Cardiolipin, alpha-D-glucopyranosyl, and L-lysylcardiolipin from gram-positive bacteria: FAB MS, monofilm and X-ray powder diffraction studies.

Cardiolipin preparations from Streptococcus B, Listeria welshimeri, Staphylococcus aureus, and a glucosyl and lysyl derivative of cardiolipin were analysed for fatty acid composition and fatty acid combinations. Three different fatty acid patterns are described and up to 17 molecular species were identified in Streptococcus B lipids by high resolution FAB MS. The physicochemical properties of these lipids were characterised in the sodium salt form by monofilm experiments and X-ray powder diffraction. All lipids formed stable monofilms. The minimal space requirement of unsubstituted cardiolipin was dictated by the fatty acid pattern. Substitution with L-lysine led to a decrease of the molecular area, substitution with D-glucopyranosyl to an increase. On self assembly at 100% relative humidity, all preparations adopted lamellar structures. They showed a high degree of order, in spite of the heterogeneous fatty acid compositions and numerous fatty acid combinations. The repeat distances in lamellar fluid phase varied between 4.99 and 5. 52 nm, the bilayer thickness between 3.70 and 4.46 nm. Surprising were the low values of sorbed water per molecule of the glucosyl and lysyl derivatives which were 58 and 60%, compared with those of the respective cardiolipin. When Na(+) was replaced as counterion by Ba(2+), the bilayer structure was retained, but the lipids were in the lamellar gel phase and the fatty acids were tilted between 32 and 53 degrees away from the bilayer normal. Wide angle X-ray diffraction studies and electron density profiles are also reported. Particular properties of glucosyl cardiolipin are discussed.

Determination of the structure of tissue samples using X-ray small angle scattering.

X-ray small angle scattering has been used in material science for about 50 years. In diagnostic medicine, it has been applied for some years. The theoretical background is the diffraction of monochromatic X-rays by the electrons of small particles. The widening of the primary beam by those samples allows a conclusion regarding particle size, size distribution, and the form of the particles. The camera requires a well-defined and small X-ray beam which has to be entrapped exactly behind the sample. To date, the medical application has been carried out mainly by the comparison of the measured curve with that of standard samples. It can be suggested that in the near future its application in medicine will increase particularly with regard to in vivo measurements. For this purpose, new cameras will have to be developed. An exact evaluation of the result requires a thorough knowledge of the theoretical basis.

Thermodynamics and Structural Studies of the Interaction of Polymyxin B with Deep Rough Mutant Lipopolysaccharides.

Deep rough mutant lipopolysaccharide (ReLPS) dissolved in aqueous solution spontaneously forms supramolecular structures which mainly consist of vesicles. Addition of Polymyxin B (PmB) to these ReLPS vesicles influence the shape of these structures as demonstrated here by electronmicroscopy and dynamic light scattering techniques. The main phase transition of the ReLPS is found at 21.3 +/- 0.1 degrees C for ReLPS from Escherichia coli and at 24.0 +/- 0.5 degrees C for ReLPS from Salmonella minnesota by differential scanning calorimetry (DSC). Using isothermal differential titration calorimetry (ITC), the thermodynamic behavior of the interaction of PmB with ReLPS vesicles has been studied. The stoichiometric ratio for the binding of PmB to ReLPS was found to lie between 0.6 and 1, as determined from ITC and monolayer experiments. No phase transition was observed for ReLPS monolayers saturated with PmB. The results indicate specific interaction of PmB with ReLPS. We propose a two-step mechanism for this interaction, which involves electrostatic attraction between charged parts of the molecules and, in the second step, hydrophobic interactions between the nonpolar parts of both compounds. Copyright 1999 Academic Press.

[Image reconstruction of computerized tomography pictures using functional algebra]. / Bildrekonstruktion von Computertomographien mit Hilfe der Funktionenalgebra.

A detailed presentation of the process for calculating computed tomograms from the measured data by means of functional algebra is given and an attempt is made to demonstrate the relationships to those inexperienced in mathematics. Suggestions are also made to the manufacturers for improving tomography software although the authors cannot exclude the possibility that some of the recommendations may have already been realized. An interpolation in Fourier space to right-angled coordinates was not employed so that additional computer time and errors resulting from the interpolation are avoided. The savings in calculation time can only be estimated but should amount to about 25%. The error-correction calculation is merely a suggestion since it depends considerably on the apparatus used. Functional algebra is introduced here because it is not so well known but does provide appreciable simplifications in comparison to an explicit presentation. Didactic reasons as well as the possibility for reducing calculation time provided the foundation for this work.

