B21-High Throughput SAXS
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Diamond Proposal Number(s):
[17118]
Abstract: A dipeptide-based synthetic amphiphile bearing a myristyl chain has been found to form hydrogels in the pH range 6.9–8.5 and organogels in various organic solvents including petroleum ether, diesel, kerosene, and petrol. These organogels and hydrogels have been thoroughly studied and characterized by different techniques including high-resolution transmission electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, and rheology. It has been found that the xerogel obtained from the peptide gelator can trap various toxic organic dyes from wastewater efficiently. Moreover, the hydrogel has been used to remove toxic heavy metal ions Pb2+ and Cd2+ from wastewater. Dye adsorption kinetics has been studied, and it has been fitted by using the Freundlich isotherm equation. Interestingly, the gelator amphiphilic peptide gels fuel oil, kerosene, diesel, and petrol in a biphasic mixture of salt water and oil within a few seconds. This indicates that these gels not only may find application in oil spill recovery but also can be used to remove toxic organic dyes and hazardous toxic metal ions from wastewater. Moreover, the gelator can be recycled several times without significant loss of activity, suggesting the sustainability of this new gelator. This holds future promise for environmental remediation by using peptide-based gelators.
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Oct 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[22925]
Open Access
Abstract: 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.
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Sep 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[18523]
Open Access
Abstract: Poly(N-substituted glycine) “peptoids” are an interesting class of peptidomimics that can resist proteolysis and mimic naturally found antimicrobial peptides (AMPs), which exhibit wide spectrum activity against bacteria. This work investigates the possibility of modifying peptoid AMP mimics (AMPMs) with aliphatic lipid “tails” to generate “lipopeptoids” that can assemble into micellar nanostructures, and evaluates their antimicrobial activities. Two families of AMPMs with different distributions of hydrophobic and cationic residues were employed—one with a uniform repeating amphiphilicity, the other with a surfactant-like head-to-tail amphiphilicity. To further evaluate the interplay between self-assembly and activity, the lipopeptoids were variously modified at the AMPM chain ends with a diethylene glycol (EG2) and/or a cationic group (Nlys-Nlys dipeptoid) to adjust amphiphilicity and chain flexibility. Self-assembly was investigated by critical aggregation concentration (CAC) fluorescence assays and dynamic light scattering (DLS). The structure of a key species was also verified by small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM). To screen for antibacterial properties, we measured the minimum inhibitory concentrations (MIC) against S. aureus, E. coli, and P. aeruginosa. We found that certain combinations of lipid tail and AMPM sequences exhibit increased antibacterial activity (i.e., decreased MICs). Perhaps counter-intuitively, we were particularly interested in increased MICs in combination with low CACs. Concealing antimicrobial interactions due to packing of AMPMs in nano-assemblies could pave the way to AMPMs that may be “inert” even if unintentionally released and prevent microbes from gaining resistance to the lipopeptoids. Overall, incorporation of EG2 significantly improved lipopeptoids packing while the hydrophobic tail length was found to have a major influence over the MIC. One particular sequence, which we named C15-EG2-(kss)4, exhibited a very low CAC of 34 μM (0.0075 wt.%) and a significantly increased MIC above values for the unmodified AMPM. With the sequence design trends uncovered from this study, future work will focus on discovering more species such as C15-EG2-(kss)4 and on investigating release mechanisms and the potency of the released lipopeptoids.
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May 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[21470, 22925]
Abstract: Morphological, spectroscopic and scattering studies of the self-assembly and aggregation process of hybrids containing gold nanoparticles (AuNPs) and the amyloid peptides [RF]4 and P[RF]4 (where R = arginine; F = phenylalanine; P = proline) in aqueous solution were performed. Two methodologies were tested for the AuNP nucleation, using sodium borohydride (NaBH4) or epigallocatechin gallate (EGCG) as a reducing agent. This led to remarkable distinct modes of assembly, AuNP decorated fibrils with NaBH4 reduction or isolated AuNPs with EGCG reduction. For both methodologies, the presence of spherical AuNPs was observed by plasmonic resonance bands in absorption spectra at ∼520 nm. Zeta potential measurements confirmed stable systems, with a similar aggregation state. Circular dichroism spectra revealed an antiparallel β-sheet conformation of the peptides. The transmission electron microscopy (TEM) images showed the coexistence of nanometer fibers and globular nanoparticles with 20 nm size. The small-angle X-ray scattering (SAXS) results show that the NaBH4 systems presented large cylindrical structures, while with increasing P[RF]4 content, a decrease in radius was observed. However, the EGCG-AuNPs were characterized by spherical particles, with a radius of 10–20 nm. Also, the colorimetric efficiency of the hybrids in the capture of Cu2+ ions in solution was monitored. Raman spectroscopy data confirmed the conformation/structure of self-assembled samples. Moreover, there are indications for a surface-enhanced Raman spectroscopy (SERS) effect for Cu2+ sites. The set of results indicates that these systems could act as a promising sensitive metal concentration probes.
