The impact of non-ideality of lipid mixing on peptide induced lipid clustering.

The influence of several antimicrobial trivalent cyclic hexapeptides on the mixing behavior of bilayer lipid membranes containing phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) with varying composition was studied using DSC and ITC. The peptides contained three arginines and three aromatic amino acids and had different sequences. All of them induce clustering of PG-rich clusters with bound peptides after binding. In a previous publication we could show that a correlation between clustering efficacy and the antimicrobial activity of the peptides exists (S. Finger et al., Biochim. Biophys. Acta 1848 (2015) 2998-3006). In the current study we investigated whether the non-ideality of the lipid mixture had any effect on the clustering efficacy and the critical peptide/lipid clustering ratio. We could show that for PG/PE membranes containing 1:1 M ratios and lipids with equal or unequal chain lengths, the amount of clustered PG depended only slightly on the absolute chain length and on the chain length difference between PG and PE. Much larger differences were observed when the PG/ PE mixing ratio was changed. In mixtures of DPPG/DPPE with high PG content, the amount of clustered PG per added peptide was much higher than in PE-rich mixtures. The ITC experiments showed that the critical peptide/lipid ratio for cluster formation is also strongly dependent on the PG/PE ratio in the mixture. In the PG/PE 3:1 mixture, the formation of clusters with bound peptide is much more likely than for mixtures with less PG. For 1:1 and 1:3 lipid mixtures, the critical peptide/lipid ratio for demixing is between 0.002 and 0.004. Therefore, even in these mixtures clustering occurs way below charge saturation of the PG in the mixture and the PG-rich clusters are not charge compensated either. The peptide concentration necessary for inducing clustering amounts to ~8 µM, a value well within the range of minimal inhibitory concentration values observed for the cyclic peptides studied here. Our results show that not only the structure of the cyclic peptide influences the clustering efficacy but also the mixing behavior of the lipids in the bilayers has an influence on the amount of clustering induced by binding of cyclic peptides.

Purification and characterization of indochrome type blue pigment produced by Pseudarthrobacter sp. 34LCH1 isolated from Atacama desert.

The interest in and demand for natural dyes has increased significantly in recent years; however, very few natural blue dyes are commercially available, because blue colored compounds in nature are relatively rare. In this study, a blue pigment-producing bacteria from Lake Chungará (Atacama Desert, Chile) was isolated, and its blue pigment was purified and chemically characterized. The pigment-producing strain was identified as Pseudarthrobacter sp. by 16S rRNA gene sequencing. The pigment was separated from the filtered culture medium by column chromatography/solid-phase extraction using different resins (ionic exchange, C-18, size exclusion). The strain produced up to 2.5 g L-1 of blue pigment, which was very soluble in water, partially soluble in methanol and insoluble in other organic solvents. The pigment was analyzed and characterized by analytical HPLC, UV-Vis, FT-IR, and H-NMR, and purified by semi-preparative HPLC. The pigment was non-toxic to brine shrimp (LD50 > 2.3 g L-1) and was stable at pH 6-10 at temperatures below 60 °C. HPLC analysis shows that the pigment is composed of four major blue fractions. The physicochemical properties and structural analysis demonstrate that this pigment belongs to the indochrome isomers, whose properties have yet to have been characterized. The high solubility in water, good stability in neutral and basic pH, and negligible toxicity of the blue pigment make it a good candidate suitable for several industrial and possibly some food applications.

Structural remodeling and oligomerization of human cathelicidin on membranes suggest fibril-like structures as active species.

Antimicrobial peptides as part of the mammalian innate immune system target and remove major bacterial pathogens, often through irreversible damage of their cellular membranes. To explore the mechanism by which the important cathelicidin peptide LL-37 of the human innate immune system interacts with membranes, we performed biochemical, biophysical and structural studies. The crystal structure of LL-37 displays dimers of anti-parallel helices and the formation of amphipathic surfaces. Peptide-detergent interactions introduce remodeling of this structure after occupation of defined hydrophobic sites at the dimer interface. Furthermore, hydrophobic nests are shaped between dimer structures providing another scaffold enclosing detergents. Both scaffolds underline the potential of LL-37 to form defined peptide-lipid complexes in vivo. After adopting the activated peptide conformation LL-37 can polymerize and selectively extract bacterial lipids whereby the membrane is destabilized. The supramolecular fibril-like architectures formed in crystals can be reproduced in a peptide-lipid system after nanogold-labelled LL-37 interacted with lipid vesicles as followed by electron microscopy. We suggest that these supramolecular structures represent the LL-37-membrane active state. Collectively, our study provides new insights into the fascinating plasticity of LL-37 demonstrated at atomic resolution and opens the venue for LL-37-based molecules as novel antibiotics.

