Unconventional Spidroin Assemblies in Aqueous Dope for Spinning into Tough Synthetic Fibers

Spider dragline silk is a remarkable fiber made by spiders from an aqueous solution of spidroins, and this feat is largely attributed to the tripartite domain architecture of the silk proteins leading to the hierarchical assembly at the nano- and microscales. Although individual amino- and carboxy-terminal domains have been proposed to relate to silk protein assembly, their tentative synergizing roles in recombinant spidroin storage and spinning into synthetic fibers remain elusive. Here,they show biosynthesis and self-assembly of a mimic spidroin composed of amino- and carboxy-terminal domains bracketing 16 consensus repeats of the core region from spider Trichonephila clavipes. The presence of both termini was found essential for self-assembly of the mimic spidroin termed N16C into fibril-like (rather than canonical micellar) nanostructures in concentrated aqueous dope and ordered alignment of these nanofibrils upon extrusion into an acidic coagulation bath. This ultimately led to continuous, macroscopic fibers with a tensile fracture toughness of 100.9 ± 13.2 MJ m–3, which is comparable to that of their natural counterparts. They also found that the recombinant proteins lacking one or both termini were unable to similarly preassemble into fibrillar nanostructures in dopes and thus yielded inferior fiber properties. This work thereby highlights the synergizing role of terminal domains in the storage and processing of recombinant analogues into tough synthetic fibers.

Spider dragline silk exhibits extraordinary toughness that combines high tensile strength and extensibility, thus outcompeting all other natural or synthetic fibers. The remarkable silk is spun by spiders from an aqueous solution of the constituent proteins, termed spidroins, and this feat is largely attributed to the underlying architecture of the spidroins enabling their ordered assembly into insoluble fibers.The two protein components, major ampullate spidroins 1 (MaSp1) and 2 (MaSp2),share a common tripartite architecture of a nonrepetitive N-terminal domain (NTD), a long repetitive core region, and a nonrepetitive C-terminal domain (CTD). The core region comprises up to 100 tandem repeats, each consisting of 40–200 amino acids with a high content of polyalanine- and glycine-rich motifs.In the process for spinning of soluble spidroins into fibers, the polyalanine motifs of the core region tend to convert into β-sheet crystallites, responsible for the strength of spider dragline silk, whereas the glycine-rich motifs form random coil/helical amorphous regions responsible for the extensibility of the fibers.

Schematic overview of the biomimetic design and recombinant biosynthesis of spidroins for fiber spinning. (a) Scheme of the recombinant spidroins. The amino acid sequences of the consensus repeat of the core domain and the amino- and carboxy-terminal domains (NTD and CTD) are derived from major ampullate spidroin 1 of spider T. clavipes. (b) Illustration of spidroin biosynthesis, purification, and concentration into dope solutions. (c) Coomassie-stained 10% SDS-PAGE gel analysis of the purified spidroins under reducing and nonreducing conditions. 2-Mercaptoethanol (a reducing agent) is omitted for protein sample preparation under the nonreducing condition, and thus, the cysteine-containing spidroins (16C and N16C) were predominantly oxidized into dimers (indicated by solid arrows), the monomers of which were indicated by dotted arrows.

The structure and function of the nonrepetitive terminal domains of spidroins have gained increasing attention in the last decade.Fundamental structural studies reveal that the highly conserved terminal domains, which are composed of approximately 100 amino acids, both show a five-helix bundle structure in solution. They have been proposed to play important roles in stabilizing the concentrated spidroins during storage in the silk gland and also triggering spidroin transformation into macroscopic fibers. Growth evidence from extensive studies shows that structural conversions of the terminal domains are smartly induced by external chemical and mechanical triggers that occur through the spinning duct of the spiders. For example, the correctly folded, dimeric state of CTD during storage is turned into a molten globulelike state upon acidification and ion exchange of sodium and chloride ions by potassium and phosphate ions within the spinning duct. This partially unfolded state of CTD further mediates the shear-induced alignment of repetitive segments into β-sheet structures. Meanwhile, acidification and depletion of sodium chloride induce conformational changes in NTD accompanied by antiparallel dimerization to lock the spidroin into large networks, whereas this domain exists in a monomeric state to inhibit premature aggregation of the spidroin during storage.Although much has been done to explore the function of terminal domains, their roles have been mostly discovered from studies on recombinant proteins containing individual terminal domains or their fusions to a short repeat region, markedly different from native spidroins with large molecular weights and the presence of both terminal domains.

