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.

In this study, we report that host defense protein-derived ten amino acid long disulfide-linked peptides self-assemble in the form of β-sheets and β-turns, and exhibit concentration-dependent self-assembly in the form...

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 <i>Argiope argentata</i> PySp1. Solution-state nuclear magnetic resonance (NMR) spectroscopy-based backbone chemical shift and dynamics analysis demonstrates 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 <i>Argiope trifasciata</i> 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.

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,...

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.