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Abstract: The activity of antimicrobial peptides stems from their interaction with bacterial membranes, which are disrupted according to a number of proposed mechanisms. Here, we investigate the interaction of a model antimicrobial peptide that contains a single arginine residue with vesicles containing model lipid membranes. The surfactant-like peptide Ala6-Arg (A6R) is studied in the form where both termini are capped (CONH-A6R-NH2, capA6R) or uncapped (NH2-A6R-OH, A6R). Lipid membranes are selected to correspond to model anionic membranes (POPE/POPG) resembling those in bacteria or model zwitterionic membranes (POPC/DOPC) similar to those found in mammalian cells. Viable antimicrobial agents should show activity against anionic membranes but not zwitterionic membranes. We find, using small-angle X-ray scattering (SAXS) and cryogenic-TEM (transmission electron microscopy) that, uniquely, capA6R causes structuring of anionic membranes due to the incorporation of the peptide in the lipid bilayer with peptide β-sheet conformation revealed by circular dichroism spectroscopy (CD). There is a preferential interaction of the peptide with POPG (which is the only anionic lipid in the systems studied) due to electrostatic interactions and bidentate hydrogen bonding between arginine guanidinium and lipid phosphate groups. At a certain composition, this peptide leads to the remarkable tubulation of zwitterionic phosphatidylcholine (PC) vesicles, which is ascribed to the interaction of the peptide with the outer lipid membrane, which occurs without penetration into the membrane. In contrast, peptide A6R has a minimal influence on the anionic lipid membranes (and no β-sheet peptide structure is observed) but causes thinning (lamellar decorrelation) of zwitterionic membranes. We also investigated the cytotoxicity (to fibroblasts) and antimicrobial activity of these two peptides against model Gram positive and Gram negative bacteria. A strong selective antimicrobial activity against Gram positive Listeria monocytogenes, which is an important food-borne pathogen, is observed for capA6R. Peptide A6R is active against all three studied bacteria. The activity of the peptides against bacteria and mammalian cells is related to the specific interactions uncovered through our SAXS, cryo-TEM, and CD measurements. Our results highlight the exquisite sensitivity to the charge distribution in these designed peptides and its effect on the interaction with lipid membranes bearing different charges, and ultimately on antimicrobial activity.

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Abstract: We investigate the self-assembly of two telechelic star polymer-peptide conjugates based on poly(ethylene oxide) (PEO) four-arm star polymers capped with oligotyrosine. The conjugates were prepared via N-carboxy anhydride (NCA)-mediated ring-opening polymerization from PEO star polymer macroinitiators. Self-assembly occurs above a critical aggregation concentration determined via fluorescence probe assays. Peptide conformation was examined using circular dichroism spectroscopy. The structure of self-assembled aggregates was probed using small-angle X-ray Scattering (SAXS) and cryogenic transmission electron microscopy (Cryo-TEM). In contrast to previous studies on linear telechelic PEO-oligotyrosine conjugates which show self-assembly into -sheet fibrils, the star architecture suppresses fibril formation and instead micelles are generally observed, a small population of fibrils only being observed upon pH adjustment. Hydrogelation is also suppressed by the polymer star architecture. However, hydrogels can be produce by addition of sodium alginate with calcium chloride. These peptide-functionalized star polymer solutions are cytocompatible at sufficiently low concentration. These polymers present tyrosine at high density and may be useful in the development of future enzyme or pH-responsive biomaterials.

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Abstract: Chirality and morphology are essential factors for protein function and interactions with other biomacromolecules. Extracellular matrix (ECM) proteins are also similar to other proteins in this sense; however, the complexity of the natural ECM makes it difficult to study these factors at the cellular level. The synthetic peptide nanomaterials harbor great promise in mimicking specific ECM molecules as model systems. In this work, we demonstrate that mechanosensory responses of stem cells are directly regulated by the chirality and morphology of ECM-mimetic peptide nanofibers with strictly controlled characteristics. Structural signals presented on L-amino acid containing cylindrical nanofibers (L-VV) favored the formation of integrin 1-based focal adhesion complexes, which increased the osteogenic potential of stem cells through the activation of nuclear YAP. On the other hand, twisted ribbon-like nanofibers (L-FF and D-FF) guided the cells into round shapes and decreased the formation of focal adhesion complexes resulting in the confinement of YAP proteins in the cytosol and a corresponding decrease in osteogenic potential. Interestingly, the D-form of twisted-ribbon like nanofibers (D-FF) increased the chondrogenic potential of stem cells more than their L-form (L-FF). Our results provide new insights into the importance and relevance of morphology and chirality of nanomaterials in their interactions with cells, and reveal that precise control over the chemical and physical properties of nanostructures can affect stem cell fate even without the incorporation of specific epitopes.

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Abstract: The self-assembly in aqueous solution of three lipopeptides comprising a bioactive motif conjugated at the N terminus to dodecyl, tetradecyl or hexadecyl lipid chains has been examined. The bioactive motif is the peptide block YEALRVANEVTLN; a C-terminal fragment of the lumican proteoglycan. This study was motivated by our previous studies on the hexadecyl homologue C16-YEALRVANEVTLN which showed aggregation into β-sheet structures above a critical aggregation concentration (cac), but most remarkably we found that these aggregates were stable to dilution below the cac. Here we find that the C12- and C14- homologues also self-assemble above a cac into β-sheet nanotapes based on bilayer packing. The cac decreases with increasing lipopeptide hydrophobicity. Unexpectedly, the β-sheet secondary structure is present upon dilution and the aggregates are thermally stable. These results indicate that the dilution trapping of β-sheet secondary structure is not associated to lipid chain melting behaviour. Instead we associate it with pH-dependent favourable inter-molecular electrostatic interactions. Investigation of the pH-dependence of aggregation led to the discovery of conditions for formation of lipopeptide hydrogels (initial sample preparation at pH 10 in NaOH solution, followed by reduction to pH ~1 by addition of HCl). The lipopeptide hydrogels comprise networks of bilayer-based peptide nanotape bundles and to our knowledge this type of hydrogel is unprecedented. These hydrogels may have future applications based on processes such as encapsulation and release that involve fast switches between solution and hydrogel nanostructures.

Open Access

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Abstract: The self-assembly of the macrophage-activating lipopeptide MALP-2 in aqueous solution has been investigated and is compared to that of the constituent peptide GNNDESNISFKEK. MALP-2 is a tolllike receptor agonist lipopeptide with diverse potential biomedical applications and its self-assembly has not previously been examined. It is found to self-assemble, above a critical aggregation concentration (cac), into remarkable “fibre raft” structures, based on lateral aggregation of β- sheet based bilayer tapes. Peptide GNNDESNISFKEK also forms β-sheet structures above a cac, although the morphology is distinct, comprising highly extended and twisted tape structures. A detailed insight into the molecular packing within the MALP-2 raft and GNNDESNISFKEK nanotape structures is obtained through X-ray diffraction and small-angle X-ray scattering. These results point to the significant influence of the attached lipid chains on the self-assembly motif, which lead to the raft structure for the lipopeptide assemblies.