Amyloid peptide mixtures: self-assembly, hydrogelation, nematic ordering and catalysts in aldol reactions

Morphological, spectroscopic and scattering studies of the self-assembly and aggregation process of mixtures of [RF]4 and P[RF]4 peptides (where: R = arginine; F = phenylalanine; P = proline), in solution and as hydrogels, were performed to obtain information about polymorphism. CD data confirmed a β-sheet secondary structure conformation for the solutions and TEM images revealed nanofibers with diameters of ~ 10 nm and micrometer lengths. SAXS curves were fitted using a mass fractal-component and a long cylinder shell form factor for the liquid samples, and only a long cylinder shell form factor for the gels. Increasing the P[RF]4 content in the systems leads to a reduction in cylinder radius and core density scattering, suggesting an increase in packing of the peptide molecules; however, the opposite effect was observed for the gels. Remarkably, the gels are birefringent, indicating nematic ordering of the gel fibrils. These compounds show potential as catalysts in the asymmetric aldol reactions, with cyclohexanone and p-nitrobenzaldehyde in aqueous media. A moderate conversion (36.9 %) and a good stereoselectivity (69:31) were observed for the system containing only [RF]4, and with the increase of the P[RF]4, a considerable decrease of the conversion was observed, suggesting differences in the self-assembly and packing factor. Rheological measurements were performed to determine the shear moduli for the soft gels. These model amyloid peptides demonstrate a range of tunable self-assembly behaviors and additionally have potential as biocatalysts

Self-assembly, tunable hydrogel properties and selective anti-cancer activity of a carnosine-derived lipidated peptide

A novel lipopeptide C16KTTβAH was designed that incorporates the KTT tripeptide sequence from “Matrixyl” lipopeptides along with the bioactive βAH (β-alanine-histidine) carnosine dipeptide motif, attached to a C16 hexadecyl lipid chain. We show that this peptide amphiphile self-assembles above a critical aggregation concentration into β-sheet nanotape structures in water, PBS and cell culture media. Nanotape bundle structures were imaged in PBS, the bundling resulting from nanotape associations due to charge screening in the buffer. In addition, hydrogelation was observed and the gel modulus was measured in different aqueous media conditions, revealing tunable hydrogel modulus depending on concentration and nature of the aqueous phase. Stiff hydrogels were observed by direct dissolution in PBS and it was also possible to prepare hydrogels with unprecedented high modulus from low concentration solutions by injection of dilute aqueous solutions into PBS. These hydrogels have exceptional stiffness compared to previously reported β-sheet peptide-based materials. In addition, macroscopic soft threads can be drawn from concentrated aqueous solutions of the lipopeptides which contain aligned nematic structures. The anti-cancer activity of the lipopeptide was assessed using two model breast cancer cell lines, compared to two fibroblast cell line controls. These studies revealed selective concentration-dependent cytotoxicity against MCF-7 cancer cells in a mM concentration range. It was shown that this occurs below the onset of lipopeptide aggregation (i.e. below the critical aggregation concentration), indicating that the cytotoxicity is not related to self-assembly but is an intrinsic property of C16KTTβAH. Finally, hydrogels of this lipopeptide demonstrated slow uptake and release of the dye Congo red, a model diagnostic compound

Peptide-stabilized emulsions and gels from an arginine-rich surfactant-like peptide with antimicrobial activity

The preparation of hydrogels and stable emulsions is important in the formulation of many functional nanostructured soft materials. We investigate the multifunctional self-assembly and bioactivity properties of a novel surfactant-like peptide (SLP) that shows antimicrobial activity, is able to form hydrogels without pH adjustment, and is able to stabilize oil-in-water emulsions. Furthermore, we demonstrate on-demand de-emulsification in response to the protease enzyme elastase. We show that SLP (Ala)9-Arg (A9R) forms β-sheet fibers above a critical aggregation concentration and that water-in-oil emulsions are stabilized by a coating of β-sheet fibers around the emulsion droplets. Furthermore, we demonstrate enzyme-responsive de-emulsification, which has potential in the development of responsive release systems. The peptide shows selective antimicrobial activity against Gram-negative pathogens including Pseudomonas aeruginosa, which causes serious infections. Our results highlight the utility of SLPs in the stabilization of oil/water emulsions and the potential for these to be used to formulate antimicrobial peptide emulsions which are additionally responsive to protease. The peptide A9R has pronounced antibacterial activity against clinically challenging pathogens, and its ability to form β-sheet fibers plays a key role in its diverse structural properties, ranging from hydrogel formation to emulsion stabilization

