Compressibility and porosity modulate the mechanical properties of giant gas vesicles.

Gas vesicles used as contrast agents for noninvasive ultrasound imaging must be formulated to be stable, and their mechanical properties must be assessed. We report here the formation of perfluoro-n-butane microbubbles coated with surface-active proteins that are produced by filamentous fungi (hydrophobin HFBI from Trichoderma reesei). Using pendant drop and pipette aspiration techniques, we show that these giant gas vesicles behave like glassy polymersomes, and we discover novel gas extraction regimes. We develop a model to analyze the micropipette aspiration of these compressible gas vesicles and compare them to incompressible liquid-filled vesicles. We introduce a sealing parameter to characterize the leakage of gas under aspiration through the pores of the protein coating. Utilizing this model, we can determine the elastic dilatation modulus, surface viscosity, and porosity of the membrane. These results demonstrate the engineering potential of protein-coated bubbles for echogenic and therapeutic applications and extend the use of the pipette aspiration technique to compressible and porous systems.

Self-Assembly and Antimicrobial Activity of Lipopeptides Containing Lysine-Rich Tripeptides.

The conformation and self-assembly of two pairs of model lipidated tripeptides in aqueous solution are probed using a combination of spectroscopic methods along with cryogenic-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS). The palmitoylated lipopeptides comprise C16-YKK or C16-WKK (with two l-lysine residues) or their respective derivatives containing d-lysine (k), i.e., C16-Ykk and C16-Wkk. All four molecules self-assemble into spherical micelles which show structure factor effects in SAXS profiles due to intermicellar packing in aqueous solution. Consistent with micellar structures, the tripeptides in the coronas have a largely unordered conformation, as probed using spectroscopic methods. The molecules are found to have good cytocompatibility with fibroblasts at sufficiently low concentrations, although some loss of cell viability is noted at the highest concentrations examined (above the critical aggregation concentration of the lipopeptides, determined from fluorescence dye probe measurements). Preliminary tests also showed antimicrobial activity against both Gram-negative and Gram-positive bacteria.

Comparison of the self-assembly and cytocompatibility of conjugates of Fmoc (9-fluorenylmethoxycarbonyl) with hydrophobic, aromatic, or charged amino acids.

The self-assembly in aqueous solution of three Fmoc-amino acids with hydrophobic (aliphatic or aromatic, alanine or phenylalanine) or hydrophilic cationic residues (arginine) is compared. The critical aggregation concentrations were obtained using intrinsic fluorescence or fluorescence probe measurements, and conformation was probed using circular dichroism spectroscopy. Self-assembled nanostructures were imaged using cryo-transmission electron microscopy and small-angle X-ray scattering (SAXS). Fmoc-Ala is found to form remarkable structures comprising extended fibril-like objects nucleating from spherical cores. In contrast, Fmoc-Arg self-assembles into plate-like crystals. Fmoc-Phe forms extended structures, in a mixture of straight and twisted fibrils coexisting with nanotapes. Spontaneous flow alignment of solutions of Fmoc-Phe assemblies is observed by SAXS. The cytocompatibility of the three Fmoc-amino acids was also compared via MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] mitochondrial activity assays. All three Fmoc-amino acids are cytocompatible with L929 fibroblasts at low concentration, and Fmoc-Arg shows cell viability up to comparatively high concentration (0.63 mM).

Tuning the Solution Self-Assembly of a Peptide-PEG (Polyethylene Glycol) Conjugate with α-Cyclodextrin.

Cyclodextrins are saccharide ring molecules which act as host cavities that can encapsulate small guest molecules or thread polymer chains. We investigate the influence of alpha-cyclodextrin (αCD) on the aqueous solution self-assembly of a peptide-polymer conjugate YYKLVFF-PEG3K previously studied by our group [Castelletto et al., Polym. Chem., 2010, 1, 453-459]. This conjugate comprises a designed amyloid-forming peptide YYKLVFF that contains the KLVFF sequence from Amyloid ß peptide, Aß16-20, along with two aromatic tyrosine residues to enhance hydrophobicity, as well as polyethylene glycol PEG with molar mass 3 kg mol-1 . The conjugate self-assembles into ß-sheet fibrils in aqueous solution. Here we show that complexation with αCD instead generates free-floating nanosheets in aqueous solution (with a ß-sheet structure). The nanosheets comprise a bilayer with a hydrophobic peptide core and highly swollen PEG outer layers. The transition from fibrils to nanosheets is driven by an increase in the number of αCD molecules threaded on the PEG chains, as determined by 1 H NMR spectroscopy. These findings point to the use of cyclodextrin additives as a powerful means to tune the solution self-assembly in peptide-polymer conjugates and potentially other polymer/biomolecular hybrids.