The efficacy of trivalent cyclic hexapeptides to induce lipid clustering in PG/PE membranes correlates with their antimicrobial activity.

Various models have been proposed for the sequence of events occurring after binding of specific antimicrobial peptides to lipid membranes. The lipid clustering model arose by the finding that antimicrobial peptides can induce a segregation of certain negatively charged lipids in lipid model membranes. Anionic lipid segregation by cationic peptides is initially an effect of charge interaction where the ratio of peptide and lipid charges is thought to be the decisive parameter in the peptide induced lipid demixing. However, the sequence of events following this initial lipid clustering is more complex and can lead to deactivation of membrane proteins involved in cell division or perturbation of lipid reorganization essential for cell division. In this study we used DSC and ITC techniques to investigate the effect of binding different cyclic hexapeptides with varying antimicrobial efficacy, to phosphatidylglycerol (PG)/phosphatidylethanolamine (PE) lipid membranes and their ability to induce lipid segregation in these mixtures. We found that these cyclic hexapeptides consisting of three charged and three aromatic amino acids showed indeed different abilities to induce lipid demixing depending on their amino acid composition and their sequence. The results clearly showed that the cationic amino acids are essential for electrostatic binding but that the three hydrophobic amino acids in the peptides and their position in the sequence also contribute to binding affinity and to the extent of induction of lipid clustering. The efficacy of these different hexapeptides to induce PG clusters in PG/PE membranes was found to be correlated with their antimicrobial activity.

Interaction of linear polyamines with negatively charged phospholipids: the effect of polyamine charge distance.

The binding of cationic polyamines to negatively charged lipid membranes is driven by electrostatic interactions and additional hydrophobic contributions. We investigated the effect of polyamines with different number of charges and charge separation on the phase transition behavior of vesicles of phosphatidylglycerols (dipalmitoylphosphatidylglycerol and dimyristoylphosphatidylglycerol) to differentiate between effects caused by the number of charges, the charge distance, and the hydrophobicity of the methylene spacer. Using differential scanning calorimetry and Fourier transform infrared spectroscopy complemented with monolayer experiments, we found that the binding constant of polyamines to negatively charged lipid vesicles depends as expected on the number of charges. However, for diamines, the effect of binding on the main phase transition of phosphatidylglycerols (PGs) is also strongly influenced by the charge distance between the ammonium groups in the backbone. Oligoamines with charges separated by two or three methylene groups bind more strongly and have larger stabilizing effects on the lipid gel phase of PGs. With multivalent polyamines, the appearance of several transition peaks points to effects of molecular crowding on the surface, i.e., binding of only two or three charges to the surface in the case of spermine, and possible concomitant domain formation.

Intracuticular wax fixes and restricts strain in leaf and fruit cuticles.

This paper investigates the effects of cuticular wax on the release of strain and on the tensile properties of enzymatically isolated cuticular membranes (CMs) taken from leaves of agave (Agave americana), bush lily (Clivia miniata), holly (Ilex aquifolium), and ivy (Hedera helix) and from fruit of apple (Malus × domestica), pear (Pyrus communis), and tomato (Lycopersicon esculentum). Biaxial strain release was quantified as the decrease in CM disc area following wax extraction. Stiffness, maximum strain and maximum force were determined in uniaxial tensile tests using strips of CM and dewaxed CMs (DCMs). Biaxial strain release, stiffness, and maximum strain, but not maximum force, were linearly related to the amount of wax extracted. Apple CM has the most wax and here the effect of wax extraction was substantially accounted for by the embedded cuticular wax. Heating apple CM to 80°C melted some wax constituents and produced an effect similar to, but smaller than, that resulting from wax extraction. Our results indicate that wax 'fixes' strain, effectively converting reversible elastic into irreversible plastic strain. A consequence of 'fixation' is increased cuticular stiffness.