Effect of lipoteichoic acid on thermotropic membrane properties.

Lipoteichoic acid, diglucosyldiacylglycerol, and phosphatidylglycerol isolated from Staphylococcus aureus were embedded in dipalmitoylglycerophosphoglycerol vesicles, and their thermotropic influence on this matrix was studied by differential scanning calorimetry. The natural fatty acids of phosphatidylglycerol effected peak broadening and a decrease in molar heat capacity. These effects were more pronounced with the glycolipid, which also increased the main transition temperature. With the lipoteichoic acid mixtures, two broad main transition peaks were observed, possibly due to different levels of lipoteichoic acid in vesicles. Both peaks showed a further upshift in transition temperatures and a pronounced decrease in molar heat capacity. Since the diacylglycerol moieties of all three amphiphiles were practically identical, the differences in the thermotropic effects have to be ascribed to the different structures of the head groups. Diglucosyldiacylglycerol is proposed to exert an additional effect by hydrogen bonding the hydroxyls of the sugar rings to their phospholipid neighbors. The stronger effect of lipoteichoic acid points to dynamic interactions of the long hydrophilic chain with the vesicle surface, which stabilize the membrane structure.

Molecular dynamics simulations of six different fully hydrated monomeric conformers of Escherichia coli re-lipopolysaccharide in the presence and absence of Ca2+.

Six previously published conformational models of Escherichia coli Re lipopolysaccharide (ReLPS) were subjected to molecular dynamics simulations using the CHARMM force field. The monomers of ReLPS were completely immersed in a water box. The dynamic behavior of the solvated models in the presence and absence of calcium cations was compared. The structure of the solvent shell was analyzed in terms of radial distribution functions. Diffusion coefficients and mean residence times were analyzed to characterize the dynamic behavior of the solvent. Order parameters and number of gauche defects were used for the description of the dynamics of the acyl chains. The cations are preferentially located between the carboxylate and phosphate groups of the headgroup. Their presence leads to a rigidification of the headgroup structure and alters the conformation of the backbone, thus influencing the structure and flexibility of the hydrophobic region as well. The effect of calcium on the backbone flexibility was measured in terms of glycosidic torsion angles. The six fatty acid chains of each ReLPS monomer adopt a highly ordered micromembrane structure. The packing parameter indicates that aggregation of these ReLPS monomers will lead to lamellar structures. Evaluation of all data enables us to present one conformation, C, which is thought to best represent the average structure of the ReLPS conformers.

Miscibility of lipoteichoic acid in dipalmitoylphosphatidylcholine studied by monofilm investigations and fluorescence microscopy.

The miscibility of the bacterial amphiphile lipoteichoic acid, a constituent of the cytoplasmic membrane of Gram-positive bacteria, in dipalmitoylphosphatidylcholine has been investigated by classic monofilm measurements and fluorescence microscopy at the air-water interface of monofilms obtained by spreading mixtures of both amphiphiles on a water subphase. The isotherms indicated miscibility of both lipids at concentrations up to 30 mol% lipoteichoic acid, whereas at higher concentrations immiscibility was detected. Increasing the lateral pressure over a certain value, lipoteichoic acid is squeezed out of the monofilm. By fluorescence microscopy the influence of lipoteichoic acid on the domain shape of condensed dipalmitoylphosphatidylcholine phases has been studied. The balance between hydrophobic and hydrophilic forces in the mixtures of both amphiphiles is discussed.

Comparison of X-ray powder-diffraction data of various bacterial lipopolysaccharide structures with theoretical model conformations.

X-ray powder-diffraction experiments have been performed on dry samples of lipid A and various rough-mutant lipopolysaccharides (LPS) of Salmonella minnesota, Salmonella typhimurium and Escherichia coli. The diffraction patterns obtained indicated exclusively lamellar, bilayered arrangements in all samples. The periodicities were found to be in the range 4.5 nm for lipid A to 8.8 nm for Ra-LPS. Upon treatment with water-saturated air, swelling of the lamellar structures was achieved, as indicated by shifts of reflections. The increase in bilayer dimensions normally was about 0.3 nm. X-ray intensities were used for the determination of the inner bilayer structure, i.e. for calculation of the one-dimensional electron-density distribution across the bilayer. For lipid A and several Re-LPS, Rd2-LPS, Rd1-LPS and Rc-LPS samples, a striking coincidence of the electron-density distributions in the lipid-A domain was found, suggesting that in all these structures the lipid-A portion is similarly arranged. For Rb1 and Ra-LPS the lipid-A domain could not be resolved due to the limited number of observed reflections. For other Re-mutant lipopolysaccharide samples, quite different X-ray patterns were obtained. Some samples yielded diffraction patterns indicating a very high state of order in the lipid-A domain, whereas, in others, a significantly reduced order in the lipid-A domain was inferred. Comparison of the X-ray data with features of a calculated three-dimensional molecular model of lipopolysaccharide revealed reasonable agreement in molecular dimensions and bilayer structure.