Effect of Glycosylation on Self-Assembly of Lipid A Lipopolysaccharides in Aqueous Solutions.

Lipopolysaccharides (LPSs) based on lipid A produced by bacteria are of interest due to their bioactivity in stimulating immune responses, as are simpler synthetic components or analogues. Here, the self-assembly in water of two monodisperse lipid A derivatives based on simplified bacterial LPS structures is examined and compared to that of a native Escherichia coli LPS using small-angle X-ray scattering and cryogenic transmission electron microscopy. The critical aggregation concentration is obtained from fluorescence probe experiments, and conformation is probed using circular dichroism spectroscopy. The E. coli LPS is found to form wormlike micelles, whereas the synthetic analogues bearing six lipid chains and with four or two saccharide head groups (Kdo2-lipid A and monophosphoryl lipid A) self-assemble into nanosheets or vesicles, respectively. These observations are rationalized by considering the surfactant packing parameter.

Cell Adhesion Motif-Functionalized Lipopeptides: Nanostructure and Selective Myoblast Cytocompatibility.

The conformation and self-assembly of four lipopeptides, peptide amphiphiles comprising peptides conjugated to lipid chains, in aqueous solution have been examined. The peptide sequence in all four lipopeptides contains the integrin cell adhesion RGDS motif, and the cytocompatibility of the lipopeptides is also analyzed. Lipopeptides have either tetradecyl (C14, myristyl) or hexadecyl (C16, palmitoyl) lipid chains and peptide sequence WGGRGDS or GGGRGDS, that is, with either a tryptophan-containing WGG or triglycine GGG tripeptide spacer between the bioactive peptide motif and the alkyl chain. All four lipopeptides self-assemble above a critical aggregation concentration (CAC), determined through several comparative methods using circular dichroism (CD) and fluorescence. Spectroscopic methods [CD and Fourier transform infrared (FTIR) spectroscopy] show the presence of ß-sheet structures, consistent with the extended nanotape, helical ribbon, and nanotube structures observed by cryogenic transmission electron microscopy (cryo-TEM). The high-quality cryo-TEM images clearly show the coexistence of helically twisted ribbon and nanotube structures for C14-WGGRGDS, which highlight the mechanism of nanotube formation by the closure of the ribbons. Small-angle X-ray scattering shows that the nanotapes comprise highly interdigitated peptide bilayers, which are also present in the walls of the nanotubes. Hydrogel formation was observed at sufficiently high concentrations or could be induced by a heat/cool protocol at lower concentrations. Birefringence due to nematic phase formation was observed for several of the lipopeptides, along with spontaneous flow alignment of the lyotropic liquid crystal structure in capillaries. Cell viability assays were performed using both L929 fibroblasts and C2C12 myoblasts to examine the potential uses of the lipopeptides in tissue engineering, with a specific focus on application to cultured (lab-grown) meat, based on myoblast cytocompatibility. Indeed, significantly higher cytocompatibility of myoblasts was observed for all four lipopeptides compared to that for fibroblasts, in particular at a lipopeptide concentration below the CAC. Cytocompatibility could also be improved using hydrogels as cell supports for fibroblasts or myoblasts. Our work highlights that precision control of peptide sequences using bulky aromatic residues within "linker sequences" along with alkyl chain selection can be used to tune the self-assembled nanostructure. In addition, the RGDS-based lipopeptides show promise as materials for tissue engineering, especially those of muscle precursor cells.

Self-Assembly and Cytocompatibility of Amino Acid Conjugates Containing a Novel Water-Soluble Aromatic Protecting Group.

