Novel non-helical antimicrobial peptides insert into and fuse lipid model membranes.

This research addresses the growing menace of antibiotic resistance by exploring antimicrobial peptides (AMPs) as alternatives to conventional antibiotics. Specifically, we investigate two linear amphipathic AMPs, LE-53 (12-mer) and LE-55 (16-mer), finding that the shorter LE-53 exhibits greater bactericidal activity against both Gram-negative (G(-)) and Gram-positive (G(+)) bacteria. Remarkably, both AMPs are non-toxic to eukaryotic cells. The heightened effectiveness of LE-53 is attributed to its increased hydrophobicity (H) compared to LE-55. Circular dichroism (CD) reveals that LE-53 and LE-55 both adopt ß-sheet and random coil structures in lipid model membranes (LMMs) mimicking G(-) and G(+) bacteria, so secondary structure is not the cause of the potency difference. X-ray diffuse scattering (XDS) reveals increased lipid chain order in LE-53, a potential key distinction. Additionally, XDS study uncovers a significant link between LE-53's upper hydrocarbon location in G(-) and G(+) LMMs and its efficacy. Neutron reflectometry (NR) confirms the AMP locations determined using XDS. Solution small angle X-ray scattering (SAXS) demonstrates LE-53's ability to induce vesicle fusion in bacterial LMMs without affecting eukaryotic LMMs, offering a promising strategy to combat antibiotic-resistant strains while preserving human cell integrity, whereas LE-55 has a smaller ability to induce fusion.

Changes in lipid membrane mechanics induced by di- and tri-phenyltins.

Organotin compounds, being biologically active, affect a variety of cellular functions due to their ability to accumulate in and penetrate biological membranes. These compounds influence the distribution of electrostatic charges, alter organization, disrupt molecular dynamics and change mechanical properties of biological membranes. It was found that the membrane/water partition coefficient equals 4, a value significantly higher than octanol/water partition coefficient. In addition, the effect of di- and tri-phenyltin chlorides on the mechanics of model lipid membranes was measured for the first time. It has been determined that phenyltins affect the global model lipid bilayer properties by reducing the membrane expansion modulus, when measured using micromanipulation technique, and elevating the bending rigidity coefficient of the lipid bilayer, as determined with the flickering noise spectroscopy. In addition, the elevated water permeability shows that phenyltins also cause the local defects formation in the lipid bilayer, i.e. lipid pores. These data shows that phenyltins may interfere indirectly with variety cellular processes by altering non-specifically the entire cellular membrane system. Accordingly, when phenyltins are added to macrophages in culture, they inflict massive alterations of cell morphology and interfere with membrane-associated processes, as visualized using fluorescence labelling of selected subcellular compartments.

Probing the position of resveratrol in lipid bilayers: A neutron reflectivity study.

The effect of the natural antioxidant resveratrol on the structure of solid supported di-palmitoyl-phosphatidyl-choline (DPPC) bilayers in their fluid state was investigated by neutron reflectometry. Results reveal an accumulation of resveratrol (up to 25%, mol/mol) inside the headgroups and they exclude its presence in the hydrophobic core. The presence of resveratrol induces an increase of the average thickness and of the interfacial roughness of the headgroup layer. This may be due to a change of the tilt angle of the phosphocholine headgroups residing next to the resveratrol to a more upright orientation and leading to a reduction of the projected area per headgroup. This effect is propagated into the hydrophobic core, where the chain packing is modified despite the absence of resveratrol. When interacting with a DPPC/cholesterol membrane, resveratrol has a similar effect on the neighboring PC headgroups as in the cholesterol free membrane. The almost precise 1:1 insertion ratio (resveratrol:cholesterol) suggests that resveratrol is most probably inserted on top of the hydroxyl group of the cholesterol in between the PC headgroups. The ordering effect of cholesterol on the hydrophobic core is absent when both cholesterol and resveratrol are present. Most probably, the interaction of resveratrol with lipid membranes is non-specific.

X-ray structure, thermodynamics, elastic properties and MD simulations of cardiolipin/dimyristoylphosphatidylcholine mixed membranes.