Molecular modelling of the three-dimensional structure and conformational flexibility of bacterial lipopolysaccharide.

Molecular modelling techniques have been applied to calculate the three-dimensional architecture and the conformational flexibility of a complete bacterial S-form lipopolysaccharide (LPS) consisting of a hexaacyl lipid A identical to Escherichia coli lipid A, a complete Salmonella typhimurium core oligosaccharide portion, and four repeating units of the Salmonella serogroup B O-specific chain. X-ray powder diffraction experiments on dried samples of LPS were carried out to obtain information on the dimensions of the various LPS partial structures. Up to the Ra-LPS structure, the calculated model dimensions were in good agreement with experimental data and were 2.4 nm for lipid A, 2.8 nm for Re-LPS, 3.5 nm for Rd-LPS, and 4.4 nm for Ra-LPS. The maximum length of a stretched S-form LPS model bearing four repeating units was evaluated to be 9.6 nm; however, energetically favored LPS conformations showed the O-specific chain bent with respect to the Ra-LPS portion and significantly smaller dimensions (about 5.0 to 5.5 nm). According to the calculations, the Ra-LPS moiety has an approximately cylindrical shape and is conformationally well defined, in contrast to the O-specific chain, which was found to be the most flexible portion within the molecule.

Molecular modelling of bacterial deep rough mutant lipopolysaccharide of Escherichia coli.

Molecular modelling techniques have been applied to compute the conformation accessible to bacterial deep rough lipopolysaccharide of Escherichia coli (Re-LPS). Analyses of the results showed that the models typically exhibit a tilt of the diglucosamine backbone with respect to the membrane normal of 53 +/- 7 degrees while both the glucosamine ring planes are oriented approximately parallel to the membrane normal. Different models were found to show compact and elongated types of acyl chain arrangements, both producing anisotropic lateral dimensions of the models of 1.0-1.1 nm and 1.7-2.0 nm for the shorter and the longer side, respectively. The conformationally allowed range of the isolated dOclA(alpha-2-4)dOclA disaccharide (dOclA = 3-deoxy-D-mannooctulosonic acid) was found to be extremely limited. It appeared that the dOclA disaccharide (dOclA)2 is centred at the top of the Re-LPS molecule preferring two orientations stabilized by hydrogen bonds involving only one phosphate group of the lipid A moiety at a time. The effect of charges on the Re-LPS conformations has been studied in separate calculations. From these calculations it was obvious that charges have no significant effects on the conformations of the isolated lipid A and (dOclA)2 moieties. However, it was found that the orientation of (dOclA)2 with respect to the lipid A part is highly sensitive to charges, i.e. in the charged models the proximity of phosphate and carboxyl groups is prevented by strong electrostatic repulsion between these negatively charged groups. In order to rationalize the acyl chain packing of the models, a simple geometrical model which correlates the tilt of the diglucosamine backbone with the energically favoured close packing of the acyl chains is proposed. Furthermore, the possibility of a chelate-like complexation of divalent cations and its contribution to head group mobility is discussed.

Architecture of bacterial lipid A in solution. A neutron small-angle scattering study.

The phase structure of isolated bacterial lipid A, the lipid anchor of the lipopolysaccharides of the outer membrane of Gram-negative bacteria, has been investigated by neutron small-angle scattering. The shape of the scattering curves obtained at different H2O/2H2O ratios revealed a lamellar organisation of the lipid A at neutral pH both above and below its main phase temperature (approximately 40-45 degrees C). Analysis of the scattering curves and interpretation of the corresponding thickness distance distribution functions of the lamellar aggregates led to a model in which the lipid A molecules form a bilayer of about 5 nm in thickness. This value for the thickness of the bilayer, as well as the neutron-scattering density profile across the bilayer, can be explained by a molecular model which shows interdigitation of the fatty acid chains of the lipid A.

On the relationships between molecular conformation, affinity towards penicillin-binding proteins, and biological activity of penicillin G-sulfoxide.