There has been considerable interest in peptides in which the Fmoc (9-fluorenylmethoxycarbonyl) protecting group is retained at the N-terminus, since this bulky aromatic group can drive self-assembly, and Fmoc-peptides are biocompatible and have applications in cell culture biomaterials. Recently, analogues of new amino acids with 2,7-disulfo-9-fluorenylmethoxycarbonyl (Smoc) protecting groups have been developed for water-based peptide synthesis. Here, we report on the self-assembly and biocompatibility of Smoc-Ala, Smoc-Phe and Smoc-Arg as examples of Smoc conjugates to aliphatic, aromatic, and charged amino acids, respectively. Self-assembly occurs at concentrations above the critical aggregation concentration (CAC). Cryo-TEM imaging and SAXS reveal the presence of nanosheet, nanoribbon or nanotube structures, and spectroscopic methods (ThT fluorescence circular dichroism and FTIR) show the presence of ß-sheet secondary structure, although Smoc-Ala solutions contain significant unaggregated monomer content. Smoc shows self-fluorescence, which was used to determine CAC values of the Smoc-amino acids from fluorescence assays. Smoc fluorescence was also exploited in confocal microscopy imaging with fibroblast cells, which revealed its uptake into the cytoplasm. The biocompatibility of these Smoc-amino acids was found to be excellent with zero cytotoxicity (in fact increased metabolism) to fibroblasts at low concentration.

Self-assembled aggregates based on cationic amphiphilic peptides: structural insight.

Short and ultra-short peptides have recently emerged as suitable building blocks for the fabrication of self-assembled innovative materials. Peptide aggregation is strictly related to the amino acids composing the sequence and their capability to establish intermolecular interactions. Additional structural and functional properties can also be achieved by peptide derivatization (e.g. with polymeric moieties, alkyl chains or other organic molecules). For instance, peptide amphiphiles (PAs), containing one or more alkyl tails on the backbone, have a propensity to form highly ordered nanostructures like nanotapes, twisted helices, nanotubes and cylindrical nanostructures. Further lateral interactions among peptides can also promote hydrogelation. Here we report the synthesis and the aggregation behaviour of four PAs containing cationic tetra- or hexa-peptides (C19-VAGK, C19-K1, C19-K2 and C19-K3) derivatized with a nonadecanoic alkyl chain. In their acetylated (Ac-) or fluorenylated (Fmoc-) versions, these peptides previously demonstrated the ability to form biocompatible hydrogels potentially suitable as extracellular matrices for tissue engineering or diagnostic MRI applications. In the micromolar range, PAs self-assemble in aqueous solution into nanotapes, or small clusters, resulting in high biocompatibility on HaCat cells up to 72 hours of incubation. Moreover, C19-VAGK also forms a gel at a concentration of 5 wt%.

Correct Identification of the Core-Shell Structure of Cell Membrane-Coated Polymeric Nanoparticles.

Transmission electron microscopy (TEM) observations of negatively stained cell membrane (CM)-coated polymeric nanoparticles (NPs) reveal a characteristic core-shell structure. However, negative staining agents can create artifacts that complicate the determination of the actual NP structure. Herein, it is demonstrated with various bare polymeric core NPs, such as poly(lactic-co-glycolic acid) (PLGA), poly(ethylene glycol) methyl ether-block-PLGA, and poly(caprolactone), that certain observed core-shell structures are actually artifacts caused by the staining process. To address this issue, fluorescence quenching was applied to quantify the proportion of fully coated NPs and statistical TEM analysis was used to identify and differentiate whether the observed core-shell structures of CM-coated PLGA (CM-PLGA) NPs are due to artifacts or to the CM coating. Integrated shells in TEM images of negatively stained CM-PLGA NPs are identified as artifacts. The present results challenge current understanding of the structure of CM-coated polymeric NPs and encourage researchers to use the proposed characterization approach to avoid misinterpretations.

Self-Assembly of Angiotensin-Converting Enzyme Inhibitors Captopril and Lisinopril and Their Crystal Structures.

The peptide angiotensin-converting enzyme inhibitors captopril and lisinopril are unexpectedly shown to exhibit critical aggregation concentration (CAC) behavior through measurements of surface tension, electrical conductivity, and dye probe fluorescence. These three measurements provide similar values for the CAC, and there is also evidence from circular dichroism spectroscopy for a possible conformational change in the peptides at the same concentration. Cryogenic transmission electron microscopy indicates the formation of micelle-like aggregates above the CAC, which can thus be considered a critical micelle concentration, and the formation of aggregates with a hydrodynamic radius of ∼6-7 nm is also evidenced by dynamic light scattering. We also used synchrotron radiation X-ray diffraction to determine the single-crystal structure of captopril and lisinopril. Our results improve the accuracy of previous data reported in the literature, obtained using conventional X-ray sources. We also studied the structure of aqueous solutions containing captopril or lisinopril at high concentrations. The aggregation may be driven by intermolecular interactions between the proline moiety of captopril molecules or between the phenylalanine moiety of lisinopril molecules.