Cardiolipins (CLs) are important biologically for their unique role in biomembranes that couple phosphorylation and electron transport like bacterial plasma membranes, chromatophores, chloroplasts and mitochondria. CLs are often tightly coupled to proteins involved in oxidative phosphorylation. The first step in understanding the interaction of CL with proteins is to obtain the pure CL structure, and the structure of mixtures of CL with other lipids. In this work we use a variety of techniques to characterize the fluid phase structure, material properties and thermodynamics of mixtures of dimyristoylphosphatidylcholine (DMPC) with tetramyristoylcardiolipin (TMCL), both with 14-carbon chains, at several mole percentages. X-ray diffuse scattering was used to determine structure, including bilayer thickness and area/lipid, the bending modulus, KC, and SXray, a measure of chain orientational order. Our results reveal that TMCL thickens DMPC bilayers at all mole percentages, with a total increase of ∼6 Å in pure TMCL, and increases AL from 64 Å(2) (DMPC at 35 °C) to 109 Å(2) (TMCL at 50 °C). KC increases by ∼50%, indicating that TMCL stiffens DMPC membranes. TMCL also orders DMPC chains by a factor of ∼2 for pure TMCL. Coarse grain molecular dynamics simulations confirm the experimental thickening of 2 Å for 20mol% TMCL and locate the TMCL headgroups near the glycerol-carbonyl region of DMPC; i.e., they are sequestered below the DMPC phosphocholine headgroup. Our results suggest that TMCL plays a role similar to cholesterol in that it thickens and stiffens DMPC membranes, orders chains, and is positioned under the umbrella of the PC headgroup. CL may be necessary for hydrophobic matching to inner mitochondrial membrane proteins. Differential scanning calorimetry, SXray and CGMD simulations all suggest that TMCL does not form domains within the DMPC bilayers. We also determined the gel phase structure of TMCL, which surprisingly displays diffuse X-ray scattering, like a fluid phase lipid. AL=40.8 Å(2) for the ½TMCL gel phase, smaller than the DMPC gel phase with AL=47.2 Å(2), but similar to AL of DLPE=41 Å(2), consistent with untilted chains in gel phase TMCL.

Two barriers or not? Dynamic force spectroscopy on the integrin α7ß1 invasin complex.

Dynamic force spectroscopy was used to test force-induced dissociation of the complex between the integrin α7ß1 and the bacterial protein invasin. Both proteins were used in truncated forms comprising the respective binding sites. Using the biomembrane force-probe, the bond system was exposed to 14 different loading rates ranging from 18 pN/s to 5.3 nN/s. At each rate, bond rupture spectra were collected. Median forces ranged from 8 to 72 pN. These showed two linear regimes when plotted against the logarithm of the force-loading rate. However, a statistical analysis of the full rupture force spectra including the detection limits of the setup showed that all measured data are well described by dissociation over a single barrier.

Caspase-8 binding to cardiolipin in giant unilamellar vesicles provides a functional docking platform for bid.

Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential activating platform for caspase-8 at the mitochondrial membrane surface. Destabilisation of this platform alters receptor-mediated apoptosis in diseases such as Barth Syndrome, which is characterised by the presence of immature cardiolipin which does not allow caspase-8 binding. We used a simplified in vitro system that mimics contact sites and/or cardiolipin-enriched microdomains at the outer mitochondrial surface in which the platform consisting of caspase-8, Bid and cardiolipin was reconstituted in giant unilamellar vesicles. We analysed these vesicles by flow cytometry and confirm previous results that demonstrate the requirement for intact mature cardiolipin for caspase-8 activation and Bid binding and cleavage. We also used confocal microscopy to visualise the rupture of the vesicles and their revesiculation at smaller sizes due to alteration of the curvature following caspase-8 and Bid binding. Biophysical approaches, including Laurdan fluorescence and rupture/tension measurements, were used to determine the ability of these three components (cardiolipin, caspase-8 and Bid) to fulfil the minimal requirements for the formation and function of the platform at the mitochondrial membrane. Our results shed light on the active functional role of cardiolipin, bridging the gap between death receptors and mitochondria.

A method for simultaneous quantification of phospholipid species by routine 31P NMR.

We report a (31)P NMR assay for quantification of aqueous phospholipid samples. Using a capillary with trimethylphosphate as internal standard, the limit of quantification is 1.30 mM. Comparison of the (31)P NMR quantification method in aqueous buffer and in organic solvent revealed that the two methods are equal within experimental error. Changing the pH of the buffer enables peak separation for different phospholipid species. This is an advantage compared to the commercial enzyme assay based on phospholipase D and choline oxidase. The reported method, using routine (31)P NMR equipment, is suitable when fast results of a limited number of samples are requested.

