The sponge phase of a mixed surfactant system.

We study the sponge phase of the mixed non-ionic/ionic surfactant system C14DMAO-TTAB-hexanol-brine. Our aim is to determine if this phase exists in this mixed system and if it preserves or changes its structure when the relative amount of the charged surfactant is increased in the mixture. SAXS, FFEM, and conductivity results show that for the same bilayer volume fraction the sponge phase preserves its global structure. We propose a method to determine the geometrical obstruction factor from electrical conductivity measurements in ionic sponge phases. Analysis of lamellar phases in the same system shows that the bilayer thickness increases when the ionic surfactant concentration is increased.

Slow Reorganization of Small Phosphatidylcholine Vesicles upon Adsorption of Amphiphilic Polymers.

Static or dynamic light scattering measurements were performed in parallel, on dilute mixtures of DPPC/DPPA vesicles (typical radius 60 nm) and hydrophobically modified polymers. This technique gave evidence of the slow kinetics involved in both the reorganization of an adsorbed polymer layer and the membrane breakage. Hours, or sometimes days, were required in order to follow the variation of both the hydrodynamic radius and the scattering intensity at intermediate stages. Images of the intermediate species were collected using freeze-fracture electron microscopy (FFEM). Comparison of different polymers (of varying molecular weight or structure) revealed the prime importance of hydrophobicity on the disruption of membranes. Although the presence of a few percent of pendant alkyl chains along the polymer backbone induced adsorption to membranes, only the association with the more hydrophobic ones (>25 mol% of pendant octyl groups) resulted in small mixed objects of micellar size (radius about 10 nm). The drop of the mean radius of intermediate structures formed upon the vesicle breakage was also sensitive to temperature. A tentative mechanism was proposed on the basis of kinetics and FFEM studies. Copyright 2001 Academic Press.

Self-assembly of regular hollow icosahedra in salt-free catanionic solutions.

Self-assembled structures having a regular hollow icosahedral form (such as those observed for proteins of virus capsids) can occur as a result of biomineralization processes, but are extremely rare in mineral crystallites. Compact icosahedra made from a boron oxide have been reported, but equivalent structures made of synthetic organic components such as surfactants have not hitherto been observed. It is, however, well known that lipids, as well as mixtures of anionic and cationic single chain surfactants, can readily form bilayers that can adopt a variety of distinct geometric forms: they can fold into soft vesicles or random bilayers (the so-called sponge phase) or form ordered stacks of flat or undulating membranes. Here we show that in salt-free mixtures of anionic and cationic surfactants, such bilayers can self-assemble into hollow aggregates with a regular icosahedral shape. These aggregates are stabilized by the presence of pores located at the vertices of the icosahedra. The resulting structures have a size of about one micrometre and mass of about 1010 daltons, making them larger than any known icosahedral protein assembly or virus capsid. We expect the combination of wall rigidity and holes at vertices of these icosahedral aggregates to be of practical value for controlled drug or DNA release.

Selective adhesion, lipid exchange and membrane-fusion processes between vesicles of various sizes bearing complementary molecular recognition groups.

Equimolar mixtures of large unilamellar vesicles (LUVs) obtained from mixtures of egg lecithin and lipids containing complementary hydrogen bonding head groups (barbituric acid (BAR) and 2,4,6-triaminopyrimidine (TAP)) were shown to aggregate and fuse. These events have been studied in detail using electron microscopy and dynamic light scattering, and by fluorimetry using membrane or water-soluble fluorescence probes. It was shown that aggregation was followed by two competitive processes: a) lipid mixing leading to redispersion of the vesicles; b) fusion events generating much larger vesicles. In order to better understand the nature of the interaction, the effects of ionic strength and surface concentration of recognition lipids on the aggregation process were investigated by dynamic light scattering. Additionally, it was possible to inhibit the aggregation kinetics through addition of a soluble barbituric acid competitor. The study was extended to giant unilamellar vesicles (GUVs) to investigate the size effect and visualise the phenomena in situ. The interactions between complementary LUVs and GUVs or GUVs and GUVs were studied by optical microscopy using dual fluorescent labelling of both vesicle populations. A selective adhesion of LUVs onto GUVs was observed by electron and optical microscopies, whereas no aggregation took place in case of a GUV/GUV mixture. Furthermore, a fusion assay of GUV and LUV using the difference of size between GUV and LUV and calceine self-quenching showed that no mixing between the aqueous pools occured.