Characterization of spidroins. (a) Far-UV CD spectra of various spidroins (0.2 mg mL–1) at pH 7.4. (b) Tryptophan fluorescence of the spidroins (2 mg mL–1). Before analyses, the spidroin dope solutions were diluted with buffers that contained 0.5 M urea, 40 mM NaCl, and 10 mM phosphate buffer at pH 7.4 or 5.0.

To make artificial spider silk fibers with the advantageous mechanical properties of their natural counterparts, it is highly desirable to synthesize recombinant spidroins with nativelike architecture and solubility for subsequent spinning into solid fibers.For example, Andersson et al. designed a synthetic spidroin composed of an NTD and a short repetitive region from spider Euprosthenops australis and a CTD from spider Araneus ventricosus (NT2RepCT). As a constitutive dimer with a molecular mass of 66 kDa, NT2RepCT could be concentrated to >500 mg mL–1 in 20 mM Tris–HCl buffer (pH 8.0) and spun into continuous fibers with a toughness of 45 ± 7 MJ m–3 when the concentrated dope was extruded into a collection bath consisting of 500 mM sodium acetate buffer and 200 mM NaCl (pH 5.0). However, the specific impacts of individual terminal domains on the minispidroin assembly and the mechanical properties of resulting fibers remain to be explored. In another study, a recombinant spidroin with 12 consensus sequences of a MaSp2 protein and both terminal domains was dialyzed against 30–50 mM sodium phosphate buffer (pH 7.2) to induce liquid–liquid phase separation (dependent on the CTD). The resulting high-density micellar phase serving as an aqueous dope could be wet-spun and poststretched in 90% isopropanol baths (pH 7.7) into fibers with a toughness of 189 ± 33 MJ m–3, which even slightly exceeds the toughness (on average) of natural fibers from A. diadematus spiders (167 ± 65 MJ m–3). However, the “locking” role of NTD was presumably unrealized because the NTD only dimerizes at acidic pH.To the best of our knowledge, the synergizing roles of terminal domains in the assembly of recombinant spidroin into synthetic fibers remain elusive.

Morphologies of various spidroins in dope and coagulation baths. (a) Representative liquid AFM images of spidroin microstructures in diluted dope at room temperature. (b) AFM images of spidroin aggregates upon coagulation in 90% ethanol at pH 7.4 or 5.0.

Here, they determine the synergizing roles of terminal domains in guiding a spidroin mimic to preassemble in concentrated aqueous solutions and to coagulate for fiber formation. Based on the major dragline silk (MaSp1) component of the golden orb-web spider Trichonephila clavipes (formerly Nephila clavipes), four spidroin variants were first designed to contain the same core region of 16 consensus repeats, yet differing in the presence of either one or both terminal domains. Next, these four variants were biosynthesized, characterized by a combination of spectroscopic and microscopic analyses, and spun into fibers in parallel. Strikingly, the biomimetic spidroin self-assembled into fibril-like nanostructures in concentrated aqueous dope (other than conventional micelles for processing into artificial fibers with native like toughness, whereas the variants lacking one or both termini were unable to similarly preassemble and yield tough synthetic fibers.

Processing of spidroin dopes into fibers. (a) Schematic illustration of microfluidic wet spinning and postspun stretching. The diagram does not follow the actual scale for an intuitive illustration of the microfluidic chip. Mechanical properties of the postdrawn fibers with coagulation at pH 5.0 or 7.4: (b) toughness, (c) extensibility, (d) strength, and (e) Young’s modulus.

Scanning electron micrographs of the postdrawn fibers. The four spidroins as indicated above the corresponding micrographs were spun into coagulation baths with pH at 5.0 (a, b) or 7.4 (c, d). (a, c) Analysis at breakpoint to examine the fiber interior core. (b, d) Analysis of the fiber surface, illustrating fibrillar structure. Scale bars, 1 μm.

  1. Unconventional Spidroin Assemblies in Aqueous Dope for Spinning into Tough Synthetic Fibers Chun-Fei Hu, Zhi-Gang Qian, Qingfa Peng, Yaopeng Zhang, and Xiao-Xia Xia ACS Biomaterials Science & Engineering 2021 7 (8), 3608-3617 DOI: 10.1021/acsbiomaterials.1c00492