Spider Dragline Silk Research Articles

Strong Silkworm Silk Fibers through CNT-Feeding and Forced Reeling.

High-performance silk fibers, with their eco-friendly degradability and renewability, have long captivated researchers as an alternative to synthetic fibers. Spider dragline silk, renowned for its exceptional strength (>1 GPa), has an extremely low yield, hindering its widespread use. While domesticated silkworms (Bombyx mori) can produce silk fibers industrially, their moderate strength (≈0.5 GPa) pales in comparison to the formidable spider dragline silk. In this study, naturally produced strong silkworm silk fibers are reported with a tensile strength of ≈1.2 GPa achieved through combining feeding carbon nanotubes (CNTs) to silkworms and in situ forced reeling for alignment. Molecular dynamics simulations confirm the interaction between the CNTs and silk fibroin, while the forced reeling process aligns these reinforcing fillers and the silk fibroin β-sheet nanocrystals along the fiber axis. Structural analysis reveals a significant enhancement in the content and alignment of β-sheet nanocrystals within the silk fibers, accounting for their superior mechanical properties, including tensile strength of ≈1.2 GPa and Young's modulus of 24.4 GPa, surpassing various types of silkworm silk and spider silk. This advancement addresses the historical trade-off between the strength and scalability of silk, potentially paving the way for eco-friendly, biodegradable, and renewable alternatives to synthetic fibers in a variety of applications.

Super‐Strong, Super‐Stiff, and Super‐Tough Fluorescent Alginate Fibers with Outstanding Tolerance to Extreme Conditions

AbstractThe combination of multiple physical properties is of great importance for widening the application scenarios of biomaterials. It remains a great challenge to fabricate biomolecules‐based fibers gaining both mechanical strength and toughness which are comparable to natural spider dragline silks. Here, by mimicking the structure of dragline silks, a high‐performance fluorescent fiber Alg‐TPEA‐PEG is designed by non‐covalently cross‐linking the polysaccharide chains of alginate with AIEgen‐based surfactant molecules as the flexible contact points. The non‐covalent cross‐linking network provides sufficient energy‐dissipating slippage between polysaccharide chains, leading to Alg‐TPEA‐PEG with highly improved mechanical performances from the plastic strain stage. By successfully transferring the extraordinary mechanical performances of polysaccharide chains to macroscopic fibers, Alg‐TPEA‐PEG exhibits an outstanding breaking strength of 1.27 GPa, Young's modulus of 34.13 GPa, and toughness of 150.48 MJ m−3, which are comparable to those of dragline silk and outperforming other artificial materials. More importantly, both fluorescent and mechanical properties of Alg‐TPEA‐PEG can be well preserved under various harsh conditions, and the fluorescence and biocompatibility facilitate its biological and biomedical applications. This study affords a new biomimetic designing strategy for gaining super‐strong, super‐stiff, and super‐tough fluorescent biomaterials.

A phenomenological theory for hydration-induced supercontraction and twist of spider dragline silk

Spider dragline silk is one promising material for producing artificial muscles, owing to its remarkable capacity for supercontraction and twist when exposed to high humidity. Based on the hydration absorption equation and the standard reinforcing model, we develop a phenomenological theory for elucidating the hydration-induced supercontraction and twist of spider dragline silk. The theory can reasonably predict the responses of softening, anisotropy, hydration-supercontraction, and twist of spider dragline silk. The theoretical predictions align with the experimental results. This study provides valuable insight into the underlying mechanisms of the hydration-induced deformation of spider dragline silk.

Structure of Spider Silk Studied with Solid‐State NMR

AbstractSpider dragline silks exhibit remarkable mechanical properties, combining both high strength and toughness. These unique characteristics arise from the intricate structure of the silk, which requires atomic‐level information to understand its origins. 13C solid‐state NMR provides this detailed structural insight into spider dragline silk. In this review, 13C CP/MAS, 13C DD/MAS and 13C INEPT NMR spectroscopies are employed to reveal the structure of spider dragline silks together with 13C conformation‐dependent chemical shifts, 2D spin‐diffusion NMR, rotational echo double resonance, dipolar‐assisted rotational resonance, and angle‐dependent NMR. The primary structure of major ampullate of spider dragline silk consists of repeated polyalanine and a glycine‐rich regions. By analyzing the 13C conformation‐dependent chemical shifts and utilizing several solid‐state NMR techniques, it has been proposed that the glycine‐rich region primarily adopts a random coil conformation, including partially β‐sheet and β‐turn structures. This contradicts the previously suggested 31 helix conformation. On the other hand, the polyalanine region exhibits an antiparallel β‐sheet structure with staggered packing arrangements. Additionally, solid‐state NMR has also revealed the structure of fragelliform spider silk. These findings contribute to the understanding of the remarkable properties of spider dragline silks and provide insights into its atomic‐level architecture.

