Structural features, intrinsic disorder, and modularity of a pyriform spidroin 1 core repetitive domain.

Orb-weaving spiders produce up to seven silk types, each with distinct biological roles, protein compositions, and mechanics. Pyriform (or piriform) silk is composed of pyriform spidroin 1 (PySp1) and is the fibrillar component of attachment discs that attach webs to substrates and to each other. Here, we characterize the 234-residue repeat unit (the "Py unit") from the core repetitive domain of Argiope argentata PySp1. Solution-state nuclear magnetic resonance (NMR) spectroscopy-based backbone chemical shift and dynamics analysis demonstrate a structured core flanked by disordered tails, structuring that is maintained in a tandem protein of two connected Py units, indicative of structural modularity of the Py unit in the context of the repetitive domain. Notably, AlphaFold2 predicts the Py unit structure with low confidence, echoing low confidence and poor agreement to the NMR-derived structure for the Argiope trifasciata aciniform spidroin (AcSp1) repeat unit. Rational truncation, validated through NMR spectroscopy, provided a 144-residue construct retaining the Py unit core fold, enabling near-complete backbone and side chain 1H, 13C, and 15N resonance assignment. A six α-helix globular core is inferred, flanked by regions of intrinsic disorder that would link helical bundles in tandem repeat proteins in a beads-on-a-string architecture.

Antibacterial activities of physiologically stable, self-assembled peptide nanoparticles.

In this study, we report that host defense protein-derived ten amino acid long disulfide-linked peptides self-assemble in the form of ß-sheets and ß-turns, and exhibit concentration-dependent self-assembly in the form of nanospheres, termed as disulfide linked nanospheres (DSNs). As expected, bare DSNs are prone to aggregation in ionic solutions and in the presence of serum proteins. To yield physiologically stable self-assembled peptide-based materials, DSNs are stabilized in the form of supramolecular assemblies using ß-cyclodextrins (ß-CD) and fucoidan, as delivery carriers. The inclusion complexes of DSNs with ß-CD (ß-CD-DSN) and electrostatic complexation of fucoidan with DSNs (FC-DSN) stabilizes the secondary structure of DSNs. Comparison of ß-CD-DSNs with FC-DSNs reveals that inclusion complexes of DSNs formed in the presence of ß-CD are highly stable under physiological conditions, show high cellular uptake, exhibit bacterial flocculation, and enhance antibacterial efficacies of DSNs in a range of Gram-positive and Gram-negative bacteria.

Material properties of disulfide-crosslinked hyaluronic acid hydrogels influence prostate cancer cell growth and metabolism.

Cells reside in vivo within three dimensional environments in which they interact with extracellular matrices (ECMs) that play an integral role in maintaining tissue homeostasis and preventing tumour growth. Thus, tissue culture approaches that more faithfully reproduce these interactions with the ECM are needed to study cancer development and progression. Many materials exist for modeling tissue environments, and the effects of differing mechanical, physical, and biochemical properties of such materials on cell behaviour are often intricately coupled and difficult to tease apart. Here, an optimized protocol was developed to generate low reaction volume disulfide-crosslinked hyaluronic acid (HA) hydrogels for use in cell culture applications to relate the properties of ECM materials to cell signalling and behaviour. Mechanically, HA hydrogels are comparable to other soft hydrogel materials such as Matrigel and agarose or to tissues lacking type I collagen and other fibrillar ECM components. The diffusion of soluble materials in these hydrogels is affected by unique mass transfer properties. Specifically, HA hydrogel concentration affects the diffusion of anionic particles above 500 kDa, whereas diffusion of smaller particles appears unimpeded by HA content, likely reflecting hydrogel pore size. The HA hydrogels have a strong exclusion effect that limits the movement of proteins into and out of the material once fully formed. Such mass transfer properties have interesting implications for cell culture, as they ultimately affect access to nutrients and the distribution of signalling molecules, affecting nutrient sensing and metabolic activity. The use of disulfide-crosslinked HA hydrogels for the culture of the model prostate cancer cell lines PC3 and LNCaP reveals correlations of protein activation linked to metabolic flux, which parallel and can thus potentially provide insights into cell survival mechanisms in response to starvation that occurs in cancer cell microenvironments.

Simultaneous Ligand and Receptor Tracking through NMR Spectroscopy Enabled by Distinct 19F Labels.

To probe ligand-receptor binding at the atomic-level, a frequent approach involves multidimensional nuclear magnetic resonance (NMR) spectroscopy experiments relying on 13C- and/or 15N-enrichment alongside 1H. Alternatively, the lack of fluorine in biomolecules may be exploited through specific incorporation of 19F nuclei into a sample. The 19F nucleus is highly sensitive to environmental changes and allows for one-dimensional NMR spectroscopic study, with perturbation to chemical shift and spin dynamics diagnostic of structural change, ligand binding, and modified conformational sampling. This was applied to the apelinergic system, which comprises a rhodopsin-like G protein-coupled receptor (the apelin receptor (AR)/APJ) and two families of cognate ligands, the apelin and apela (ELABELA/toddler) peptides. Specifically, AR fragments consisting of either the N-terminal tail and first transmembrane (TM) α-helix (AR55) or the first three transmembrane α-helices (TM1-3) were prepared with biosynthetic fluorotryptophan incorporation. Interactions of each AR fragment with a high-affinity, 2,4,5-trifluorophenylalanine labeled apelin analogue were compared by 19F NMR. Distinct ranges of 19F chemical shifts for ligand and receptor provide unambiguous tracking of both species, with distinct binding behaviour observed for each AR fragment implying that AR55 is not sufficient to recapitulate the physiological binding event. Site-specific perturbation was also apparent for the apelin analogue as a function of substitution site, indicating an orientational binding preference. As a whole, this strategy of distinctive 19F labelling for ligand and receptor provides a relatively fast (i.e., employing 1D NMR experiments) and highly sensitive method to simultaneously and definitively track binding in both species.

Recombinant Pyriform Silk Fiber Mechanics Are Modulated by Wet-Spinning Conditions.

Pyriform silk is a critically important fiber in spider web construction. It functions in a glue-coated attachment disc to form junctions in spider webs, connecting the web to disparate materials. Despite the biological importance of this silk, both natural and recombinant pyriform silk have yet to be structurally or mechanically characterized. In this study, we demonstrate recombinant production of a 477 amino acid protein based on Argiope argentata pyriform silk. This pyriform silk protein shows α-helicity in both an aqueous buffer and in a fluorinated acid- and alcohol-based spinning dope. Wet-spinning produced fibers having no visible defects in surface or cross-sectional analysis, with mechanical behavior varying as a function of postspin stretching conditions and correlating to loss of α-helicity in the fibrous state. Multiple conditions gave rise to fibers that are both strong and extensible, contrasting with some other silks that are biased toward being strong or extensible. This behavior is strikingly similar to recombinant aciniform silk, despite distinct primary structuring and composition.