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Start of funding 01.07.2019

Understanding the Natural Spinning Process of Spider Silk

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Natural spider silks reveal outstanding mechanical properties such as strength and extensibility resulting in a great toughness rivaling that of Kevlar and other man-made fibers. Due to limitations in natural availability, biomimetic alternatives are currently under investigation for several technical and medical applications. The natural process of spinning silk is highly controlled and involves changes in pH, salt, osmotic balance, and shear. One gap in knowledge is the conditions in the gland (i.e. the storage conditions of the spinning dope) and the role of silk protein pre-structuring in helping to achieve maximum toughness in the spun fibers. Our proposed work seeks to answer the question: How are the toughest fibers created? Therefore, we aim to produce recombinant spider silk proteins and investigate them at storage-mimicking conditions and in the final fiber using NMR. Understanding the structural properties of spider silk will help to produce artificial fibers for application in biomedical and technological approaches.

Final report:
Natural spider silks reveal outstanding mechanical properties such as strength and extensibility resulting in a great toughness rivaling that of Kevlar and other man-made fibers. Due to limitations in natural availability, biomimetic alternatives are currently under investigation for several technical and medical applications. The natural process of spinning silk is highly controlled and involves changes in pH, salt, osmotic balance, and shear.

Previously published work has shown that recombinant spider silk fibers spun from dopes with phosphate-induced pre-assembly (biomimetic dopes) display a toughness approaching native spider silks far exceeding the mechanical properties of fibers spun from dopes without pre-assembly (classical dopes). Dynamic light scattering experiments comparing the two dopes reveal that biomimetic dopes display a systematic increase in assembly size over time, while light microscopy indicates liquid-liquid phase separation (LLPS) as evidenced by the formation of micron scale liquid droplets.

LLPS is a phenomenon relevant in multi-component settings of many biological processes, including compartmentation, pathological conditions like Alzheimer’s disease, and protein assemblies. LLPS plays also a key role in spider silk fiber formation. A thorough understanding of the natural silk spinning process is thus vital to transfer it to artificial spinning techniques to achieve biomimetic fibers with superior properties outperforming other fibrous materials. Ex vivo studies and experiments utilizing recombinant spider silk proteins fortified in our project the relevance of LLPS during spider silk assembly. We provided a concerted, transitional concept for technical silk spinning [1].

Solution NMR showed that the structural state in classical and biomimetic dopes displays a general random coil conformation in both cases however, some subtle but, distinct differences are observed including a more ordered state for the biomimetic dope and small chemical shift perturbations indicating differences in hydrogen bonding of the protein in the different dopes with notable changes occurring for Tyr residues [2].

Solid-state NMR demonstrated that the final wet-spun fibers from the two dopes display no structural differences of the poly(Ala) stretches but, biomimetic fibers display a significant difference in Tyr ring packing in non-beta-sheet, disordered helical domains that can be traced back to differences in dope preparations. We concluded that phosphate pre-orders the recombinant silk protein in biomimetic dopes resulting in LLPS and fibers that exhibit vastly improved toughness that could be due to aromatic ring packing differences in non-beta-sheet domains that contain Tyr [2].

References of our joint work in PubMed- or Scopus- or similar listed journals:

[1] Zeußel L, Bargel, H., Holland GP., Scheibel T, Liquid-liquid Phase Separation of Spider Silk Proteins. Polymer J. 2025, Accepted 03.04.2025.

[2] Stengel D, Saric M, Johnson HR, Schiller T, Diehl J, Chalek K, Onofrei D, Scheibel T, Holland GP. Tyrosine's Unique Role in the Hierarchical Assembly of Recombinant Spider Silk Proteins: From Spinning Dope to Fibers. Biomacromolecules. 2023, 24:1463-1474. PubMed PMID: 36791420.