Structural conversion of the spidroin C-terminal domain during assembly of spider silk fibers

The major ampullate Spidroin 1 (MaSp1) is the main protein of the dragline spider silk. The C-terminal (CT) domain of MaSp1 is crucial for the self-assembly into fibers but the details of how it contributes to the fiber formation remain unsolved. Here we exploit the fact that the CT domain can form silk-like fibers by itself to gain knowledge about this transition. Structural investigations of fibers from recombinantly produced CT domain from E. australis MaSp1 reveal an α-helix to β-sheet transition upon fiber formation and highlight the helix No4 segment as most likely to initiate the structural conversion. This prediction is corroborated by the finding that a peptide corresponding to helix No4 has the ability of pH-induced conversion into β-sheets and self-assembly into nanofibrils. Our results provide structural information about the CT domain in fiber form and clues about its role in triggering the structural conversion of spidroins during fiber assembly.

Sequence-based data-constrained deep learning framework to predict spider dragline mechanical properties

Spider dragline silk is known for its exceptional strength and toughness; hence understanding the link between its primary sequence and mechanics is crucial. Here, we establish a deep-learning framework to clarify this link in dragline silk. The method utilizes sequence and mechanical property data of dragline spider silk as well as enriching descriptors such as residue-level mobility (B-factor) predictions. Our sequence representation captures the relative position, repetitiveness, as well as descriptors of amino acids that serve to physically enrich the model. We obtain high Pearson correlation coefficients (0.76–0.88) for strength, toughness, and other properties, which show that our B-factor based representation outperforms pure sequence-based models or models that use other descriptors. We prove the utility of our framework by identifying influential motifs and demonstrating how the B-factor serves to pinpoint potential mutations that improve strength and toughness, thereby establishing a validated, predictive, and interpretable sequence model for designing tailored biomaterials.

Superstrong and tough silk fibers cross-linked with functionalized graphene

Natural silkworm silks with an intriguing integration of good strength, modulus, toughness and biocompatibility, have been widely used in textile and medical industry. However, the strength of silkworm silks is inferior to that of spider dragline silks, which are the strongest natural silks but extremely low yield. Herein, we develop a feasible strategy to continuously produce superstrong and tough regenerated silk fibers (RSFs) through the reconstruction of hierarchical and cross-linked architectures. Amine functionalized graphene (NH2-G) has been introduced to improve the content of β-sheet and crystalline orientation, and glutaraldehyde (GA) has been used to bridge NH2-G and silk micro/nanofibrils (SNFs) to form a highly interconnected and closely packed network, resulting in the remarkably improvement of mechanical properties. The resultant fibers exhibit an excellent tensile strength of 1029.6 ± 144.8 MPa, a Young's modulus of 16.8 ± 1.5 GPa, and a toughness of 277.4 ± 17.0 MJ/m3, exceeding those of the natural silkworm silks, spider dragline silks and most other previously reported regenerated silk fibers. We believe that the high-performance silk-based fibers are promising applications in lightweight and advanced structural materials.

Modulating Polyalanine Motifs of Synthetic Spidroin for Controllable Preassembly and Strong Fiber Formation.

Spider dragline (major ampullate) silk is one of the toughest known fibers in nature and exhibits an excellent combination of high tensile strength and elasticity. Increasing evidence has indicated that preassembly plays a crucial role in facilitating the proper assembly of silk fibers by bridging the mesoscale gap between spidroin molecules and the final strong fibers. However, it remains challenging to control the preassembly of spidroins and investigate its influence on fiber structural and mechanical properties. In this study, we explored to bridge this gap by modulating the polyalanine (polyA) motifs in repetitive region of spidroins to tune their preassemblies in aqueous dope solutions. Three biomimetic silk proteins with varying numbers of alanine residues in polyA motif and comparable molecular weights were designed and biosynthesized, termed as N16C-5A, N15C-8A, and N13C-12A, respectively. It was found that all three proteins could form nanofibril assemblies in the concentrated aqueous dopes, but the size and structural stability of the fibrils were distinct from each other. The silk protein N15C-8A with 8 alanine residues in polyA motif allowed for the formation of stable nanofibril assemblies with a length of approximately 200 nm, which were not prone to disassemble or aggregate as that of N16C-5A and N13C-12A. More interestingly, the stable fibril assembly of N15C-8A enabled spinning of simultaneously strong (623.3 MPa) and tough (107.1 MJ m-3) synthetic fibers with fine molecular orientation and close interface packing of fibril bundles. This work highlights that modulation of polyA motifs is a feasible way to tune the morphology and stability of the spidroin preassemblies in dope solutions, thus controlling the structural and mechanical properties of the resulting fibers.