The binding capacity of penicillin G-sulfoxide towards the penicillin-binding proteins (PBP) of Staphylococcus aureus H was studied. The sulfoxide and its parent compound, penicillin G, differ only in two aspects, the sulfur-bound oxygen and an altered conformation of the five-membered thiazolidine-ring system. These minor alterations of the penicillin structure resulted in a drastical decrease of binding activity (about two orders of magnitude) of the sulfoxide derivative towards its target enzymes. Furthermore, the sulfoxide did not exhibit the selectivity of subinhibitory doses for PBP 3, as could be observed for penicillin G. The biological consequences of this behaviour were monitored via growth curves, uptake of cell wall label, and analysis of the cell wall. Binding studies revealed that comparable growth inhibition and impairment of cell wall label uptake were achieved by at least a 100-fold higher penicillin G-sulfoxide concentration, compared to its parent compound. In cell wall analysis, the application of high doses of the antibiotics, i.e. nearly saturated PBP, verified the above mentioned observation. Surprisingly, small but significant differences in cell wall composition occurred using subinhibitory doses, probably due to the altered affinity towards PBP 3, supporting the hypothesis of an important role of this PBP in peptidoglycan transpeptidation.

High state of order of isolated bacterial lipopolysaccharide and its possible contribution to the permeation barrier property of the outer membrane.

The conformational properties of the isolated S form of Salmonella sp. lipopolysaccharide (LPS), of Re mutant LPS, and of free lipid A were investigated by using X-ray diffraction and conformational energy calculations. The data obtained showed that LPS in a dried, in a hydrated, and probably also in an aqueous dispersion state is capable of forming bilayered lamellar arrangements similar to phospholipids. From the bilayer packing periodicities, a geometrical model of the extensions of the LPS regions lipid A, 2-keto-3-deoxyoctulosonic acid, and O-specific chain along the membrane normal could be calculated. Furthermore, the lipid A component was found to assume a remarkably high ordered conformation: its fatty acid chains were tightly packed in a dense hexagonal lattice with a center-to-center distance of 0.49 nm. The hydrophilic backbone of lipid A showed a strong tendency to form domains in the membrane, resulting in a more or less parallel arrangement of lipid A units. According to model calculations, the hydrophilic backbone of lipid A appears to be oriented approximately 45 degrees to the membrane surface, which would lead to a shed roof-like appearance of the surface structure in the indentations of which the 2-keto-3-deoxyoctulosonic acid moiety would fit. In contrast, the O-specific chains assume a low ordered, heavily coiled conformation. Comparison of these structural properties with those known for natural phospholipids in biological membranes indicates that the high state of order of the lipid A portion of LPS might be an important factor in the structural role and permeation barrier functions of LPS in the outer membrane of gram-negative bacteria.

A new proposal for the primary and secondary structure of the glycan moiety of pseudomurein. Conformational energy calculations on the glycan strands with talosaminuronic acid in 1C conformation and comparison with murein.

Using the method of conformational energy calculations, favoured conformations of a pseudomurein sugar strand built up from beta 1,3-linked N-acetyl-D-glucosamine and N-acetyl-L-talosaminuronic acid were obtained. Such a completely beta 1,3-linked polysaccharide primary structure, although contrasting with the originally proposed alternating beta 1,3-alpha 1,3-linked structure [König, H., Kandler, O., Jensen, M. and Rietschel, E. Th. (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 627-636] would be in agreement with all experimental data hitherto known. Starting from an analysis of favoured conformations of the monosaccharide building blocks and those obtained for disaccharide parts, the favoured helical conformations of the complete polysaccharide chains could be explored. Our completely beta 1,3-linked chain could adopt two types of conformation: extended and hollow; the latter was discarded as unsuitable for cell wall assembly. The extended conformation type was shown to exhibit a remarkable similarity if compared to the secondary structures accessible to murein-type polysaccharide chains. In contrast to the conformations accessible for the beta 1,3-alpha 1,3-linked primary structure, the new hypothetical pseudomurein structure was found to be more extended, possessed a flexible peptide attachment site at every second sugar residue and led to an orientation of consecutive peptide attachment sites analogous to the data known for murein-type chains. From the two possibilities compatible with the experimental data available, the completely beta 1,3-linked sugar strand structure could well be realized in the native pseudomurein network of methanobacteria. In this case the hypothesis for a similar three-dimensional architecture for murein and pseudomurein would be supported.

Structural studies on the bacterial cell wall peptidoglycan pseudomurein. I. Conformational energy calculations on the glycan strands in C1 conformation and comparison with murein.

