Developing an in situ LED irradiation system for small-angle X-ray scattering at B21, Diamond Light Source.

Beamline B21 at the Diamond Light Source synchrotron in the UK is a small-angle X-ray scattering (SAXS) beamline that specializes in high-throughput measurements via automated sample delivery systems. A system has been developed whereby a sample can be illuminated by a focused beam of light coincident with the X-ray beam. The system is compatible with the highly automated sample delivery system at the beamline and allows a beamline user to select a light source from a broad range of wavelengths across the UV and visible spectrum and to control the timing and duration of the light pulse with respect to the X-ray exposure of the SAXS measurement. The intensity of the light source has been characterized across the wavelength range enabling experiments where a quantitative measure of dose is important. Finally, the utility of the system is demonstrated via measurement of several light-responsive samples.

Amino acid containing amphiphilic hydrogelators with antibacterial and antiparasitic activities.

Nanoscale self-assembly of peptide constructs represents a promising means to present bioactive motifs to develop new functional materials. Here, we present a series of peptide amphiphiles which form hydrogels based on ß-sheet nanofibril networks, several of which have very promising anti-microbial and anti-parasitic activities, in particular against multiple strains of Leishmania including drug-resistant ones. Aromatic amino acid based amphiphilic supramolecular gelators C14-Phe-CONH-(CH2)n-NH2 (n = 6 for P1 and n = 2 for P3) and C14-Trp-CONH-(CH2)n-NH2 (n = 6 for P2 and n = 2 for P4) have been synthesized and characterized, and their self-assembly and gelation behaviour have been investigated in the presence of ultrapure water (P1, P2, and P4) or 2% DMSO(v/v) in ultrapure water (P3). The rheological, morphological and structural properties of the gels have been comprehensively examined. The amphiphilic gelators (P1 and P3) were found to be active against both Gram-positive bacteria B. subtilis and Gram-negative bacteria E. coli and P. aeruginosa. Interestingly, amphiphiles P1 and P3 containing an L-phenylalanine residue show both antibacterial and antiparasitic activities. Herein, we report that synthetic amphiphiles with an amino acid residue exhibit a potent anti-protozoan activity and are cytotoxic towards a wide array of protozoal parasites, which includes Indian varieties of Leishmania donovani and also kill resistant parasitic strains including BHU-575, MILR and CPTR cells. These gelators are highly cytotoxic to promastigotes of Leishmania and trigger apoptotic-like events inside the parasite. The mechanism of killing the parasite is shown and these gelators are non-cytotoxic to host macrophage cells indicating the potential use of these gels as therapeutic agents against multiple forms of leishmaniasis in the near future.

Beamline B21: high-throughput small-angle X-ray scattering at Diamond Light Source.

B21 is a small-angle X-ray scattering (SAXS) beamline with a bending magnet source in the 3 GeV storage ring at the Diamond Light Source Ltd synchrotron in the UK. The beamline utilizes a double multi-layer monochromator and a toroidal focusing optic to deliver 2 × 1012 photons per second to a 34 × 40 µm (FWHM) focal spot at the in-vacuum Eiger 4M (Dectris) detector. A high-performance liquid chromatography system and a liquid-handling robot make it possible to load solution samples into a temperature-controlled in-vacuum sample cell with a high level of automation. Alternatively, a range of viscous or solid materials may be loaded manually using a range of custom sample cells. A default scattering vector range from 0.0026 to 0.34 Å-1 and low instrument background make B21 convenient for measuring a wide range of biological macromolecules. The beamline has run a full user programme since 2013.

Peptide-Based Gel in Environmental Remediation: Removal of Toxic Organic Dyes and Hazardous Pb2+ and Cd2+ Ions from Wastewater and Oil Spill Recovery.

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.

Amyloid Peptide Mixtures: Self-Assembly, Hydrogelation, Nematic Ordering, and Catalysts in Aldol Reactions.

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 ß-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.

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 ß-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.

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.

Self-Assembly of Telechelic Tyrosine End-Capped PEO Star Polymers in Aqueous Solution.

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-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 and cryogenic transmission electron microscopy. In contrast to previous studies on linear telechelic PEO-oligotyrosine conjugates that show self-assembly into ß-sheet fibrils, the star architecture suppresses fibril formation and micelles are generally observed instead, a small population of fibrils only being observed upon pH adjustment. Hydrogelation is also suppressed by the polymer star architecture. These peptide-functionalized star polymer solutions are cytocompatible at sufficiently low concentration. These systems present tyrosine at high density and may be useful in the development of future enzyme or pH-responsive biomaterials.

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.

Self-assembly of bioactive peptides, peptide conjugates, and peptide mimetic materials.

Molecular self-assembly is a multi-disciplinary field of research, with potential chemical and biological applications. One of the main driving forces of self-assembly is molecular amphiphilicity, which can drive formation of complex and stable nanostructures. Self-assembling peptide and peptide conjugates have attracted great attention due to their biocompatibility, biodegradability and biofunctionality. Understanding assembly enables the better design of peptide amphiphiles which may form useful and functional nanostructures. This review covers self-assembly of amphiphilic peptides and peptide mimetic materials, as well as their potential applications.

Structure-dynamics correlations in composite PF127-PEG-based hydrogels; cohesive/hydrophobic interactions determine phase and rheology and identify the role of micelle concentration in controlling 3D extrusion printability.

