Amyloid and Hydrogel Formation of a Peptide Sequence from a Coronavirus Spike Protein

We demonstrate that a conserved coronavirus spike protein peptide forms amyloid structures, differing from the native helical conformation and not predicted by amyloid aggregation algorithms. We investigate the conformation and aggregation of peptide RSA­IED­LLF­DKV, which is a sequence common to many animal and human coronavirus spike proteins. This sequence is part of a native α-helical S2 glycoprotein domain, close to and partly spanning the fusion sequence. This peptide aggregates into β-sheet amyloid nanotape structures close to the calculated pI = 4.2, but forms disordered monomers at high and low pH. The β-sheet conformation revealed by FTIR and circular dichroism (CD) spectroscopy leads to peptide nanotape structures, imaged using transmission electron microscopy (TEM) and probed by small-angle X-ray scattering (SAXS). The nanotapes comprise arginine-coated bilayers. A Congo red dye UV–vis assay is used to probe the aggregation of the peptide into amyloid structures, which enabled the determination of a critical aggregation concentration (CAC). This peptide also forms hydrogels under precisely defined conditions of pH and concentration, the rheological properties of which were probed. The observation of amyloid formation by a coronavirus spike has relevance to the stability of the spike protein conformation (or its destabilization via pH change), and the peptide may have potential utility as a functional material. Hydrogels formed by coronavirus peptides may also be of future interest in the development of slow-release systems, among other applications

Cyclodextrin-Induced Suppression of the Crystallization of Low-Molar-Mass Poly(ethylene glycol)

We examine the effect of alpha-cyclodextrin (αCD) on the crystallization of poly­(ethylene glycol) (PEG) [poly­(ethylene oxide), PEO] in low-molar-mass polymers, with Mw = 1000, 3000, or 6000 g mol–1. Differential scanning calorimetry (DSC) and simultaneous synchrotron small-/wide-angle X-ray scattering (SAXS/WAXS) show that crystallization of PEG is suppressed by αCD, provided that the cyclodextrin content is sufficient. The PEG crystal structure is replaced by a hexagonal mesophase of αCD-threaded polymer chains. The αCD threading reduces the conformational flexibility of PEG and, hence, suppresses crystallization. These findings point to the use of cyclodextrin additives as a powerful means to tune the crystallization of PEG (PEO), which, in turn, will impact bulk properties including biodegradability

Altering Peptide Fibrillization by Polymer Conjugation

A strategy is presented that exploits the ability of synthetic polymers of different nature to disturb the strong self-assembly capabilities of amyloid based β-sheet forming peptides. Following a convergent approach, the peptides of interest were synthesized via solid-phase peptide synthesis (SPPS) and the polymers via reversible addition–fragmentation chain transfer (RAFT) polymerization, followed by a copper­(I) catalyzed azide–alkyne cycloaddition (CuAAC) to generate the desired peptide–polymer conjugates. This study focuses on a modified version of the core sequence of the β-amyloid peptide (Aβ), Aβ(16–20) (KLVFF). The influence of attaching short poly­(<i>N</i>-isopropylacrylamide) and poly­(hydroxyethylacrylate) to the peptide sequences on the self-assembly properties of the hybrid materials were studied via infrared spectroscopy, TEM, circular dichroism and SAXS. The findings indicate that attaching these polymers disturbs the strong self-assembly properties of the biomolecules to a certain degree and permits to influence the aggregation of the peptides based on their β-sheets forming abilities. This study presents an innovative route toward targeted and controlled assembly of amyloid-like fibers to drive the formation of polymeric nanomaterials

Slow-Release RGD-Peptide Hydrogel Monoliths

We report on the formation of hydrogel monoliths formed by functionalized peptide Fmoc-RGD (Fmoc: fluorenylmethoxycarbonyl) containing the RGD cell adhesion tripeptide motif. The monolith is stable in water for nearly 40 days. The gel monoliths present a rigid porous structure consisting of a network of peptide fibers. The RGD-decorated peptide fibers have a β-sheet secondary structure. We prove that Fmoc-RGD monoliths can be used to release and encapsulate material, including model hydrophilic dyes and drug compounds. We provide the first insight into the correlation between the absorption and release kinetics of this new material and show that both processes take place over similar time scales

A Direct Comparison of One- and Two-Component Dendritic Self-Assembled Materials: Elucidating Molecular Recognition Pathways