Conformational energy calculations have been used to explore the conformations which may be realized for the sugar moiety of murein and pseudomurein. For the building blocks of the pseudomurein sugar strands, i.e. for the monosaccharides beta-D-N-acetylglucosamine (NAG) and alpha-L-N-acetyltalosaminuronic acid (NAT), both in C1 ring conformation, as well as for their 1,3 and 1,4 linked disaccharides, the favoured conformations were obtained. The helical parameters of sugar strands of both linkage types, which describe the regular structure of the corresponding polysaccharides, poly-(1,3-NAT-NAG) and poly-(1,4-NAT-NAG), were calculated. Both types of polysaccharides poly-(NAG-NAT) considered in this study favoured extended conformations, which in the case of 1,3 linked polymers showed less gain of length per saccharide unit compared to 1,4 linked poly-(NAG-NAT) residues. For a 1,3 linked sugar moiety of pseudomurein every pair of neighbouring peptides attached to glycan chain pointed in favoured conformations approximately to opposite sides of the strands, whereas in a 1,4 linked poly-(NAG-NAT) the peptides protruded approximately to the same side of the glycan moiety. A comparison between pseudomurein and murein revealed that the sugar moieties of both peptidoglycans have similar features in respect to their overall structure, i.e. both favoured more or less extended structures. In contrast to these data the shapes of the resulting polysaccharide moieties were remarkably different. In poly-(1,3-NAG-NAT) the glycan chains possessed a zig-zag-like arrangement, whereas for glycan chains of the murein type relatively flat structures were preferred. These remaining differences in the conformational arrangement between both peptidoglycans depend strongly on the C1 chair conformation of NAT. It is, therefore, attractive to speculate about an hypothetical pseudomurein sugar chain configuration comprising beta-L-N-acetyltalosaminuronic acid in its 1C conformation.

Study of the Zn-containing DD-carboxypeptidase of Streptomyces albus G by small-angle X-ray scattering in solution.

Study of the Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase of Streptomyces albus G by small-angle X-ray scattering in solution yielded the following molecular parameters: radius of gyration R = 1.82 +/- 0.05 nm; largest diameter D = 5.9 +/- 0.2 nm; relative molecular mass Mr = 17000 +/- 2000; volume V approximately equal to 35 +/- 2 nm3; degree of hydration: 0.25 +/- 0.02 g water/g protein. By reference to theoretical scattering curves of rigid triaxial homogeneous bodies, a model which fits all experimental data is an elliptical cylinder. Such a model is compatible with that observed in the crystal structure. At those high concentrations necessary to form inactive enzyme-ligand associations the non-competitive beta-lactam inhibitors, cephalothin and cephalosporin C, drastically altered the scattering behaviour of the protein.

Infrared spectroscopy, a tool for probing bacterial peptidoglycan. Potentialities of infrared spectroscopy for cell wall analytical studies and rejection of models based on crystalline chitin.

Infrared spectroscopic measurements are used to obtain insights into the three-dimensional architecture of peptidoglycan (murein), the rigid component of almost all bacterial cell walls. The infrared spectra of various types of peptidoglycans (including all chemotypes and examples of the so called A and B groups) were compared to each other and to those obtained from crystalline chitin. All peptidoglycans investigated exhibited very similar infrared spectra. In particular the conformationally sensitive amide A, I and II absorption bands were found to be constantly centered around 3300 cm-1, 1657 cm-1 and 1534 cm-1 respectively; furthermore, the spectral region between 1200 cm-1 and 800 cm-1, characterized by several strong absorption bands connected to complex sugar ring modes, proved to be remarkably uniform. Additionally the infrared spectra remained significantly constant between -175 degrees C and + 75 degrees C and turned out to be rather independent of sample preparation (solvent replacement, freeze-drying and film producing). An analysis of band half-widths revealed no high crystalline state of order of peptidoglycan. On the basis of band positions and half-widths of amide bands, regular conformations like alpha helices of beta pleated sheets could be excluded. Several distinctive, fingerprint-like spectral features of the various murein samples permitted a facile identification of individual peptidoglycans. Moreover, infrared spectroscopy seems to be very promising as an analytical tool, e.g. for tracing variations of cell wall structure, detecting conformational changes and estimating crosslinking indices in a quick and simple way. The comparative analysis of amide band positions and band half-widths yielded substantial differences between infrared spectra of chitin and murein, thus rejecting previous models based on the assumption of a nearly crystalline chitin-like structure of the glycan chains of murein.