Dragline silk fibers from golden orb‐web spider Trichonephila clavata ensure structural and mechanical robustness against ultraviolet radiation

AbstractA diurnal spider, Trichonephila clavata (T. clavata), is often called a golden orb‐weaver because its dragline or lifeline silk fibers exhibit a golden color. However, little is known about the biological roles of the golden pigment. Meanwhile, Araneus ventricosus, which is a nocturnal spider, spins colorless silk fibers. Here, we investigated the effect of ultraviolet (UV) irradiation on physical properties of silk fibers obtained from these diurnal and nocturnal spiders. After UV exposure, the dragline silk of the nocturnal spiders appeared abraded and became insoluble due to gelation, whereas the diurnal spider silk remained intact. A dynamic mechanical thermal analysis under different humidity conditions revealed that the storage modulus of the silk from the nocturnal spider dropped markedly at low humidity levels after UV irradiation. This indicated that the molecular weight of this silk degraded under UV exposure and that the number of terminal amino and carboxyl groups increased due to the breakage of the main protein chains. In contrast, the structural and mechanical properties of the diurnal spider silk were maintained regardless of the UV irradiation. Therefore, the golden pigments in the dragline silk of T. clavata most likely played a role in protecting the silk from UV damage.

A review of the mechanics of dragline spider silk

Spider silks demonstrate extraordinary mechanical performance. They rely on an intricate hierarchical structure that gives rise to unique properties. Of the many types of spider silks that are produced, dragline spider silk has attracted the most research attention due its extremely high strength. Since data on dragline spider silk is readily available, much can be understood about the nature of spider silk by analyzing the structure of dragline spider silk. Moreover, the study of spider silk can inspire the design of new materials. Here, we review the structure of dragline silk, present a particular material model to explain their behavior, and discuss the potential outlook in the area.

A review of the mechanics of dragline spider silk

Spider silks demonstrate extraordinary mechanical performance. They rely on an intricate hierarchical structure that gives rise to unique properties. Of the many types of spider silks that are produced, dragline spider silk has attracted the most research attention due its extremely high strength. Since data on dragline spider silk is readily available, much can be understood about the nature of spider silk by analyzing the structure of dragline spider silk. Moreover, the study of spider silk can inspire the design of new materials. Here, we review the structure of dragline silk, present a particular material model to explain their behavior, and discuss the potential outlook in the area.

The role of flow in the self-assembly of dragline spider silk proteins

Hydrodynamic flow in the spider duct induces conformational changes in dragline spider silk proteins (spidroins) and drives their assembly, but the underlying physical mechanisms are still elusive. Here we address this challenging multiscale problem with a complementary strategy of atomistic and coarse-grained molecular dynamics simulations with uniform flow. The conformational changes at the molecular level were analyzed for single-tethered spider silk peptides. Uniform flow leads to coiled-to-stretch transitions and pushes alanine residues into β sheet and poly-proline II conformations. Coarse-grained simulations of the assembly process of multiple semi-flexible block copolymers using multi-particle collision dynamics reveal that the spidroins aggregate faster but into low-order assemblies when they are less extended. At medium-to-large peptide extensions (50%–80%), assembly slows down and becomes reversible with frequent association and dissociation events, whereas spidroin alignment increases and alanine repeats form ordered regions. Our work highlights the role of flow in guiding silk self-assembly into tough fibers by enhancing alignment and kinetic reversibility, a mechanism likely relevant also for other proteins whose function depends on hydrodynamic flow.

