Faraday Discussions最新文献

Fibroblast Growth Factor 2 (FGF2) is a potent mitogen secreted from mammalian cells through an unconventional secretory pathway. This process is mediated by direct translocation of FGF2 across the plasma membrane into the extracellular space. It requires several components that are asymmetrically distributed between the two leaflets of the plasma membrane. At the inner plasma membrane leaflet, FGF2 undergoes sequential interactions with the Na,K-ATPase, Tec kinase, and the phosphoinositide PI(4,5)P₂. While the Na,K-ATPase, and Tec kinase are auxiliary factors, interactions of FGF2 with PI(4,5)P₂ trigger the core mechanism of FGF2 membrane translocation, inducing FGF2-oligomerization-dependent formation of lipidic membrane pores. At the outer plasma membrane leaflet, membrane-inserted FGF2 oligomers are captured and disassembled by Glypican-1 (GPC1), resulting in translocation of FGF2 to the cell surface. In a cellular context, a single FGF2 membrane translocation event occurs within 200 milliseconds. In contrast, in an in vitro system, which uses a fully reconstituted liposomal inside-out system with FGF2 added from the outside and luminal encapsulation of high-affinity heparin molecules, FGF2 membrane translocation takes several minutes. Here, we hypothesize that the observed difference is, at least in part, due to the asymmetrical membrane lipid distribution and the spatial organization of the FGF2 translocation machinery in native plasma membranes. We suggest that the molecular machinery mediating FGF2 membrane translocation assembles in ordered nanodomains, characterized by sphingomyelin (SM), cholesterol and phosphoinositide PI(4,5)P₂ coupled together. The transbilayer asymmetry of these lipids likely plays a crucial role in regulating the thermodynamics and kinetics of FGF2-induced membrane pore formation. Therefore, succeeding in reconstituting the FGF2 translocation machinery in artificial membranes with an asymmetric transbilayer distribution of SM, PI(4,5)P₂ and other membrane lipids may reveal a direct impact on pore-opening kinetics. Similarly, disrupting lipid asymmetry in cells may significantly impact FGF2 secretion rates, a finding that would underscore the importance of the spatial organization of lipids in membrane dynamics. Testing this hypothesis may advance our understanding of how membrane asymmetry and ordered lipid nanodomains regulate critical biological processes, such as the unconventional secretion of FGF2.

Surfactant-like peptides, in which hydrophilic and hydrophobic residues are encoded within different domains in the peptide sequence, undergo facile self-assembly in aqueous solution to form supramolecular hydrogels. These peptides have been explored extensively as substrates for the creation of functional materials since a wide variety of amphipathic sequences can be prepared from commonly available amino acid precursors. The self-assembly behavior of surfactant-like peptides has been compared to that observed for small molecule amphiphiles in which nanoscale phase separation of the hydrophobic domains drives the self-assembly of supramolecular structures. Here, we investigate the relationship between sequence and supramolecular structure for a pair of bola-amphiphilic peptides, Ac-KLIIIK-NH₂ (L2) and Ac-KIIILK-NH₂ (L5). Despite similar length, composition, and polar sequence pattern, L2 and L5 form morphologically distinct assemblies, nanosheets and nanotubes, respectively. Cryo-EM helical reconstruction was employed to determine the structure of the L5 nanotube at near-atomic resolution. Rather than displaying self-assembly behavior analogous to conventional amphiphiles, the packing arrangement of peptides in the L5 nanotube displayed steric zipper interfaces that resembled those observed in the structures of β-amyloid fibrils. Like amyloids, the supramolecular structures of the L2 and L5 assemblies were sensitive to conservative amino acid substitutions within an otherwise identical amphipathic sequence pattern. This study highlights the need to better understand the relationship between sequence and supramolecular structure to facilitate the development of functional peptide-based materials for biomaterials applications.