VAMP (synaptobrevin) is present in the plasma membrane of nerve terminals.

Synaptic vesicle docking and exocytosis require the specific interaction of synaptic vesicle proteins (such as VAMP/synaptobrevin) with presynaptic plasma membrane proteins (such as syntaxin and SNAP 25). These proteins form a stable, SDS-resistant, multimolecular complex, the SNARE complex. The subcellular distribution of VAMP and syntaxin within Torpedo electric organ nerve endings was studied by immunogoldlabeling of SDS-digested freeze-fracture replicas (Fujimoto, 1995). This technique allowed us to visualize large surface areas of the presynaptic plasma membrane and numerous synaptic vesicles from rapidly frozen nerve endings and synaptosomes. VAMP was found associated with synaptic vesicles, as also shown by conventional electron microscopy immunolabeling, and to the presynaptic plasma membrane (P leaflet). Syntaxin was also detected in the nerve ending plasma membrane, without gold labeling of synaptic vesicles. Comparison of gold particle densities suggests that the presynaptic plasma membrane contains 3 VAMP molecules per molecule of syntaxin. After biotinylation of intact synaptosomes, the synaptosomal plasma membrane was isolated on Streptavidin coated magnetic beads. Its antigenic content was compared to that of purified synaptic vesicles. VAMP was present in both membranes whereas syntaxin and SNAP 25 were highly enriched in the synaptosomal plasma membrane. This membrane has a low content of classical synaptic vesicle proteins (synaptophysin, SV2 and the vesicular acetylcholine transporter). The VAMP to syntaxin stoichiometry in the isolated synaptosomal membrane was estimated by comparison with purified antigens and close to 2, in accordance with morphological data. SDS-resistant SNARE complexes were detected in the isolated presynaptic membrane but absent in purified synaptic vesicles. Taken together, these results show that the presence of VAMP in the plasma membrane of nerve endings cannot result from exocytosis of synaptic vesicles, a process which could, as far as SNAREs are concerned, very much resemble homotypic fusion.

Macromolecular Colloids of Diblock Poly(amino acids) That Bind Insulin.

The diblock polymer poly(l-leucine-block-l-glutamate), bLE, was synthesized by acid hydrolysis of the ester poly(l-leucine-block-l-methyl glutamate). During the hydrolysis reaction the leucine block precipitates from the reaction mixture, forming nanosized particulate structures. These particles can be purified and further suspended in water or in 0.15 M phosphate saline buffer (PBS) to give stable, colloidal dispersions. TEM analysis shows the predominant particle form to be that of platelets with a diameter of 200 nm. Smaller cylindrical or spherical particles form a relatively minor fraction of the sample. After fractionation, analysis shows the platelets to be compositionally rich in leucine, while the spheres are glutamate-rich. (1)H NMR, CD, and X-ray diffraction indicate that the core of the platelets is composed of crystalline, helical leucine segments. The poly(l-glutamate) polyelectrolyte brush extending out from the two faces of the disk stabilizes individual particles from flocculation. At pH 7.4, the nanoparticles (platelets and cylinders) spontaneously adsorb proteins, such as insulin, directly from solution. Partial desorption of the protein in its native configuration can be induced by simple dilution. The reversibility of the insulin-nanoparticle complex is the basis for a potential new delivery system. Copyright 1999 Academic Press.

Self-assembly of chlorophenols in water.

In saturated solutions of some di- and trichlorophenols, structures with complex morphologies, consisting of thin, transparent sheets often coiling into helices and ultimately twisting into filaments, were observed under the optical microscope. Freeze-fracture electron microscopy, x-ray diffraction, phase diagrams, and molecular modeling were performed to elucidate the observed phenomena. Here, we present evidence that the chlorophenols studied, when interacting with water, self-assemble into bilayers. The fact that some chlorophenols form the same supramolecular structures as those described previously for structurally nonrelated surfactants sheds light on the mechanisms of self-assembly.

Self-assembly of flat nanodiscs in salt-free catanionic surfactant solutions

Discs of finite size are a very rare form of stable surfactant self-assembly. It is shown that mixing of two oppositely charged single-chain surfactants can produce rigid nanodiscs as well as swollen lamellar liquid crystals with frozen bilayers. The crucial requirement for obtaining nanodisc self-assembly is the use of H+ and OH- as counterions. These counterions then form water and lower the conductivity to 10 microsiemens per centimeter. In the case of cationic component excess, a dilute solution of nanodiscs is in thermodynamic equilibrium with a lamellar phase. The diameter of the cationic nanodiscs is continuously adjustable from a few micrometers to 30 nanometers, with the positive charge located mainly around the edges.