Highly strong and tough silk by feeding silkworms with rare earth ion-modified diets

Nature-derived silk fibers possess excellent biocompatibility, sustainability, and mechanical properties, yet producing strong and tough silk fibers in a facile and large-scale manner remains a significant challenge. Herein, we report a simple method for preparing strong and tough silk fibers by feeding silkworms rare earth ion-modified diets. The resulting silk fibers exhibit significantly increased tensile strength and toughness, with average values of 0.85 ± 0.07 GPa and 156 ± 13 MJ m−3, respectively, and maximum values of 0.97 ± 0.04 GPa and 188 ± 19 MJ m−3, approaching those of spider dragline silk. Our findings suggest that the incorporation of rare earth ions (La3+ or Eu3+) into the silk fibers contributes to this enhancement. Structure analysis reveals a reduction in content and an improvement in orientation of β-sheet nanocrystals in silk fibers. X-ray photoelectron spectroscopy analysis confirms the chemical interaction between rare earth ions with β-sheet nanocrystals. The structural evolution and chemical interactions lead to the simultaneous enhancement in both strength and toughness. This work presents a simple, scalable, and effective strategy for producing ultra-strong and tough silk fibers with potential applications in areas requiring super structural materials, such as personal protection and aerospace.

NMR assignment and dynamics of the dimeric form of soluble C-terminal domain major ampullate spidroin 2 from Latrodectus hesperus.

Spider dragline silk has attracted great interest due to its outstanding mechanical properties, which exceed those of man-made synthetic materials. Dragline silk, which is composed of at least major ampullate spider silk protein 1 and 2 (MaSp1 and MaSp2), contains a long repetitive domain flanked by N-terminal and C-terminal domains (NTD and CTD). Despite the small size of the CTD, this domain plays a crucial role as a molecular switch that regulates and directs spider silk self-assembly. In this study, we report the 1H, 13C, and 15N chemical shift assignments of the Latrodectus hesperus MaSp2 CTD in dimeric form at pH 7. Our solution NMR data demonstrated that this protein contains five helix regions connected by a flexible linker.

Spider dragline silk fibres maintain rectangular columnar liquid crystalline phase

ABSTRACT The liquid crystalline phase of spider silk is thought to enable for the silk protein to be stored at high concentration in the gland without gelation, facilitating the instant fibre formation at the spinning duct. Nevertheless, it has never been revealed whether the liquid crystalline state of the silk protein maintains after the fibre formation. In this study, we conducted the X-ray liquid crystal structure analysis to reveal the details of molecular assemblies of the native spider silk proteins. Based on the wide- and small angle X-ray scattering data using the uniaxial bundle of spider draglines, the spider dragline silk fibres maintain a rectangular columnar liquid crystalline phase with a P21/a (p2gg) symmetry. The liquid crystalline state of the spider silk fibre is not nematic, cholesteric, nor hexagonal columnar, as previously proposed, but rectangularly packed columnar liquid crystal. The movable silk molecular chains along the long fibre axis in the rectangular columnar liquid crystalline structure help to explain the mechanical strength of the native spider dragline silk fibres. The results obtained in this study will contribute to understanding the biological basis underlying the silk fibre formation and will be useful to the biomimicry of spider silk fibres.

C-Terminal Domain Controls Protein Quality and Secretion of Spider Silk in Tobacco Cells.

The remarkable mechanical strength and extensibility of spider dragline silk spidroins are attributed to the major ampullate silk proteins (MaSp). Although fragmented MaSp molecules have been extensively produced in various heterologous expression platforms for biotechnological applications, complete MaSp molecules are required to achieve instinctive spinning of spidroin fibers from aqueous solutions. Here, a plant cell-based expression platform for extracellular production of the entire MaSp2 protein is developed, which exhibits remarkable self-assembly properties to form spider silk nanofibrils. The engineered transgenic Bright-yellow 2 (BY-2) cell lines overexpressing recombinant secretory MaSp2 proteins yield 0.6-1.3 µg L-1 at 22 days post-inoculation, which is four times higher than those of cytosolic expressions. However, only 10-15% of these secretory MaSp2 proteins are discharged into the culture media. Surprisingly, expression of functional domain-truncated MaSp2 proteins lacking the C-terminal domain in transgenic BY-2 cells increases recombinant protein secretion incredibly, from 0.9 to 2.8mg L-1 per day within 7 days. These findings demonstrate significant improvement in the extracellular production of recombinant biopolymers such as spider silk spidroins using plant cells. In addition, the results reveal the regulatory roles of the C-terminal domain of MaSp2 proteins in controlling their protein quality and secretion.

