Spider wrapping silk fibre architecture arising from its modular soluble protein precursor

Abstract

Spiders store spidroins in their silk glands as high concentration aqueous solutions, spinning these dopes into fibres with outstanding mechanical properties. Aciniform (or wrapping) silk is the toughest spider silk and is devoid of the short amino acid sequence motifs characteristic of the other spidroins. Using solution-state NMR spectroscopy, we demonstrate that the 200 amino acid Argiope trifasciata AcSp1 repeat unit contrasts with previously characterized spidroins, adopting a globular 5-helix bundle flanked by intrinsically disordered N- and C-terminal tails. Split-intein-mediated segmental NMR-active isotope-enrichment allowed unambiguous demonstration of modular and malleable “beads-on-a-string” concatemeric behaviour. Concatemers form fibres upon manual drawing with silk-like morphology and mechanical properties, alongside secondary structuring and orientation consistent with native AcSp1 fibres. AcSp1 structural stability varies locally, with the fifth helix denaturing most readily. The structural transition of aciniform spidroin from a mostly α-helical dope to a mixed α-helix/β-sheet-containing fibre can be directly related to spidroin architecture and stability.

Characterizing Aciniform Silk Repetitive Domain Backbone Dynamics and Hydrodynamic Modularity

“…During fibre formation, AcSp1 undergoes a partial conversion from α-helical to β-sheet structuring [21], putatively seeded at helix 5 in the W unit [19,22]. This transition is recapitulated in recombinant W 2 between the soluble and fibrous forms [19].…”

“…In our previous structural studies [19], we performed segmental-labelling using split intein trans -splicing [49,50], whereby either the first (W 2-1 ) or second (W 2-2 ) W unit in W 2 was enriched with NMR-active 13 C and/or 15 N nuclei while the other W unit was at natural abundance. Although we demonstrated differential dynamics between the globular core and linker/tail regions through variation in the observed heteronuclear [ 1 H]- 15 N NOE, a more in-depth analysis of backbone dynamics is necessary to compare and contrast the behaviour of isolated W units vs. concatemers and to provide insight into more subtle variations in dynamics within the W unit.…”

Identification of Wet-Spinning and Post-Spin Stretching Methods Amenable to Recombinant Spider Aciniform Silk

“…We have previously expressed recombinant AcSp1-derived constructs with varying numbers of W units (nomenclature: W n , n= number of repeats) in Escherichia coli 36 and demonstrated that W 2 , W 3 , and W 4 constructs can be hand-pulled from low-concentration buffered aqueous solutions to form multi-centimeter fibers 32,36 . However, lowconcentration solutions are inefficient for high-yield fiber production and we have found that increasing W n concentration in aqueous solution promotes non-productive aggregation (results not shown).…”

“…AcSp1 undergoes a conceptually similar structural transition, but this results in a highly distinct fibrous structuring. In solution, AcSp1 from A. trifasciata is composed of compactly structured ~138 residue globular helical/turn containing domains connected together by 62 residue intrinsically disordered linkers 32 . Upon fiber formation, the protein retains a similar proportion of disorder and turn content alongside a mixture of moderately oriented β-sheet (~30%) and α-helical (~24%) domains 33,34 .…”

Development of New Smart Materials and Spinning Systems Inspired by Natural Silks and Their Applications

“…Acriniform silk is also used to form the soft inner layer of egg case. Tremblay et al (2015) found that the high toughness of aciniform silk is related to the primary sequence of spidroin and the distinctive mixture of α-helical, β-sheet, and non-canonical secondary structures. We expect that well understanding of the hierarchical architecture about these silks will inspire to the designs and development of advanced materials.…”