Crystallographic analysis of freeze-fractured three-dimensionally ordered specimens.

We describe here an original approach for solving the structure of three-dimensionally ordered specimens at low and medium resolutions. It combines freeze-fracture electron microscopy and quantitative image processing and has been first successfully applied to the crystallographic study of different lipid-containing cubic phases. The structure preservation during cryofixation is controlled by recording X-ray diffraction before and after freezing. Well frozen cubic phases show fracture planes which look like well defined cleavage planes of 3-D crystals. These fracture planes (domains) reveal a mosaic of 2D ordered sub-domains which are geometrically related to each other by simple crystallographic operations. The symmetry properties of the images mirror faithfully the symmetry of the space groups. The shifts and rotations observed between adjacent sub-domains are related to this symmetry. Different cubic phases display different fracture behavior, highly characteristic for a given space group. Interpretation of the averaged images of different domains in terms of molecular structure is done by the comparison of the averaged periodic motifs either with the corresponding sections of the electron density map (from X-ray diffraction data) or with the corresponding sections of a 3-D-space filling model. We show here that the same procedure may be applied to other three-dimensionally ordered specimens such as 3-D crystals of membrane proteins or of other proteins, including naturally occurring protein crystals of some secretory organelles. Finally, the same approach could also provide a powerful tool for the study of membrane protein crystallogenesis, particularly for the formation of 3-D crystals.

Perfluoroalkylphosphocholines are poor protein-solubilizing surfactants, as tested with neutrophil plasma membranes.

We have tested the membrane-protein solubilizing properties of two perfluoroalkylphosphocholines. These compounds belong to a series of fluorinated amphiphiles which are being investigated as potential stabilizing agents for a variety of fluorocarbon-based systems. We are particularly interested in cytochrome b558 from phagocytes, the redox component of NADPH oxidase. Its heavy subunit is believed to carry binding sites for NADPH and FAD. Nevertheless, when the cytochrome is purified in the presence of classical detergents, it carries no FAD. This could be due to a delipidating, denaturing effect of these detergents (octyl glucoside, Triton, etc). The first perfluoroalkyphosphocholine, C8F17(CH2)2O-P(O2-)-O(CH2)2N+(CH3)3(F8C2PC), extracted about as much protein from neutrophil plasma membranes into a 100,000 g supernatant as octyl glucoside. The second compound, C8F17(CH2)11O-P(O2-)-O(CH2)2N+(CH3)3(F8C11PC), was less efficient. We found that flavin was still protein-bound in the crude F8C2PC extract at a FAD to heme ratio of about 1, and a good NADPH oxidase activity was obtained without addition of exogenous FAD, even after dialysis or gel filtration, whereas dialysis eliminated most of the FAD from the octyl glucoside extracts. These experiments appeared to make F8C2PC an interesting membrane-solubilizing agent. Nevertheless, no protein in the F8C2PC extract could be adsorbed on the chromatographic supports normally used for purification. After dilution of the extract and addition of 15 mM octyl glucoside, some of the proteins, such as myeloperoxidase, could be adsorbed (and eluted), but not cytochrome b558. Freeze-fracture electron microscopy showed that the F8C2PC extracts contained numerous vesicles and aggregates of small shapeless particles. Higher centrifugal forces sedimented most proteins of the 100,000 g supernatant. As a check, the effect of F8C2PC was tested on sarcoplasmic reticulum vesicles, the behavior of which with respect to the usual non-denaturating detergents has been well studied. There was little, if any, solubilization. We conclude that, although supernatants of F8C2PC extracts of neutrophil membranes are optically clear, proteins are not really solubilized. This result is in keeping with the absence of lytic effects of F8C2PC on erythrocyte membranes.

Molecular biology of S-layers.