Alpha helical surfactant-like peptides self-assemble into pH-dependent nanostructures.

A designed surfactant-like peptide is shown, using a combination of cryogenic-transmission electron microscopy and small-angle X-ray scattering, to have remarkable pH-dependent self-assembly properties. Peptide Arg3-Leu12 (R3L12) forms a network of peptide nanotubes at pH 9 and below. These are associated with α-helical conformation in a "cross-α" nanotube structure, in which peptide dimers lie perpendicular to the nanotube axis, with arginine coated inner and outer nanotube walls. In contrast, this peptide forms decorated vesicular aggregates at higher pH values, close to the pKa of the arginine residues. These structures are associated with a loss of α-helical order as detected through X-ray scattering, circular dichroism and FTIR spectroscopy, the latter technique also revealing a loss of ordering of leucine side chains. This suggests a proposed model for the decorated or patchy vesicular structures that comprises disordered peptide as the matrix of the membrane, with small domains of ordered peptide dimers forming the minority domains. We ascribe this to a lipid-raft like phase separation process, due to conformational disordering of the leucine hydrophobic chains. The observation of the self-assembly of a simple surfactant-like peptide into these types of nanostructure is remarkable, and peptide R3L12 shows unique pH-dependent morphological and conformational behaviour, with the potential for a range of future applications.

Directed Assembly of Cellulose Nanocrystals in Their Native Solid-State Template of a Processed Fiber Cell Wall.

Nanoparticle assembly is intensely surveyed because of the numerous applications within fields such as catalysis, batteries, and biomedicine. Here, directed assembly of rod-like, biologically derived cellulose nanocrystals (CNCs) within the template of a processed cotton fiber cell wall, that is, the native origin of CNCs, is reported. It is a system where the assembly takes place in solid state simultaneously with the top-down formation of the CNCs via hydrolysis with HCl vapor. Upon hydrolysis, cellulose microfibrils in the fiber break down to CNCs that then pack together, resulting in reduced pore size distribution of the original fiber. The denser packing is demonstrated by N2 adsorption, water uptake, thermoporometry, and small-angle X-ray scattering, and hypothetically assigned to attractive van der Waals interactions between the CNCs.

Native Structure of the Plant Cell Wall Utilized for Top-Down Assembly of Aligned Cellulose Nanocrystals into Micrometer-Sized Nanoporous Particles.

Despite their sustainable appeal, biomass components are currently undervalued in nanotechnology because means to control the assembly of bio-based nanoparticles are lagging behind the synthetic counterparts. Here, micrometer-sized particles consisting of aligned cellulose nanocrystals (CNCs) are prepared by crosslinking cellulose in cotton linter fibers that are prehydrolyzed with gaseous HCl, resulting in chemical cleavage necessary for CNC formation but retaining the morphology of the native fibers. That way, the intrinsic alignment of cellulose microfibrils within the fiber cell wall can be retained and utilized for top-down CNC alignment. Subsequent crosslinking with citric acid cements the alignment and preserves it, following the dispersion of CNCs trapped end-to-end, connected, and crosslinked within the colloidally stable micrometer-sized particles. Furthermore, thermoporosimetry and cryogenic transmission electron microscopy (Cryo TEM) shows that the particles possess mainly nanoporous (<2 nm) character in water. The approach challenges the current paradigm of predominantly bottom-up methods for nanoparticle assembly.

Complementary substrate specificity and distinct quaternary assembly of the Escherichia coli aerobic and anaerobic ß-oxidation trifunctional enzyme complexes.