Self-assembly, nematic phase formation and organocatalytic behaviour of a proline-functionalized lipopeptide

The self-assembly of the amphiphilic lipopeptide PAEPKI-C16 (P = proline, A = alanine, E = glutamic acid, K = lysine, I = isoleucine, C16 = hexadecyl) was investigated using a combination of spectroscopic, microscopic and scattering methods and compared to C16-IKPEAP with the same (reversed) peptide sequence and the alkyl chain positioned N-terminally and which lacks a free N-terminal proline residue. The catalytic activity of these peptides were then compared using a model aldol reaction system. For PAEPKI-C16, Cryo-TEM images showed the formation of micrometer length fibers, which by Small-angle X-ray scattering (SAXS) were found to have a radius of 2.5 - 2.6 nm. Spectroscopic analysis shows these fibers are built from -sheets. This behaviour is in complete contrast to that of C16-IKPEAP which forms spherical micelles with peptides in a disordered conformation [Hutchinson, J. A. et al. J. Phys. Chem. B 2019, 123, 613]. For PAEPKI-C16, the spontaneous alignment of fibers was observed upon increasing pH, which was accompanied by observed birefringence and anisotropy of SAXS patterns. This shows the formation of a nematic liquids and unprecedented nematic hydrogel formation was also observed these lipopeptides at sufficiently high concentrations. SAXS shows retention of an ultrafine (1.7 nm core radius) fibrillar network within the hydrogel. PAEPKI-C16 with free N-terminal proline shows enhanced anti:syn diastereoselectivity and better conversion compared to C16-IKPEAP. The cytotoxicity of PAEPKI-C16 was also lower than C16-IKPEAP for both fibroblast and cancer cell lines. These results highlight the sensitivity of lipopeptide properties to the presence of a free proline residue. The spontaneous nematic phase formation by PAEPKI-C16 points to the highly anisotropy of its ultrafine fibrillar structure and the formation of such a phase at low concentration in aqueous solution may be valuable for future applications

Selective antibacterial activity and lipid membrane interactions of arginine-rich amphiphilic peptides

The self-assembly behaviour and antimicrobial activity of two new designed amphiphilic peptides, R3F3 and R4F4, containing short hydrophobic phenylalanine (F) cationic arginine (R) sequences are investigated. The conformation of the peptides was examined using circular dichroism and FTIR spectroscopy, which show that they have a disordered secondary structure. Concentration-dependent fluorescence assays show the presence of a critical aggregation concentration (cac) for each peptide. Above the cac, small angle x-ray scattering (SAXS) and transmission electron microscopy (TEM) show the presence of a population of twisted tapes for R3F3 and nanosheets for R4F4. The interaction of the peptides with model bacterial membranes comprising mixtures of the lipids DPPG [1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol)] and DPPE [1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine], was studied using SAXS and cryogenic-TEM. Analysis of the SAXS structure factor indicates that R3F3 interacts with lipid bilayers by inducing correlation between bilayers, whereas R4F4 interacts with the bilayers causing an increase in polydispersity of the vesicle wall thickness. Both peptides break vesicles with a 1:3 DPPG:DPPE composition, which is close to the ratio of PG and PE lipids observed in the lipid membrane of Pseudomonas aeruginosa, a pathogen responsible for serious infections and which has developed antimicrobial resistant strains. Both peptides show activity against this bacterium in planktonic form. Peptide R4F4 shows particularly strong bioactivity against this microbe, with a minimum inhibitory concentration (MIC) value in the range of concentrations where the peptide is cytocompatible. It was further shown to have activity against other Pseudomonas species including the common plant pathogen P. syringae. Finally, we show that R4F4 inhibits the development of P. aeruginosa biofilms. This was examined in detail and a proposed mechanism involving binding of the signalling molecule c-di-GMP is suggested, based on circular dichroism spectroscopy studies and Congo red assays of extracellular polysaccharides produced by the stressed bacteria. Thus, R4F4 is a promising candidate antimicrobial peptide with activity against Pseudomonas species