In this chapter we report on the molecular biology of crystalline surface layers of different bacterial groups. The limited information indicates that there are many variations on a common theme. Sequence variety, antigenic diversity, gene expression, rearrangements, influence of environmental factors and applied aspects are addressed. There is considerable variety in the S-layer composition, which was elucidated by sequence analysis of the corresponding genes. In Corynebacterium glutamicum one major cell wall protein is responsible for the formation of a highly ordered, hexagonal array. In contrast, two abundant surface proteins from the S-layer of Bacillus anthracis. Each protein possesses three S-layer homology motifs and one protein could be a virulence factor. The antigenic diversity and ABC transporters are important features, which have been studied in methanogenic archaea. The expression of the S-layer components is controlled by three genes in the case of Thermus thermophilus. One has repressor activity on the S-layer gene promoter, the second codes for the S-layer protein. The rearrangement by reciprocal recombination was investigated in Campylobacter fetus. 7-8 S-layer proteins with a high degree of homology at the 5' and 3' ends were found. Environmental changes influence the surface properties of Bacillus stearothermophilus. Depending on oxygen supply, this species produces different S-layer proteins. Finally, the molecular bases for some applications are discussed. Recombinant S-layer fusion proteins have been designed for biotechnology.

The S-layer protein of Corynebacterium glutamicum is anchored to the cell wall by its C-terminal hydrophobic domain.

PS2 is the S-layer protein of Corynebacterium glutamicum. The S-layer may be detached from the cell as organized sheets by detergents at room temperature. The solubilization of PS2 in the form of monomers requires detergent treatment at high temperature (70 degrees C), conditions under which the protein is denatured. Treatment of the cells with proteinase K or trypsin results in the detachment of the organized S-layer, which remains organized. Because we show that trypsin cleaves the C-terminal part of the protein, we conclude that this domain is involved in the association of the S-layer to the cell but is not essential in the interaction between individual PS2 proteins within the S-layer. A modified form of PS2, deleted of its C-terminal hydrophobic sequence, was constructed. The protein is almost unable to form an organized S-layer and is mainly released into the medium. We suggest that PS2 is anchored via its C-terminal hydrophobic sequence to a hydrophobic layer of the wall of the bacterium located some distance above the cytoplasmic membrane.

Freeze fracture electron microscopy of lyotropic lipid systems: quantitative analysis of the inverse micellar cubic phase of space group Fd3m (Q227).

An inverse micellar cubic phase of cubic aspect 15 formed by dioleoylglycerol/dioleoylphosphatidylcholine mixtures has been studied by freeze fracture electron microscopy. The structure was well preserved after freezing samples which had been hydrated either in pure water or in 30 vol% aqueous glycerol solutions. Electron microscopy images of high quality and resolution have been obtained. Four types of fracture planes, perpendicular to the [111], [110], [311] and [100] crystallographic axes, were identified by optical diffraction of the images from selected areas of the replicas. This is the largest number of different fracture planes yet observed in any lipid mesophase by electron microscopy. These planes are also perpendicular to the directions of the lowest order, and most intense reflections in the X-ray patterns from this cubic phase. The images were filtered using correlation averaging techniques, and they revealed the presence of mirror planes, which establishes that the space group is Fd3m (Q227) rather than Fd3. The interpretation of the images was aided by the novel use of standard deviation (s.d.) information obtained from the averaging procedures. The results are easily interpreted with the structure model deduced from X-ray diffraction and consisting of a complex packing of two different sizes of quasi-spherical inverse micelles located at positions (a) and (d) of the Fd3m unit cell. The results also show clearly that the fracture pathways always coincide with the regions of high CH3 concentration, located between the crystallographic planes containing the larger inverse micelles located at positions (a).

Organization of the outer layers of the cell envelope of Corynebacterium glutamicum: a combined freeze-etch electron microscopy and biochemical study.

The cell surface of Corynebacterium glutamicum grown on solid medium was totally covered with a highly ordered, hexagonal surface layer. Also, freeze-fracture revealed two fracture surfaces which were totally covered with ordered arrays displaying an hexagonal arrangement and the same unit cell dimension as the surface layer. The ordered arrays on the concave fracture surface, closest to the cell surface, were due to the presence of particles while those on the convex fracture surface were their imprints. The same cells grown on liquid medium displayed a cell surface and fracture surfaces only partially covered with ordered arrays. In this case, the ordered regions had the same relative position on the cell surface and on the fracture surfaces. All ordered arrays were totally absent in a mutant for cspB, the gene encoding PS2, one of the two major cell wall proteins. Treatment of the cells with proteinase K caused the gradual alteration of PS2 into a slightly lower molecular mass form. This was accompanied by a concomitant disappearance of the ordered fracture surfaces followed by the detachment of the ordered surface layer from the cell as large ordered patches displaying the same lattice symmetry and dimensions as those of the surface layer. The ordered patches were isolated. They contained the totality of PS2 initially associated with the cell. We conclude that the highly ordered surface layer of the intact cell was composed of PS2 interacting strongly with some cell wall material leading to its organization. This organized cell wall material produced fracture surfaces. We show that in the absence of intact PS2 protein on the cell wall, the same cell wall material was not organized and formed a structureless smooth layer.