A library of composite polymer networks (CPNs) were formed by combining Pluronic F127, as the primary gelator, with a range of di-acrylate functionalised PEG polymers, which tune the rheological properties and provide UV crosslinkability. A coarse-grained sol-gel room temperature phase diagram was constructed for the CPN library, which identifies PEG-dependent disruption of micelles as leading to liquefication. Small angle X-ray scattering and rheological measurements provide detailed insight into; (i) micelle-micelle ordering; (ii) micelle-micelle disruption, and; (iii) acrylate-micelle disruption; with contributions that depend on composition, including weak PEG chain length and end group effects. The influence of composition on 3D extrusion printability through modulation of the cohesive/hydrophobic interactions was assessed. It was found that only micelle content provides consistent changes in printing fidelity, controlled largely by printing conditions (pressure and feed rate). Finally, the hydrogels were shown to be UV photo-crosslinkable, which further improves fidelity and structural integrity, and usefully reduces the mesh size. Our results provide a guide for design of 3D-printable CPN inks for future biomedical applications.

Micro/nano-patterns for enhancing differentiation of human neural stem cells and fabrication of nerve conduits via soft lithography and 3D printing.

Topographical cues on materials can manipulate cellular fate, particularly for neural cells that respond well to such cues. Utilizing biomaterial surfaces with topographical features can effectively influence neuronal differentiation and promote neurite outgrowth. This is crucial for improving the regeneration of damaged neural tissue after injury. Here, we utilized groove patterns to create neural conduits that promote neural differentiation and axonal growth. We investigated the differentiation of human neural stem cells (NSCs) on silicon dioxide groove patterns with varying height-to-width/spacing ratios. We hypothesize that NSCs can sense the microgrooves with nanoscale depth on different aspect ratio substrates and exhibit different morphologies and differentiation fate. A comprehensive approach was employed, analyzing cell morphology, neurite length, and cell-specific markers. These aspects provided insights into the behavior of the investigated NSCs and their response to the topographical cues. Three groove-pattern models were designed with varying height-to-width/spacing ratios of 80, 42, and 30 for groove pattern widths of 1 µm, 5 µm, and 10 µm and nanoheights of 80 nm, 210 nm, and 280 nm. Smaller groove patterns led to longer neurites and more effective differentiation towards neurons, whereas larger patterns promoted multidimensional differentiation towards both neurons and glia. We transferred these cues onto patterned polycaprolactone (PCL) and PCL-graphene oxide (PCL-GO) composite 'stamps' using simple soft lithography and reproducible extrusion 3D printing methods. The patterned scaffolds elicited a response from NSCs comparable to that of silicon dioxide groove patterns. The smallest pattern stimulated the highest neurite outgrowth, while the middle-sized grooves of PCL-GO induced effective synaptogenesis. We demonstrated the potential for such structures to be wrapped into tubes and used as grafts for peripheral nerve regeneration. Grooved PCL and PCL-GO conduits could be a promising alternative to nerve grafting.

Molecular and cellular insight into Escherichia coli SslE and its role during biofilm maturation.

Escherichia coli is a Gram-negative bacterium that colonises the human intestine and virulent strains can cause severe diarrhoeal and extraintestinal diseases. The protein SslE is secreted by a range of pathogenic and commensal E. coli strains. It can degrade mucins in the intestine, promotes biofilm maturation and it is a major determinant of infection in virulent strains, although how it carries out these functions is not well understood. Here, we examine SslE from the commensal E. coli Waksman and BL21 (DE3) strains and the enterotoxigenic H10407 and enteropathogenic E2348/69 strains. We reveal that SslE has a unique and dynamic structure in solution and in response to acidification within mature biofilms it can form a unique aggregate with amyloid-like properties. Furthermore, we show that both SslE monomers and aggregates bind DNA in vitro and co-localise with extracellular DNA (eDNA) in mature biofilms, and SslE aggregates may also associate with cellulose under certain conditions. Our results suggest that interactions between SslE and eDNA are important for biofilm maturation in many E. coli strains and SslE may also be a factor that drives biofilm formation in other SslE-secreting bacteria.

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.

Selective Antibacterial Activity and Lipid Membrane Interactions of Arginine-Rich Amphiphilic Peptides.

The self-assembly behavior and antimicrobial activity of two designed amphiphilic peptides, R3F3 and R4F4, containing short hydrophobic phenylalanine (F) and 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) reveal 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-phosphoglycerol] 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 Pseudomonas 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 signaling 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.

Self-assembled gold nanoparticles and amphiphile peptides: a colorimetric probe for copper(II) ion detection.

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 ß-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.

Magnetic Field-Induced Alignment of Nanofibrous Supramolecular Membranes: A Molecular Design Approach to Create Tissue-like Biomaterials.

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 have 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 the ability to adopt distinct conformations. Zeta potential measurements and circular dichroism confirm their positive charge and variable conformation (random coil, ß-sheet, or α-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 fiberlike 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.

Model self-assembling arginine-based tripeptides show selective activity against Pseudomonas bacteria.

Three model arginine-rich tripeptides RXR (X = W, F or non-natural residue 2-napthylalanine) were investigated as antimicrobial agents, with a specific focus to target Pseudomonas aeruginosa through membrane lysis. Activity against biofilms was related to binding of the second messenger molecule, nucleotide bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). Strong selective activity against P. aeruginosa in planktonic form was observed for RFR and RWR.

Self-Assembly, Nematic Phase Formation, and Organocatalytic Behavior 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, 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 ß-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.