecropin A was not significantly altered if the content of aminoacylated phosphatidylethanolamines did not exceed 20 mol.%, which is the concentration found in bacterial membranes. However, a lysyl-phosphatidylethanolamine content of 20 mol% significantly inhibits dye release from lipid vesicles, to a degree that depends on the peptide. In the case of mastoparan X, dye release is essentially abolished at 20 mol.% lysyl-phosphatidylethanolamine, whereas cecropin A is less sensitive to the presence of lysyl-phosphatidylethanolamine. These observations are understood through the complex interplay between peptide binding and membrane stabilization as a function of the aminoacylated lipid content. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Effect of cationic lipids in the formation of asymmetries in supported bilayers

We have studied the formation of a supported bilayer containing both cationic and zwitterionic lipids by fusion of small unilamellar vesicles (SUV) onto the solid surface at low salt conditions using a combination of attenuated total reflection infrared (ATR-IR) and deuterium NMR spectroscopy with microcalorimetry. The data suggest that a significant cationic lipid asymmetry between the outer (distal) and the inner (proximal) monolayer of a supported bilayer results under conditions of prolonged incubation times of the solid support with the SUV coating solution. For a SUV composition of DPPC/DHDAB (4:1) we observed an enrichment of the cationic component in the proximal monolayer of up to 200% compared to the distal monolayer after 12 h incubation. It is suggested that the electrostatic potential arising from the solid surface is the driving force for the creation of this asymmetry by means of directed flip-flop between the monolayers and/or by temporary fusion between SUV from the bulk with the supported bilayer.     

Effect of surface treatment on diffusion and domain formation in supported lipid bilayers

Supported lipid bilayers are widely used as model systems due to their robustness. Due to the solid support, the properties of supported lipid bilayers are different from those of freestanding bilayers. In this article, we examine whether different surface treatments affect the properties of supported lipid bilayers. It will be shown that depending on the treatment method, the diffusion of the lipids can be adjusted approximately threefold without altering the composition. Additionally, as the bilayer-support interaction decreases, it becomes easier to form coexisting liquid-ordered and liquid-disordered domains. The physical/chemical alterations that result from the different treatment methods will be discussed.     

Structure of the full-length major pilin from Streptococcus pneumoniae: implications for isopeptide bond formation in gram-positive bacterial pili

The surface of the pneumococcal cell is adorned with virulence factors including pili. The major pilin RrgB, which forms the pilus shaft on pathogenic Streptococcus pneumoniae, comprises four immunoglobulin (Ig)-like domains, each with a common CnaB topology. The three C-terminal domains are each stabilized by internal Lys-Asn isopeptide bonds, formed autocatalytically with the aid of an essential Glu residue. The structure and orientation of the crucial N-terminal domain, which provides the covalent linkage to the next pilin subunit in the shaft, however, remain incompletely characterised. We report the crystal structure of full length RrgB, solved by X-ray crystallography at 2.8 Å resolution. The N-terminal (D1) domain makes few contacts with the rest of the RrgB structure, and has higher B-factors. This may explain why D1 is readily lost by proteolysis, as are the N-terminal domains of many major pilins. D1 is also found to have a triad of Lys, Asn and Glu residues in the same topological positions as in the other domains, yet mass spectrometry and the crystal structure show that no internal isopeptide bond is formed. We show that this is because β-strand G of D1, which carries the Asn residue, diverges from β-strand A, carrying the Lys residue, such that these residues are too far apart for bond formation. Strand G also carries the YPKN motif that provides the essential Lys residue for the sortase-mediated intermolecular linkages along the pilus shaft. Interaction with the sortase and formation of the intermolecular linkage could result in a change in the orientation of this strand, explaining why isopeptide bond formation in the N-terminal domains of some major pilins appears to take place only upon assembly of the pili.     

Phospholipid exchange shows insulin receptor activity is supported by both the propensity to form wide bilayers and ordered raft domains

Insulin receptor (IR) is a membrane tyrosine kinase that mediates the response of cells to insulin. IR activity has been shown to be modulated by changes in plasma membrane lipid composition, but the properties and structural determinants of lipids mediating IR activity are poorly understood. Here, using efficient methyl-alpha-cyclodextrin mediated lipid exchange, we studied the effect of altering plasma membrane outer leaflet phospholipid composition upon the activity of IR in mammalian cells. After substitution of endogenous lipids with lipids having an ability to form liquid ordered (Lo) domains (sphingomyelins) or liquid disordered (Ld) domains (unsaturated phosphatidylcholines (PCs)), we found that the propensity of lipids to form ordered domains is required for high IR activity. Additional substitution experiments using a series of saturated PCs showed that IR activity increased substantially with increasing acyl chain length, which increases both bilayer width and the propensity to form ordered domains. Incorporating purified IR into alkyl maltoside micelles with increasing hydrocarbon lengths also increased IR activity, but more modestly than by increasing lipid acyl chain length in cells. These results suggest that the ability to form Lo domains as well as wide bilayer width contributes to increased IR activity. Inhibition of phosphatases showed that some of the lipid dependence of IR activity upon lipid structure reflected protection from phosphatases by lipids that support Lo domain formation. These results are consistent with a model in which a combination of bilayer width and ordered domain formation modulates IR activity via IR conformation and accessibility to phosphatases.     

