Thaddée Gulik-Krzywicki

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.

Morphological changes of phosphatidylcholine bilayers induced by melittin: vesicularization, fusion, discoidal particles

Morphological changes induced by the melittin tetramer on bilayers of egg phosphatidylcholine and dipalmitoylphosphatidylcholine have been studied by quasi-elastic light scattering, gel filtration and freeze-fracture electron microscopy. It is concluded that melittin similarly binds and changes the morphology of both single and multilamellar vesicles, provided that their hydrocarbon chains have a disordered conformation, i.e., at temperatures higher than that of the transition, Tm. When the hydrocarbon chains are ordered (gel phase), only small unilamellar vesicles are morphologically affected by melittin. However after incubation at T greater than Tm, major structural changes are detected in the gel phase, regardless of the initial morphology of the lipids. Results from all techniques agree on the following points. At low melittin content, phospholipid-to-peptide molar ratios, Ri greater than 30, heterogeneous systems are observed, the new structures coexisting with the original ones. For lipids in the fluid phase and Ri greater than 12, the complexes formed are large unilamellar vesicles of about 1300 +/- 300 A diameter and showing on freeze-fracture images rough fracture surfaces. For lipids in the gel phase, T less than Tm after passage above Tm, and for 5 less than Ri less than 50, disc-like complexes are observed and isolated. They have a diameter of 235 +/- 23 A and are about one bilayer thick; their composition corresponds to one melittin for about 20 +/- 2 lipid molecules. It is proposed that the discs are constituted by about 1500 lipid molecules arranged in a bilayer and surrounded by a belt of melittin in which the mellitin rods are perpendicular to the bilayer. For high amounts of melittin, Ri less than 2, much smaller and more spherical objects are observed. They are interpreted as corresponding to lipid-peptide co-micelles in which probably no more bilayer structure is left. It is concluded that melittin induces a reorganization of lipid assemblies which can involve different processes, depending on experimental conditions: vesicularization of multibilayers; fusion of small lipid vesicles; fragmentation into discs and micelles. Such processes are discussed in connexion with the mechanism of action of melittin: the lysis of biological membranes and the synergism between melittin and phospholipases.

Biomimetic organization: Octapeptide self-assembly into nanotubes of viral capsid-like dimension

The controlled self-assembly of complex molecules into well defined hierarchical structures is a promising route for fabricating nanostructures. These nanoscale structures can be realized by naturally occurring proteins such as tobacco mosaic virus, capsid proteins, tubulin, actin, etc. Here, we report a simple alternative method based on self-assembling nanotubes formed by a synthetic therapeutic octapeptide, Lanreotide in water. We used a multidisciplinary approach involving optical and electron microscopies, vibrational spectroscopies, and small and wide angle x-ray scattering to elucidate the hierarchy of structures exhibited by this system. The results revealed the hexagonal packing of nanotubes, and high degree of monodispersity in the tube diameter (244 Å) and wall thickness (≈18 Å). Moreover, the diameter is tunable by suitable modifications in the molecular structure. The self-assembly of the nanotubes occurs through the association of β-sheets driven by amphiphilicity and a systematic aromatic/aliphatic side chain segregation. This original and simple system is a unique example for the study of complex self-assembling processes generated by de novo molecules or amyloid peptides.

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 65000, 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 W‐terminal segment of 30‐amino‐acid residues reminiscent of eukaryotic and prokaryotic signal pep‐tides, 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 Slayer protein is the product of the cspB gene.

Kinetic, binding and ultrastructural properties of the beef heart adenine nucleotide carrier protein after incorporation into phospholipid vesicles.