Peptide nanotubes self-assembled from leucine-rich alpha helical surfactant-like peptides

The designed arginine-rich surfactant-like peptide R3L12 (arginine3–leucine12) is shown to form a remarkable diversity of self-assembled nanostructures in aqueous solution, depending on pH, including nanotubes, mesh-like tubular networks in three-dimensions and square planar arrays in two-dimensions. These structures are built from α-helical antiparallel coiled–coil peptide dimers arranged perpendicular to the nanotube axis, in a “cross-α” nanotube structure. The aggregation behavior is rationalized based on the effects of dimensionality, and the balance of hydrophobic and electrostatic interactions. The nanotube and nanomesh structures display arginine at high density on their surfaces, which may be valuable for future applications

Ugi multicomponent reaction to prepare peptide–peptoid hybrid structures with diverse chemical functionalities

Monodisperse sequenced peptides and peptoids present unique nano-structures based on their self-assembled secondary and tertiary structures. However, the generation of peptide and peptoid hybrid oligomers in a sequence-defined manner via Ugi multicomponent reaction has not yet been studied. Herein, we report a synthetic strategy that enables both the modification of peptides as well as the generation of sequence-defined peptide–peptoid hybrid structures. Our synthetic methodology rests on the fusion of solid phase peptide synthesis with Ugi multicomponent reactions. We evidence that a diversity of chemical functionalities can be inserted into peptides or used in the design of peptide–peptoid hybrids exploiting a wide functional array including amines, carboxylic acids, hydrocarbons, carbohydrates as well as polymers, introducing a sequence-defined synthetic platform technology for precision peptoid hybrids

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 Lβ (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

Polymorphism of asymmetric catalysts based on amphiphilic lipopeptides in solution

The self-assembly of model [P]RWG lipopeptides (P: L-proline, R: L-arginine, W: L-tryptophan, G: L-glycine), containing one or two aliphatic octadecyl (C18) chains in water and cyclohexanone/water solutions was examined. The self-assembly of mixtures of these RWG and PRWG lipopeptides was also investigated. These materials presented a similar critical aggregation concentration of ∼4.0 × 10−4 wt% and were characterized by unordered secondary structures with some β-sheet content. TEM and cryo-TEM revealed the presence of mainly nanotape structures with micelles observed for systems rich in PRWG(C18H37). Analysis of detailed SAXS form factor measurements revealed the presence of bilayers 3–4 nm thick while the PRWG(C18H37) micelles have a core radius of approximately 3 nm, and a shell thickness of 2 nm. For the cyclohexanone/water systems polymorphs containing cluster aggregates (with radius of 0.25 nm to 0.50 nm) and some elongated structures (with radius of 5.7 nm to 26.1 nm) were seen. Longer structures were formed with the increase of the proline-containing lipopeptide content. The catalytic activity of these peptides was assessed using a model nitro-aldol reaction. The concentration of water in the reaction system influenced the conversion, higher content promoted better efficiency for the water systems, but the opposite was observed for the cyclohexanone/water samples

Impact of subtle change in branched amino acid on the assembly and properties of perylene bisimides hydrogels

We investigate how apparent slight changes to the chemical structure of amino acid-functionalised perylene bisimides (PBIs) affect the self-assembled aggregates formed and their resulting physical and optical properties. PBIs functionalised with L-valine (PBI-V), L-leucine (PBI-L) and L-isoleucine (PBI-I) are investigated due to their similarly branched structure and their assemblies in water were studied using UV-vis absorption spectroscopy, cyclic voltammetry (CV), small angle X-ray scattering (SAXS) and viscosity at different pHs. It was seen that each PBI behaved differently. Each of the PBIs were then used to prepare hydrogels, and their properties again assessed, with PBI-I forming different hydrogels than the other PBIs. By understanding how slight changes in chemical structure can affect bulk properties we become a step closer to designing gels with specific physical and electrical properties