Interaction of polycationic polymers with supported lipid bilayers and cells: nanoscale hole formation and enhanced membrane permeability

Interactions of polycationic polymers with supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers and live cell membranes (KB and Rat2) have been investigated using atomic force microscopy (AFM), cytosolic enzyme assays, confocal laser scanning microscopy (CLSM), and a fluorescence-activated cell sorter (FACS). Polycationic polymers poly-L-lysine (PLL), polyethylenimine (PEI), and diethylaminoethyl-dextran (DEAE-DEX) and sphere-like poly(amidoamine) (PAMAM) dendrimers are employed because of their importance for gene and drug delivery. AFM studies indicate that all the polycationic polymers cause the formation and/or expansion of preexisting defects in supported DMPC bilayers in the concentration range of 1-3 microg/mL. By way of contrast, hydroxyl-containing neutral linear poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA) do not induce hole formation or expand the size of preexisting defects in the same concentration range. All polymers tested are not toxic to KB or Rat2 cells up to a 12 microg/mL concentration (XTT assay). In the concentration range of 6-12 microg/mL, however, significant amounts of the cytosolic enzymes lactate dehydrogenase (LDH) and luciferase (LUC) are released. PEI, which possesses the greatest density of charged groups on its chain, shows the most dramatic increase in membrane permeability. In addition, treatment with polycationic polymers allows the small dye molecules propidium idodide (PI) and fluorescein (FITC) to diffuse in and out of the cells. CLSM images also show internalization of PLL labeled with FITC dye. In contrast, controls of membrane permeability using the neutral linear polymers PEG and PVA show dramatically less LDH and LUC leakage and no enhanced dye diffusion. Taken together, these data are consistent with the hypothesis that polycationic polymers induce the formation of transient, nanoscale holes in living cells and that these holes allow a greatly enhanced exchange of materials across the cell membrane.     

Ripples and the formation of anisotropic lipid domains: imaging two-component supported double bilayers by atomic force microscopy

Direct visualization of the fluid-phase/ordered-phase domain structure in mica-supported bilayers composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-distearoyl-sn-glycero-3-phosphocholine mixtures is performed with atomic force microscopy. The system studied is a double bilayer supported on a mica surface in which the top bilayer (which is not in direct contact with the mica) is visualized as a function of temperature. Because the top bilayer is not as restricted by the interactions with the surface as single supported bilayers, its behavior is more similar to a free-standing bilayer. Intriguing straight-edged anisotropic fluid-phase domains were observed in the fluid-phase/ordered-phase coexistence temperature range, which resemble the fluid-phase/ordered-phase domain patterns observed in giant unilamellar vesicles composed of such phospholipid mixtures. With the high resolution provided by atomic force microscopy, we investigated the origin of these anisotropic lipid domain patterns, and found that ripple phase formation is directly responsible for the anisotropic nature of these domains. The nucleation and growth of fluid-phase domains are found to be directed by the presence of ripples. In particular, the fluid-phase domains elongate parallel to the ripples. The results show that ripple phase formation may have implications for domain formation in biological systems.     

Structure and function of lipopolysaccharides

The lipopolysaccharides of Gram-negative bacteria have a profound effect on the mammalian immune system and are of great significance in the pathophysiology of many disease processes. Consideration is given in this review to the relationship between structure and function of these lipopolysaccharides.     

Electrophoretic and mass spectrometric strategies for profiling bacterial lipopolysaccharides

Capillary electrophoresis (CE) is a high-resolution separation technique that has been widely used for trace analysis in biological samples. On-line capillary electrophoresis-electrospray mass spectrometry (CE-MS) was developed for the analysis of lipopolysaccharide (LPS) glycoforms from the gram-negative bacteria, Haemophilus influenzae. In this paper, we report on the application of CE-MS to characterize structural differences in O-deacylated LPS samples from H. influenzae strains Rd 11.7 and 375.1. The resolution capability of on-line CE-MS was first demonstrated by analysis of a complex LPS mixture from H. influenzae strain Rd 11.7. This strain contains a mixture of isomeric glycoforms differing in the number and positions of hexose moieties. Sialic acid containing glycoforms were also determined. Structural features of LPS from a lic1 mutant of H. influenzae strain 375 (375.1) were studied using on-line CE-MS/MS. With the separation provided by CE, two isomeric glycoforms differing in the location of phosphoethanolamine substituents were characterized by tandem mass spectrometry.     

NMR studies of membranes composed of glycolipids and phospholipids

Lipid membranes composed of monogalactosyldiacylglycerol (MGDG) and dimyristoylphosphatidylcholine (DMPC) were studied by means of NMR spectroscopy. The macroscopic phase behaviour was investigated by (31)P NMR under stationary conditions, whereas microscopic properties such as segmental ordering were probed by two-dimensional (1)H-(13)C separated local field experiments under magic-angle spinning conditions. Our results clearly show that ordering/disordering effects occur for the headgroups as well as for the acyl chains when the sample composition is varied. In particular, the (1)H-(13)C dipolar couplings within the galactose headgroup of MGDG exhibited significant concentration dependence.     

NMR studies of membranes composed of glycolipids and phospholipids

Lipid membranes composed of monogalactosyldiacylglycerol (MGDG) and dimyristoylphosphatidylcholine (DMPC) were studied by means of NMR spectroscopy. The macroscopic phase behaviour was investigated by ³¹P NMR under stationary conditions, whereas microscopic properties such as segmental ordering were probed by two-dimensional ¹H-¹³C separated local field experiments under magic-angle spinning conditions. Our results clearly show that ordering/disordering effects occur for the headgroups as well as for the acyl chains when the sample composition is varied. In particular, the ¹H-¹³C dipolar couplings within the galactose headgroup of MGDG exhibited significant concentration dependence.