Many articles describe the use of enzymes to induce the formation of a supramolecular hydrogel. These enzymes catalyze the transformation of water-soluble precursors, often short peptides, into hydrogelators. The use of non-enzymatic proteins to induce or stabilize peptide self-assembly is a rarely reported phenomenon, which raises fundamental questions: how can a protein induce peptide self-assembly? How is the peptide recognized and how does it, or the peptide assembly, interact with the protein? The heptapeptide Fmoc-GFFYE-NH-(CH₂)₂-s-s-(CH₂)₂-NH-CO-(CH₂)₂-CO-EE-OH, called L-1 (L = natural chiral amino acids), is a water-soluble compound leading to an increasingly viscous solution over time due to the formation of nanofibers, but does not result in hydrogel (at least not within 3 months). When bovine serum albumin (BSA) is added to a freshly prepared solution of L-1, a hydrogel is obtained in less than 10 min. The variation in the L-1/BSA ratio has an impact on the gelation rate and the mechanical properties of the resulting hydrogel. Thus, the protein appears to act as (i) a catalyst and (ii) a cross-linking point. Strikingly, if the enantiomer D-1 (D = unnatural chiral amino acids) is used instead of L-1, the mixture with BSA remains liquid and non-viscous. Similar behavior is also observed for other proteins. Spectroscopic analyses (CD, fluorescence) and electronic microscopy images confirm that the L-1 peptide self-assembles in nanofibers of 10 nm diameter through β-sheet organization, which is not the case for the peptide D-1. A molecular dynamics study shows that BSA is capable of interacting with both enantiomer peptides L-1 and D-1. However, interaction with L-1 tends to unfold the peptide backbone, making the interaction with the protein more stable and promoting the assembly of L-1 peptides. Conversely, the interaction between BSA and D-1 is more dynamic and appears to be less spatially localized on the BSA. Furthermore, in this interaction, the D-1 peptide keeps its globular conformation. These results highlight the impact of a short peptide's chirality on protein-triggered supramolecular hydrogelation.

Lyotropic liquid crystal gels of phytantriol and monoolein are well known examples of self-assembled systems in water, which have multiple applications across biomedical and materials science. However aqueous systems can be restricted by rapid solvent evaporation, and the limited solubility of some species in water. Here we explore the formation of liquid crystalline phases of phytantriol and monoolein in mixtures of water with two protic ionic liquids, ethylammonium nitrate (EAN) and ethanolammonium nitrate (EtAN), and three deep eutectic solvents (DES) formed from mixtures of choline chloride with urea, fructose or citric acid. The structures of the gel phase in excess solvent were measured using small angle X-ray scattering for a fixed lipid concentration (5% w/w) as a function of temperature. The phase diagrams of both lipids in DES-water mixtures and the non-amphiphilic ionic liquid, EtAN, indicate that higher negative curvature inverse hexagonal structures are favoured by addition of water. However, the amphiphilic ionic liquid EAN swells and stabilises the cubic Pn3m structure. The interplay of solvent structure, polarity and molecular size are key to understanding the formation and stability of lyotropic liquid crystalline gels in these systems.

We present five automated descriptors: Aggregate Detection Index (ADI); Sheet Formation Index (SFI); Vesicle Formation Index (VFI); Tube Formation Index (TFI); and Fibre Formation Index (FFI), that have been designed for analysing peptide self-assembly in molecular dynamics simulations. These descriptors, implemented as Python modules, enhance analytical precision and enable the development of screening methods tailored to specific structural targets rather than general aggregation. Initially tested on the FF dipeptide, the descriptors were validated using a comprehensive dipeptide dataset. This approach facilitates the identification of promising self-assembling moieties with nanoscale properties directly linked to macroscale functions, such as hydrogel formation.

Modifying the salt form of active pharmaceutical ingredients is a common method to enhance their physicochemical and biological properties, whilst improving their ability to be formulated into medicines that can be effectively delivered to patients. Salts and counterions are especially relevant to peptide therapies, given that the majority of low molecular weight peptides synthesised by solid-phase protocols form a trifluoroacetate (TFA) salt due to the use of trifluoroacetic acid in resin cleaving and follow-on purification methods. TFA salts are not viewed as favourably by medicine regulators and can be defined as a new chemical entity entirely due to their different biological and physicochemical properties. Despite some exceptions, the vast majority of therapeutic peptides are marketed as hydrochloride (HCl) or acetate salts, even though most early research and development is centred on TFA salts. The aim of the study was to compare the impact of salt form (TFA vs. HCl) on the biostability, cell cytotoxicity, drug release and rheological properties of a Napffky(p)G-OH peptide hydrogel platform that demonstrates promise as a long-acting drug delivery system. This study demonstrated no significant difference between the salt forms for properties important to its intended use. This paper also raises important points for discussion relating to the environmental and regulatory status of peptide salts and their use as pharmaceuticals.