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Apr 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[18523]
Abstract: A molecular design approach to fabricate nanofibrous membranes by self-assembly of aromatic cationic peptides with hyaluronic acid (HA) and nanofiber alignment under a magnetic field is reported. Peptides are designed to contain a block composed of four phenylalanine residues at the C-terminus, to drive their self-assembly by hydrophobic association and aromatic stacking, and a positively charged domain of lysine residues for electrostatic interaction with HA. These two blocks are connected by a linker with a variable number of amino acids and ability to adopt distinct conformations. Zeta potential measurements and circular dichroism confirm their positive charge and variable conformation (random coil, beta-sheet or alpha-helix), which depend on the pH and sequence. Their self-assembly, examined by fluorescence spectroscopy, small-angle X-ray scattering and transmission electron microscopy, show the formation of fiber-like nanostructures in the micromolar range. When the peptides are combined with HA, hydrogels or flat membranes are formed. The molecular structure tunes the mechanical behavior of the membranes and the nanofibers align in the direction of magnetic field due to the high diamagnetic anisotropy of phenylalanine residues. Mesenchymal stem cells cultured on magnetically-aligned membranes elongate in direction of the nanofibers supporting their application for soft tissue engineering.
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Apr 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[21470, 22925]
Abstract: The self-assembly of the amphiphilic lipopeptide PAEPKI-C16 (P = proline, A = alanine, E = glutamic acid, K = lysine, I = isoleucine, and C16 = hexadecyl) was investigated using a combination of microscopy, spectroscopy, and scattering methods and compared to that of C16-IKPEAP with the same (reversed) peptide sequence and the alkyl chain positioned at the N-terminus and lacking a free N-terminal proline residue. The catalytic activity of these peptides was then compared using a model aldol reaction system. For PAEPKI-C16, the cryo-TEM images showed the formation of micrometer-length fibers, which by small-angle X-ray scattering (SAXS) were found to have radii of 2.5–2.6 nm. Spectroscopic analysis shows that these fibers are built from β-sheets. This behavior is in complete contrast to that of C16-IKPEAP, which forms spherical micelles with peptides in a disordered conformation [Hutchinson J. Phys. Chem. B 2019, 123, 613]. In PAEPKI-C16, spontaneous alignment of fibers was observed upon increasing pH, which was accompanied by observed birefringence and anisotropy of SAXS patterns. This shows the ability to form a nematic phase, and unprecedented nematic hydrogel formation was also observed for 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 that of 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 high anisotropy of its ultrafine fibrillar structure, and the formation of such a phase at low concentrations in aqueous solution may be valuable for future applications.
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Mar 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[18523, 21470]
Open Access
Abstract: Morphological, spectroscopic, and scattering studies of the self-assembly and aggregation 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 in aqueous solution, 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 scattering density, suggesting an increase in packing of the peptide molecules; however, the opposite effect is observed for the gels, where the scattering density is higher in the shell for the systems containing higher P[RF]4 content. 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. With increasing P[RF]4 content, 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.
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Mar 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[17118, 18523]
Abstract: 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.
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Jan 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[18523]
Abstract: 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.
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Aug 2019
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[21470]
Open Access
Abstract: The self-assembly and biocatalytic activity of the proline-functionalized lipopeptide PRW-NH-C16 are examined, and compared to that of the related PRW-O-C16 lipopeptide, which differs in having an ester linker between the lipid chain and tripeptide headgroup instead of an amide linker. Lipopeptide PRW-NH-C16 self-assembles into spherical micelles above a critical aggregation concentration, similar to the behaviour of PRW-O-C16 reported previously [B.M. Soares et al. Phys. Chem. Chem. Phys., 2017, 19, 1181—1189]. However, PRW-NH-C16 shows improved catalytic activity in a model aldol reaction. In addition, we explore incorporation of the biocatalytic lipopeptide into lipid cubosomes. SAXS shows that increasing lipopeptide concentration leads to an expansion of the monoolein cubosome lattice spacing, and loss of long-range cubic order as the lipopeptide is encapsulated in the cubosomes. At higher loadings of lipopeptide, reduced cubosome formation is observed at the expense of vesicle formation. Our results show that the peptide-lipid chain linker does not influence self-assembly but does impart improved biocatalytic activity. Furthermore, we show that lipopeptides can be incorporated into lipid cubosomes, leading to restructuring into vesicles at high loadings. These findings point the way towards the future development of bioactive lipopeptide assemblies and slow release cubosome-based delivery systems.
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Jul 2019
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