From Soluble Protein to Anchoring Filament: Understanding the Structural and Mechanical Foundations of Pyriform Spider Silk
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Spider silks are biomaterials used by spiders for many diverse adaptations, with toughness comparable to Kevlar and strength comparable to high strength steel. Orb-weaver spiders produce up to seven distinct classes of silk containing spider proteins (spidroins), with specific protein structural changes upon fibrillogenesis remaining unknown for most classes. The work detailed in this thesis focuses on the pyriform spidroin 1 (PySp1) from Argiope argentata. Based on the central pyriform silk repetitive domain of PySp1, I successfully engineered representative recombinant pyriform silk proteins. Silk fibres formed from a two-repeat-containing protein referred to as HPy2 showed a relatively high combination of strength and extensibility, in contrast to extremes of one mechanical property vs. the other as seen in most silks. This was the first reporting of recombinant pyriform silk mechanical properties. Refinement of these expression methods in M9 media increased yield, qualitatively increased purity, and increased extensibility in spun fibers. To understand the structure of this protein in the solution state and how it changes during the fibre-forming process, I performed atomic-level solution and fibre structural studies. My results show that the repetitive domain units of PySp1 are modular and contain a highly ordered, 6-helical bundle, including an internal bridging helix, and long disordered linkers at each terminus. While the solution state structure is devoid of β-sheets, recombinant pyriform silk fibres show evidence of a partial α-to-β transition and supramolecular structure. As a whole, this work provided a benchmark for recombinant pyriform silk mechanical properties, the protein solution-state properties, and some understanding of the structural changes and assembly upon fibre formation.