The trifunctional enzyme (TFE) catalyzes the last three steps of the fatty acid ß-oxidation cycle. Two TFEs are present in Escherichia coli, EcTFE and anEcTFE. EcTFE is expressed only under aerobic conditions, whereas anEcTFE is expressed also under anaerobic conditions, with nitrate or fumarate as the ultimate electron acceptor. The anEcTFE subunits have higher sequence identity with the human mitochondrial TFE (HsTFE) than with the soluble EcTFE. Like HsTFE, here it is found that anEcTFE is a membrane-bound complex. Systematic enzyme kinetic studies show that anEcTFE has a preference for medium- and long-chain enoyl-CoAs, similar to HsTFE, whereas EcTFE prefers short chain enoyl-CoA substrates. The biophysical characterization of anEcTFE and EcTFE shows that EcTFE is heterotetrameric, whereas anEcTFE is purified as a complex of two heterotetrameric units, like HsTFE. The tetrameric assembly of anEcTFE resembles the HsTFE tetramer, although the arrangement of the two anEcTFE tetramers in the octamer is different from the HsTFE octamer. These studies demonstrate that EcTFE and anEcTFE have complementary substrate specificities, allowing for complete degradation of long-chain enoyl-CoAs under aerobic conditions. The new data agree with the notion that anEcTFE and HsTFE are evolutionary closely related, whereas EcTFE belongs to a separate subfamily.

Lignans in Knotwood of Norway Spruce: Localisation with Soft X-ray Microscopy and Scanning Transmission Electron Microscopy with Energy Dispersive X-ray Spectroscopy.

Lignans are bioactive compounds that are especially abundant in the Norway spruce (Picea abies L. Karst.) knotwood. By combining a variety of chromatographic, spectroscopic and imaging techniques, we were able to quantify, qualify and localise the easily extractable lignans in the xylem tissue. The knotwood samples contained 15 different lignans according to the gas chromatography-mass spectrometry analysis. They comprised 16% of the knotwood dry weight and 82% of the acetone extract. The main lignans were found to be hydroxymatairesinols HMR1 and HMR2. Cryosectioned and resin-embedded ultrathin sections of the knotwood were analysed with scanning transmission X-ray microscopy (STXM). Cryosectioning was found to retain only lignan residues inside the cell lumina. In the resin-embedded samples, lignan was interpreted to be unevenly distributed inside the cell lumina, and partially confined in deposits which were either readily present in the lumina or formed when OsO4 used in staining reacted with the lignans. Furthermore, the multi-technique characterisation enabled us to obtain information on the chemical composition of the structural components of knotwood. A simple spectral analysis of the STXM data gave consistent results with the gas chromatographic methods about the relative amounts of cell wall components (lignin and polysaccharides). The STXM analysis also indicated that a torus of a bordered pit contained aromatic compounds, possibly lignin.

Restructuring of Lipid Membranes by an Arginine-Capped Peptide Bolaamphiphile.

We study the self-assembly of arginine-capped bolaamphiphile peptide RA3R (A: alanine, R: arginine) together with its binding to model membranes and its cytotoxicity and antimicrobial activity. Anionic 2-oleoyl-1-palmitoyl- sn-glycero-3-phospho-rac-(1-glycerol) sodium salt/2-oleoyl-1-palmitoyl- sn-glycero-3-phosphoethanolamine (POPG/POPE) vesicles and zwitterionic 1,2-dioleoyl- sn-glycero-3-phosphocholine/2-oleoyl-1-palmitoyl- sn-glycero-3-phosphocholine (POPC/DOPC) vesicles are used as model membranes to mimic bacterial and mammalian cell membranes, respectively. We show that RA3R adopts a polyproline-II collagen-like conformation in water. Binding of RA3R to POPG/POPE vesicles induces a strong correlation between the lipid bilayers, driven by RA3R/POPG attractive electrostatic interaction together with a shift of the intramolecular POPE zwitterionic interaction toward an attractive electrostatic interaction with the RA3R. Populations of RA3R/POPG/POPE vesicles comprise different bilayer spacings, dA and dB, controlled by the conformation of the lipid chains corresponding to the Lß (gel-like) and Lα (liquid-crystal) phases, respectively. Cryo-TEM images reveal the presence of vesicles with no internal structure, compartmentalized thin-wall vesicles, or multilayer vesicles with uncorrelated layers and compartmentalization depending on the RA3R/POPG/POPE composition. In contrast, the interaction of RA3R with multilamellar POPC/DOPC vesicles leads to the decorrelation of the lipid bilayers. RA3R was tolerated by skin fibroblast cells for a concentration up to 0.01 wt %, while 0.25 wt % RA3R proved to be an efficient antibacterial agent against Gram-positive bacteria L. monocytogenes. Our results highlight the ability of RA3R to distinguish between bacterial and mammalian cells and establish this peptide as a candidate to reduce the proliferation of L. monocytogenes bacteria.

Melanin production by tyrosinase activity on a tyrosine-rich peptide fragment and pH-dependent self-assembly of its lipidated analogue.