Insights into the material properties of dragline spider silk affecting Schwann cell migration

Dragline silk of Trichonephila spiders has attracted attention in various applications. One of the most fascinating uses of dragline silk is in nerve regeneration as a luminal filling for nerve guidance conduits. In fact, conduits filled with spider silk can measure up to autologous nerve transplantation, but the reasons behind the success of silk fibers are not yet understood.In this study dragline fibers of Trichonephila edulis were sterilized with ethanol, UV radiation, and autoclaving and the resulting material properties were characterized with regard to the silk's suitability for nerve regeneration. Rat Schwann cells (rSCs) were seeded on these silks in vitro and their migration and proliferation were investigated as an indication for the fiber's ability to support the growth of nerves. It was found that rSCs migrate faster on ethanol treated fibers. To elucidate the reasons behind this behavior, the fiber's morphology, surface chemistry, secondary protein structure, crystallinity, and mechanical properties were studied. The results demonstrate that the synergy of dragline silk's stiffness and its composition has a crucial effect on the migration of rSCs. These findings pave the way towards understanding the response of SCs to silk fibers as well as the targeted production of synthetic alternatives for regenerative medicine applications.

Unusual pKa Values Mediate the Self-Assembly of Spider Dragline Silk Proteins.

Spider dragline silk is a remarkably tough biomaterial and composed primarily of spidroins MaSp1 and MaSp2. During fiber self-assembly, the spidroin N-terminal domains (NTDs) undergo rapid dimerization in response to a pH gradient. However, obtaining a detailed understanding of this mechanism has been hampered by a lack of direct evidence regarding the protonation states of key ionic residues. Here, we elucidated the solution structures of MaSp1 and MaSp2 NTDs from Trichonephila clavipes and determined the experimental pKa values of conserved residues involved in dimerization using NMR. Surprisingly, we found that the Asp40 located on an acidic cluster protonates at an unusually high pH (∼6.5-7.1), suggesting the first step in the pH response. Then, protonation of Glu119 and Glu79 follows, with pKas above their intrinsic values, contributing toward stable dimer formation. We propose that exploiting the atypical pKa values is a strategy to achieve tight spatiotemporal control of spider silk self-assembly.

Spider Silk as a Flexible Light Waveguide for Temperature Sensing

We propose an approach to utilize the natural spider dragline silk (SDS) as a flexible light waveguide integrated into an optic device. SDS is utilized as an air-clad biocompatible light waveguide in the fabricated multimode interferometer. Based on the evanescent field coupling theory, the light propagating in the microfiber can be coupled into SDS, and then coupled to another microfiber connected with a spectrometer. The difference in the effective refractive index of the modes propagating in the silk fiber changes in different external environments, thus the output multimode interference spectrum shifts with the change of environment. Accordingly, the multimode interferometer is encapsulated in polydimethylsiloxane (PDMS), a kind of flexible and temperature-sensitive material, which makes the interferometer have competitive temperature sensitivity. With the increase in temperature, the refractive index of PDMS decreases, and thus the output multimode interference spectrum blue shifts. The interferometer shows repeatability, reversibility and stability in temperature sensing, with a sensitivity of −1.15 nm/°C. The presented interferometer proves that the SDS is a natural independent light guide with good mechanical properties to be applied in the fabrication of integrated optic devices.

Silk-in-Silk Nerve Guidance Conduits Enhance Regeneration in a Rat Sciatic Nerve Injury Model.

Advanced nerve guidance conduits can provide an off-the-shelf alternative to autografts for the rehabilitation of segmental peripheral nerve injuries. In this study, the excellent processing ability of silk fibroin and the outstanding cell adhesion quality of spider dragline silk are combined to generate a silk-in-silk conduit for nerve repair. Fibroin-based silk conduits (SC) are characterized, and Schwann cells are seeded on the conduits and spider silk. Rat sciatic nerve (10mm) defects are treated with an autograft (A), an empty SC, or a SC filled with longitudinally aligned spider silk fibers (SSC) for 14weeks. Functional recovery, axonal re-growth, and re-myelination are assessed. The material characterizations determine a porous nature of the conduit. Schwann cells accept the conduit and spider silk as growth substrate. The in vivo results show a significantly faster functional regeneration of the A and SSC group compared to the SC group. In line with the functional results, the histomorphometrical analysis determines a comparable axon density of the A and SSC groups, which is significantly higher than the SC group. These findings demonstrate that the here introduced silk-in-silk nerve conduit achieves a similar regenerative performance as autografts largely due to the favorable guiding properties of spider dragline silk.