Mapping the location of grafted PNIPAAM in mesoporous SBA-15 silica using gas adsorption analysis.

The thermoresponsive polymer poly-N-isopropylacrylamide (PNIPAAM) was grafted in mesoporous SBA-15 silica. The grafting process consists of three steps: (i) increasing the amount of surface silanol groups of SBA-15 by hydroxylation, (ii) attachment of an anchor (1-(trichlorosilyl)-2-(m/p-(chloromethylphenyl)ethane) and finally (iii) the polymerization of the monomers (NIPAAM) onto the anchor. After each step, the materials were characterized regarding the porosity, using inert gas (argon, nitrogen) physisorption measurements. Also, the structure was investigated by small-angle X-ray diffraction analysis and thermogravimetric analysis was used for determination of the amount of grafted material. A total of 17% by weight of organic material was introduced in the porous host and the structure was preserved during the grafting process. Physisorption measurements revealed that the anchor is mainly located in the intrawall pores present in SBA-15. Consequently, the polymer is preferentially located in the intrawall pores or in the vicinity thereof. The final mesopore volume is 0.47 cm(3) g(-1) as compared to 0.96 cm(3) g(-1) for the pure SBA-15. The surprisingly large loss of mesopore volume and an almost constant mesopore diameter is consistent with a partial sealing of the mesopore volume in the composite materials. The potential thermocontrol combined with the large mesoporosity and the possible "storage space" provided by the sealed mesopore volume leads to a material with possibilities for various applications.

Mechanically enforced bond dissociation reports synergistic influence of Mn2+ and Mg2+ on the interaction between integrin α7ß1 and invasin.

Integrins require the divalent ions magnesium and manganese for ligand recognition. Here we mechanically enforced bond dissociation to explore the influence of these ions on the mechanical strength of the specific bond between α(7) ß(1) integrin and its pathologically relevant ligand invasin. Upon addition of these cations to the measurement buffer, we observe a pronounced increase in the force necessary to separate integrin and invasin coated beads. Both ions were found to work synergistically. With free invasin in the measurement buffer we furthermore observe that competitive blocking of binding sites overrides the increase in binding strength of individual beads. We show that this is due to a very strong dependence of bond affinity on divalent ions. Our study illustrates the importance of divalent ions for the regulation of force transmission by integrin ligand bonds on the molecular level.

New insights into the mucoadhesion of pectins by AFM roughness parameters in combination with SPR.

The object of this study was to assess the mucoadhesion of the three main commercially available types of pectin by atomic force microscopy (AFM) and surface Plasmon resonance (SPR). Polyacrylic acid and polyvinyl pyrrolidone were used as positive and negative control, respectively. Image analysis of the AFM scans revealed a significant change of roughness parameters when low-ester pectin was introduced to mica supported bovine submaxillarymucin, indicating a high mucoadhesion for this type of pectin. Only minor changes were observed with high-ester and amidated pectin. The same ranking order of adhesion affinity was confirmed by SPR. In conclusion, a high specific mucin interaction of pectin with a high charge density was demonstrated directly on a molecular scale without interference from the viscoelastic properties or the intra-molecular interactions between the polymer chains themselves, using two independent methods.

Nanobubbles give evidence of incomplete wetting at a hydrophobic interface.

The appearance of a hydrophobic surface, namely a crystalline (111) Si wafer coated with a thick soft polystyrene film, and the morphological changes along this interface depending on the polarity of an adjoining liquid phase were studied with magnetic tapping mode atomic force microscopy. Interfacially associated nanobubbles of decreasing size and number are observed as the hydrophobicity of the subphase increases. The disturbance of the water structure in the contact region induces the formation of nanobubbles. The topology of the interface is visualized, starting with the dry polymer under normal atmosphere conditions and observing the changes as air is replaced by a series of liquids. With water, the surface coverage of the substrate with bubbles is almost a close-packed configuration. The bubble shape is well approximated by spherical caps of a rather low aspect ratio. The Gaussian size distributions of bubble shape parameters are discussed. The contact angle of the nanobubbles is substantially smaller than the corresponding number measured for a macroscopic droplet. This apparent discrepancy might be resolved if the nanobubbles were assumed to exist along the interface as a connecting sublayer between a depleted water film at the hydrophobic polymer surface and an adsorbed macrodroplet.