We investigate the self-assembly of a palmitoylated (C16-chain at the N terminus) peptide fragment in comparison to the unlipidated peptide EELNRYY, a fragment of the gut hormone peptide PYY3-36. The lipopeptide C16-EELNRYY shows remarkable pH-dependent self-assembly above measured critical aggregation concentrations, forming fibrils at pH 7, but micelles at pH 10. The parent peptide does not show self-assembly behaviour. The lipopeptide forms hydrogels at sufficiently high concentration at pH 7, the dynamic mechanical properties of which were measured. We also show that the tyrosine functionality at the C terminus of EELNRYY can be used to enzymatically produce the pigment melanin. The enzyme tyrosinase oxidises tyrosine into 3,4-dihydroxyphenylalanine (DOPA), DOPA-quinone and further products, eventually forming eumelanin. This is a mechanism of photo-protection in the skin, for this reason controlling tyrosinase activity is a major target for skin care applications and EELNRYY has potential to be developed for such uses.

The Effect of Lipidation on the Self-Assembly of the Gut-Derived Peptide Hormone PYY3-36.

Lipidation is a powerful strategy to improve the stability in vivo of peptide drugs. Attachment of a lipid chain to a hydrophilic peptide leads to amphiphilicity and the potential for surfactant-like self-assembly. Here, the self-assembly and conformation of three lipidated derivatives of the gastrointestinal peptide hormone PYY3-36 is examined using a comprehensive range of spectroscopic, scattering, and electron microscopy methods and compared to those of the parent PYY3-36 peptide. The peptides are lipidated at Ser(11), Arg(17), or Arg(23) in the peptide; the former is within the ß-turn domain (based on the published solution NMR structure), and the latter two are both within the α-helical domain. We show that it is possible to access a remarkable diversity of nanostructures ranging from micelles to nanotapes and fibrillar hydrogels by control of assembly conditions (concentration, pH, and temperature). All of the lipopeptides self-assemble above a critical aggregation concentration (cac), determined through pyrene fluorescence probe measurements, and they all have predominantly α-helical secondary structure at their native pH. The pH and temperature dependence of the α-helical conformation were probed via circular dichroism spectroscopy experiments. Lipidation was found to provide enhanced stability against changes in temperature and pH. The self-assembled structures were investigated using small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM). Distinct differences in nanostructure were observed for lipidated and unlipidated peptides, also depending on the position of lipidation. Remarkably, micelles containing lipopeptides with α-helical peptide conformation were observed. Gelation was observed at higher concentrations in certain pH intervals for the lipidated peptides, but not for unlipidated PYY3-36. Thus, lipidation, in addition to enhancing stability against pH and temperature variation, also provides a route to prepare PYY peptide hydrogels. These findings provide important insights into the control of PYY3-36 conformation and aggregation by lipidation, relevant to the development of future therapeutics based on this peptide hormone, for example, in treatments for obesity.

Conformation and Aggregation of Selectively PEGylated and Lipidated Gastric Peptide Hormone Human PYY3-36.

The gastric peptide hormone human PYY3-36 is a target for the development of therapeutics, especially for treatment of obesity. The conformation and aggregation behavior of PEGylated and lipidated derivatives of this peptide are examined using a combination of fluorescence dye assays, circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AUC) measurements, small-angle X-ray scattering (SAXS) and cryogenic-transmission electron microscopy (cryo-TEM). The behavior of two PYY3-36 derivatives lipidated (with octyl chains) in different positions is compared to that of two derivatives with PEG attached at different residues and to that of the native peptide. We find that, unexpectedly, PYY3-36 forms amyloid fibril structures above a critical aggregation concentration. Formation of these structures is suppressed by PEGylation or lipidation. PEGylation significantly reduces the (reversible) loss of α-helix content observed on heating PYY3-36. The PEG conjugates form mainly monomeric structures in solution- coiled-coil formation, and other aggregation presumably being sterically hindered by swollen PEG chains. However, some small aggregates are detected by AUC. In complete contrast, both of the two lipidated peptides show the formation of spherical micelle-like structures which are small oligomeric aggregates. Our findings show that PEGylation and lipidation are complementary strategies to tune the conformation and aggregation of the important gastric peptide hormone human PYY3-36.

Arginine-Containing Surfactant-Like Peptides: Interaction with Lipid Membranes and Antimicrobial Activity.

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 ß-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 ß-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.