This paper compares and contrasts, for the first time, one- and two-component gelation systems that are direct structural analogues and draws conclusions about the molecular recognition pathways that underpin fibrillar self-assembly. The new one-component systems comprise l-lysine-based dendritic headgroups covalently connected to an aliphatic diamine spacer chain via an amide bond. One-component gelators with different generations of headgroup (from first to third generation) and different length spacer chains are reported. The self-assembly of these dendrimers in toluene was elucidated using thermal measurements, circular dichroism (CD) and NMR spectroscopies, scanning electron microscopy (SEM), and small-angle X-ray scattering (SAXS). The observations are compared with previous results for the analogous two-component gelation system in which the dendritic headgroups are bound to the aliphatic spacer chain noncovalently via acid−amine interactions. The one-component system is inherently a more effective gelator, partly as a consequence of the additional covalent amide groups that provide a new hydrogen bonding molecular recognition pathway, whereas the two-component analogue relies solely on intermolecular hydrogen bond interactions between the chiral dendritic headgroups. Furthermore, because these amide groups are important in the assembly process for the one-component system, the chiral information preset in the dendritic headgroups is not always transcribed into the nanoscale assembly, whereas for the two-component system, fiber formation is always accompanied by chiral ordering because the molecular recognition pathway is completely dependent on hydrogen bond interactions between well-organized chiral dendritic headgroups

Nanoscale Structure of Poly(Ethylene Glycol) Hybrid Block Copolymers containing Amphiphilic β-Strand Peptide Sequences

This paper discusses the solid state and melt nanoscale structure of a series of novel poly(ethylene glycol) (PEG) hybrid di- and triblock copolymers, which contain amphiphilic β-strand peptide sequences. The block copolymers have been prepared via solid-phase synthesis, affording perfectly monodisperse peptide segments with a precisely defined α-amino acid sequence. Attenuated total reflection Fourier transform infrared spectroscopy and X-ray scattering experiments indicate that the self-assembly properties of the peptide sequences are retained upon conjugation to PEG and mediate the formation of an ordered superstructure consisting of alternating PEG layers and peptide domains with an highly organized antiparallel β-sheet structure. The results suggest that combination of biological structural motifs with synthetic polymers may be a versatile strategy for the development of novel self-assembled materials with complex internal structures and the potential to interface with biology

Interaction between a Cationic Surfactant-like Peptide and Lipid Vesicles and Its Relationship to Antimicrobial Activity

We investigate the properties of an antimicrobial surfactant-like peptide (Ala)₆(Arg), A₆R, containing a cationic headgroup. The interaction of this peptide with zwitterionic (DPPC) lipid vesicles is investigated using a range of microscopic, X-ray scattering, spectroscopic, and calorimetric methods. The β-sheet structure adopted by A₆R is disrupted in the presence of DPPC. A strong effect on the small-angle X-ray scattering profile is observed: the Bragg peaks from the DPPC bilayers in the vesicle walls are eliminated in the presence of A₆R and only bilayer form factor peaks are observed. All of these observations point to the interaction of A₆R with DPPC bilayers. These studies provide insight into interactions between a model cationic peptide and vesicles, relevant to understanding the action of antimicrobial peptides on lipid membranes. Notably, peptide A₆R exhibits antimicrobial activity without membrane lysis

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 β-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-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 β-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

A Rheological and SAXS Study of the Lamellar Order in a Side-on Liquid Crystalline Block Copolymer

We study the structure and shear flow behavior of a side-on liquid crystalline triblock copolymer, named PBA-b-PA444-b-PBA (PBA is poly(butyl acrylate) and PA444 is a poly(acrylate) with a nematic liquid crystal side-on mesogen), in the self-assembled lamellar phase and in the disordered phase. Simultaneous oscillatory shear and small-angle X-ray scattering experiments show that shearing PBA-b-PA444-b-PBA at high frequency and strain amplitudes leads to the alignment of the lamellae with normals perpendicular to the shear direction and to the velocity gradient direction, i.e., in the perpendicular orientation. The order-to-disorder transition temperature (TODT) is independent of the applied strain, in contrast to results reported in the literature for coil−coil diblock copolymers, which show an increase in TODT with shear rate. It is possible that in our system, TODT does not depend on the applied strain because the fluctuations are weaker than those present in coil−coil diblock copolymer systems