Polymorphic phase behavior of perfluoroalkylated phosphatidylcholines.

The polymorphic phase behavior of the F-alkyl modified phosphatidylcholines FnCmPC with Fn = CnF2n + 1 and Cm = -(CH2)m- and the physicochemical properties of their aqueous dispersions have been investigated. We show that the supramolecular assemblies formed by F4C4PC, F6C4PC, F8C4PC and F4C10PC dispersed in water consist of liposomes. F6C10PC forms, as does F8C10PC, a ribbon-like phase (two-dimensional centered rectangular lattice) at 25 degrees C, but on heating, it forms a lamellar phase. Upon cooling, the lamellar gel phase is metastable and converts slowly back into the ribbon-like phase. Analyses of the dispersions before and after heat sterilization and upon storage at 25 degrees C reveal an exceptional stability of the FnCmPC-based liposomes which contrasts strongly with that of DPPC vesicles. This enhanced stability most likely arises from the increased hydrophobic character resulting from the presence of the perfluoroalkyl tails. The gel to fluid phase transition temperature of the FnCmPCs is found to be related to the total length of the hydrophobic chain and more markedly to the length of the perfluoroalkyl tail. This phase transition is first induced by the melting of the fluorocarbon chain. Each portion of the Fn tail and of the hydrocarbon spacer experiences intrinsic changes of molecular motion with temperature. The partitioning of a lipophilic/hydrophilic paramagnetic probe between the aqueous and lipidic phases present in the FnCmPC dispersions shows that an increase in fluorophilic character results in a lower solubility of the probe in the membrane, thus reflecting a dramatic decrease of the membrane's lipophilicity.

The eggshell of Drosophila melanogaster. VIII. Morphogenesis of the wax layer during oogenesis.

Utilizing freeze-fracturing and conventional electron microscopy methods, we have studied the details of morphogenesis and construction of the wax layer envelope from Oregon R and mutants of Drosophila melanogaster eggs during oogenesis. The wax layer is synthesized and secreted by the follicular cells in the form of lipid vesicles during stage 10b. During secretion (stages 10b, 11 and 12) the lipid vesicles are accumulated on the vitelline membrane surface and become flat. At the late stages of choriogenesis (stages 13, 14) the lipid vesicles are compressed tightly between the vitelline membrane and the other already constructed eggshell layers, so the wax layer becomes very thin and is hardly seen in cross-fractured views.

Characterization of the cspB gene encoding PS2, an ordered surface-layer protein in Corynebacterium glutamicum.

PS2 is one of two major proteins detected in the culture media of various Corynebacterium glutamicum strains. The coding and promoter regions of the cspB gene encoding PS2 were cloned in lambda gt11 using polyclonal antibodies raised against PS2 for screening. Expression of the cspB gene in Escherichia coli led to the production of a major anti-PS2 labelled peptide of 63,000 Da, corresponding presumably to the mature form of PS2. It was detected in the cytoplasm, periplasm and surrounding medium of E. coli. Three other slower migrating bands of 65,000 68,000 and 72,000 Da were detected. The largest one probably corresponds to the precursor form of PS2 in E. coli. Analysis of the nucleotide sequence revealed an open reading frame (ORF) of 1533 nucleotides. The deduced 510-amino-acid polypeptide had a calculated molecular mass of 55,426 Da. According to the predicted amino acid sequence, PS2 is synthesized with a N-terminal segment of 30-amino-acid residues reminiscent of eukaryotic and prokaryotic signal peptides, and a hydrophobic domain of 21 residues near the C-terminus. Although no significant homologies were found with other proteins, it appears that some characteristics and the amino acid composition of PS2 share several common features with surface-layer proteins. The cspB gene was then disrupted in C. glutamicum by gene replacement. Freeze-etching electron microscopy performed on the wild-type strain indicated that the cell wall of C. glutamicum is covered with an ordered surface of proteins (surface layer, S-layer) which is in very close contact with other cell-wall components. These structures are absent from the cspB-disrupted strain but are present after reintroduction of the cspB gene on a plasmid into this mutant. Thus we demonstrate that the S-layer protein is the product of the cspB gene.