Molecular self-assembly enables the formation of intricate networks through non-covalent interactions, serving as a key strategy for constructing structures ranging from molecules to macroscopic forms. While zero-dimensional and one-dimensional nanostructures have been widely achieved, two-dimensional nanostrip structures present unique advantages in biomedical and other applications due to their high surface area and potential for functionalization. However, their efficient design and precise regulation remain challenging. This study systematically explores how different hydrophobic amino acid linkers impact the microscopic morphology in two-component co-assembly systems with strong electrostatic interactions. The introduction of the AA linker resulted in distinctive 2D nanostrips, which stacked to form bilayer sheets, whereas VV, LL, and NleNle linkers formed one-dimensional fibers. In contrast, GG and PP linkers did not produce stable aggregates. Our findings highlight the role of intermolecular interactions in the development of 2D assemblies, providing new insights into the design and application of 2D materials.

This work presents a strategy for generating composite hydrogels bearing photoconductive conduits held by supramolecular interactions that are compatible with digital light processing (DLP) printing. Conductive polymers are typically processed with organic solvents as the film, yet if used as biomaterials, excitable cells often require matching with the mechanical and structural properties of their native, aqueous three-dimensional (3-D) microenvironment. Here, we utilize peptide-functionalized porphyrin units capable of self-assembling into photoconductive nanostructures with defined nanomorphologies under aqueous conditions. In addition to the DXXD peptide arms (X = V, F), the sequence variants studied here include a peptidic moiety bearing allyloxycarbonyl (alloc) groups that can serve as crosslinking sites of the acrylate-based monomers that ultimately form the base 3-D covalent network for the hydrogels. We investigate the impact of pre-templating polymeric gelators with supramolecular assemblies vs. printing a dispersed peptide-porphyrin in a polymer composite, specifically, the potential impact of the morphologies of the supramolecular additives or "dopants" on the resulting mechanical property, conductivity, and printability of the hydrogels, comprised of a hybrid between acrylated polymers and supramolecular peptide-porphyrin assemblies. Lastly, we demonstrate the role of photophysical properties that emerge from peptide-tuned porphyrin assemblies as a photoabsorber additive that influences the printing outcomes of the composite hydrogel. Overall, we present a covalent-supramolecular composite hydrogelator system where the self-assembled networks offer a pathway for energy transport and mechanical reinforcement/dissipation at the same time, leading to the formation of a hydrogel with optoelectronic, mechanical, and printable behavior that can be influenced by self-assembled dopants.

While there have been great advances in the design and synthesis of supramolecular gels, their characterization methods have largely stayed the same, with electron microscopy of dried samples, or small-angle scattering and spectroscopy dominating the approaches used. Although these methods provide valuable insights into structural properties, they are unable to unambiguously generate reliable atomic models that can further guide the site-specific modification of supramolecular gelators. Cryogenic electron microscopy (cryo-EM), allowing the high-resolution imaging of the sample in a hydrated state, has emerged as the dominant technique in structural biology, but has yet to become a routine method in materials science. Here, we describe the use of cryo-EM to determine the atomic structure of the tubular micelle formed by the dipeptide CarbIF, revealing the mechanism of assembly and gelation. Using the CarbIF micelle as an example, we highlight some of the challenges in using cryo-EM to study such materials, and how determination of the helical symmetry can be the most difficult aspect of such a project.

Herein, we study the role that hydrophobicity plays in the temperature-dependent self-assembly of a family of β-hairpin peptide amphiphiles through the lens of thermally folding a protein from its cold-denatured state. This was facilitated by the development of new computational tools to measure solvent-accessible charge (SAC) and solvent-accessible hydrophobicity (SAH) at the resolution of atomic groups. Peptides in their disordered states are characterized by large SAH values that shift their thermal assembly transitions to observable temperatures, which is not possible for most native proteins, allowing comparisons amongst peptides to be made. We find that peptides with large SAH values assemble into β-sheet-rich fibers at lower temperatures and at faster rates than peptides having smaller SAH values. This is consistent with peptide assembly being driven by the hydrophobic effect, which involves the release of ordered water from hydrophobic moieties during assembly. We also find that peptide SAH values correlate linearly with T_g, the midpoint of the transition defining monomeric peptide transitioning to fibrils, for peptides of similar charge. Interestingly, the data also suggest that although entropy drives assembly, the exact temperature at which the assembly transition takes place is likely influenced by additional thermodynamic considerations.