1. ADP/ATP transport has been reconstituted by incorporation of the purified carrier protein in liposomes filled with ATP. The transport was assayed by uptake of [14C]ADP into the liposomes, and by release of ATP as determined by a luminescence technique. [14C]ADP uptake was strictly dependent on internal ATP. 2. The simplest phospholipid system capable of yielding high rates of ADP/ATP transport was a mixture of phosphatidylethanolamine and cariolipin (92: 8, w/w). 3. ADP/ATP transport in the reconstituted system proceeded by exchange-diffusion with a 1/1 stoichiometry. The specificity for aDP and ATP was absolute. The capacity and the rate of exchange depended on the concentration of ATP present in liposomes. The rate of transport at 20 degrees C, at 20 mM internal ATP, routinely ranged between 300 and 1000 nmol of nucleotide exchanged per min/mg of added carrier protein. The apparent Km value for external ADP was around 10 microM. 4. The ADP/ATP exchange in the reconstituted system was rather stable to ageing. It dropped by only 20% after 1 day of ageing at 20 degrees C. Divalent cations (Mg2+, Mn2+, Ca2+) at concentrations higher than 1 to 2 mM had a deleterious effect on ADP/ATP transport, concomitant with the release of internal ATP and accumulation of multilamellar vesicles. 5. Atractyloside behaved as a competitive inhibitor and carboxyatractyloside as a non-competitive inhibitor. Bongkrekic acid required a slightly acidic pH to be inhibitory. The data concerning atractyloside, carboxyatractyloside and bongkrekic acid were similar to those obtained with whole mitochondria, suggesting that the carrier protein in liposomes has the same asymmetrical arrangement as in the mitochondria. 6. The percentage of competent carrier protein in liposomes was calculated from dose-response data concerning the inhibition of ADP/ATP transport by atractyloside or carboxyatractyloside, and from the amount of bound [3H]-atractyloside removable by ADP. By both methods, 3 to 6% of the added carrier protein was found to be competent in ADP/ATP transport, based on the assumption that the binding of one atractyloside or carboxyatractyloside molecule per 30000 molecular weight carrier unit results in complete inhibition of transport. 7. Freeze-fracture electron microscopy showed that the ADP/ATP carrier protein-lipid preparations are formed by small vesicles, most of which give rise to smooth fracture faces (probably pure lipid vesicles). Only a small percentage of the vesicles (2 to 4% depending on the amount of carrier protein added) were clearly particulated. About 90% of the particulated vesicles showed no more than 2 particles per vesicle and only 5% more than 5 particles per vesicle. The distribution of the particles between convex and concave fracture faces was asymmetric; about 2/3 of the protein molecules were anchored at the external surface of the vesicles and only 1/3 at the internal one...

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°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.

Correlations between fluorescence, X-ray diffraction, and physiological properties in cytoplasmic membrane vesicles isolated from Escherichia coli

The catalytic properties (i.e. transport) of cytoplasmic membrane vesicles isolated from Escherichia coli ML strain can be dissociated from their barrier properties. Initial rates of sugar and amino acid transport exhibit sharp activity transitions with respect to temperature at 15 to 20 °C, and optima at 45 to 50 °C. On the other hand, accumulation of maximal intramembranal concentrations of sugar phosphates via the phosphoenolpyruvate-phosphotransferase system exhibits temperature optima from 30 to 46 °C depending on the carbon source used for the growth of the parent cells. Moreover, the temperature optimum for accumulation of sugar phosphate in a particular membrane preparation is correlated with an abrupt increase in passive membrane permeability that occurs at that same temperature. By means of X-ray diffraction and fluorescence techniques applied to membrane vesicles into which dansyl phosphatidylethanolamine has been incorporated, two transitions are detected as functions of temperature. One transition is correlated with the transition in transport activity that occurs at around 20 °C, and coincides with the “melting” of paraffin chains of the membrane phospholipids. The second transition is correlated with the abrupt increases in passive membrane permeability.

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

Summary— 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 dimension 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 the ordered 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.

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.

Freeze‐fracture electron microscopic analysis of solutions of biological molecules

Freeze‐fracture electron microscopy was used to study the morphology of proteins in solution. The sizes of the particles appearing on the fractured surfaces, replicated with tungsten‐tantalum, were measured in a direction perpendicular to the shadowing angle. The distributions of the measured particle sizes could be correlated with the known shape and dimensions for each protein. It is concluded that freeze‐fracture electron microscopy is a useful technique to study the morphology of biological molecules in solution, particularly hydrophobic proteins which may be difficult to study by other microscopic techniques.