Spider Silk Research Articles

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.

Auditory Neurons on the Silk Road – spider silk for bridging the nerve-electrode-gap

Abstract The cochlear implant (CI) restores hearing to patients with severe to profound sensorineural hearing loss by stimulating the spiral ganglion neurons (SGN). Due to the inner ear anatomy, a fluid-filled gap remains between the SGN and the electrode array. This gap impedes focused stimulation and thus optimized performance with the CI. Regenerating neurites may bridge this fluid-filled gap for a direct nerveelectrode- link but require a supportive matrix. Spider silk might be a suitable candidate for this support. Therefore, silk fibers were tested for biocompatibility with inner ear neuronal tissue, suitability as neurite outgrowth matrix and applicability to the CI-electrode array. Dragline silk from female Trichonephila spiders was woven around a frame and autoclaved. Spiral ganglia of young rats were prepared, cut and placed in a drop of medium on the parallel silk fibers for pre-cultivation to support adherence before remaining medium was added. After 5d of cultivation, cells were fixed and immunocytologically stained. For CIapplication, silk was wrapped around a silicone dummy and a one-sided spacer. Opposite the spacer, alginate-hydrogel was applied for silk-fixation and -shielding. After gelation, silk loops were cut in the middle to form protruding threads. Cells showed a high degree of migration and neurite regeneration along the silk fibers and in some cases the outgrown neurites directly contacted the silk. It was possible to fix the silk to the dummy as protruding fibers. The tested spider silk is compatible with the inner ear tissue in culture, supports cell growth and seems to be an attractive material for neurite contacting. Application to the CI as protruding fibers is possible, making it a promising candidate for structural support to bridge the nerve-electrode-gap.

Inspired by spider silk: Ultra-tough and exceptionally stretchable coating

Inspired by spider silk: Ultra-tough and exceptionally stretchable coating

Natural spider silk nanofibrils produced by assembling molecules or disassembling fibers.

Spider silk is biocompatible, biodegradable, and rivals some of the best synthetic materials in terms of strength and toughness. Despite extensive research, comprehensive experimental evidence of the formation and morphology of its internal structure is still limited and controversially discussed. Here, we report the complete mechanical decomposition of natural silk fibers from the golden silk orb-weaver Trichonephila clavipes into ≈10 nm-diameter nanofibrils, the material's apparent fundamental building blocks. Furthermore, we produced nanofibrils of virtually identical morphology by triggering an intrinsic self-assembly mechanism of the silk proteins. Independent physico-chemical fibrillation triggers were revealed, enabling fiber assembly from stored precursors "at-will". This knowledge furthers the understanding of this exceptional material's fundamentals, and ultimately, leads toward the realization of silk-based high-performance materials. STATEMENT OF SIGNIFICANCE: Spider silk is one of the strongest and toughest biomaterials, rivaling the best man-made materials. The origins of these traits are still under debate but are mostly attributed to the material's intriguing hierarchical structure. Here we fully disassembled spider silk into 10 nm-diameter nanofibrils for the first time and showed that nanofibrils of the same appearance can be produced via molecular self-assembly of spider silk proteins under certain conditions. This shows that nanofibrils are the key structural elements in silk and leads toward the production of high-performance future materials inspired by spider silk.

Engineering SilkCatcher/Tag pairs for production of native-sized spider silk proteins

Engineering SilkCatcher/Tag pairs for production of native-sized spider silk proteins

Factors Influencing Properties of Spider Silk Coatings and Their Interactions within a Biological Environment.

Biomaterials are an indispensable part of biomedical research. However, although many materials display suitable application-specific properties, they provide only poor biocompatibility when implanted into a human/animal body leading to inflammation and rejection reactions. Coatings made of spider silk proteins are promising alternatives for various applications since they are biocompatible, non-toxic and anti-inflammatory. Nevertheless, the biological response toward a spider silk coating cannot be generalized. The properties of spider silk coatings are influenced by many factors, including silk source, solvent, the substrate to be coated, pre- and post-treatments and the processing technique. All these factors consequently affect the biological response of the environment and the putative application of the appropriate silk coating. Here, we summarize recently identified factors to be considered before spider silk processing as well as physicochemical characterization methods. Furthermore, we highlight important results of biological evaluations to emphasize the importance of adjustability and adaption to a specific application. Finally, we provide an experimental matrix of parameters to be considered for a specific application and a guided biological response as exemplarily tested with two different fibroblast cell lines.

Spider‐Silk‐Inspired Heterogeneous Supramolecular Network with Strain‐Stiffening, High Damping Capacity, and Supercontraction

AbstractNatural materials are adaptive with intriguing mechanical properties such as strain‐stiffening, high damping, and stimuli‐responsive actuation under ambient conditions, which are evolutionarily derived to adapt to variable environments. Such adaptabilities are highly desirable for advanced smart materials in soft robots and bionic applications yet rarely achieved all in one synthetic material. Inspired by the molecular chemistry and structure of spider silk, a structurally heterogeneous supramolecular network with all‐round adaptabilities is developed by evaporation‐induced self‐assembly of hydrogen‐bonded macromolecules. The supramolecular network consists of both hard (crystallites) and soft (amorphous) phases with dynamic hydrogen bonds, exhibiting strong strain, time, and hydration dependency with adaptive mechanical and structural responses to varying strain, deformation rate as well as moisture. Through multifunctional crosslinking, the network exhibits silk‐like attributes with an extensibility of >130%, intense strain‐stiffening (sixfold modulus enhancement), high damping capacity (>80%) over a wide range of strain rate, and moisture‐triggered large supercontraction (contraction ratio of >50%). Artificial materials with such combined adaptiveness under bio‐benign conditions are promising for applications in biomimetic and biomedical fields.

Self-Assembly of RGD-Functionalized Recombinant Spider Silk Protein into Microspheres in Physiological Buffer and in the Presence of Hyaluronic Acid.

Biomaterials made of self-assembling protein building blocks are widely explored for biomedical applications, for example, as drug carriers, tissue engineering scaffolds, and functionalized coatings. It has previously been shown that a recombinant spider silk protein functionalized with a cell binding motif from fibronectin, FN-4RepCT (FN-silk), self-assembles into fibrillar structures at interfaces, i.e., membranes, fibers, or foams at liquid/air interfaces, and fibrillar coatings at liquid/solid interfaces. Recently, we observed that FN-silk also assembles into microspheres in the bulk of a physiological buffer (PBS) solution. Herein, we investigate the self-assembly process of FN-silk into microspheres in the bulk and how its progression is affected by the presence of hyaluronic acid (HA), both in solution and in a cross-linked HA hydrogel. Moreover, we characterize the size, morphology, mesostructure, and protein secondary structure of the FN-silk microspheres prepared in PBS and HA. Finally, we examine how the FN-silk microspheres can be used to mediate cell adhesion and spreading of human mesenchymal stem cells (hMSCs) during cell culture. These investigations contribute to our fundamental understanding of the self-assembly of silk protein into materials and demonstrate the use of silk microspheres as additives for cell culture applications.

High-strength, self-reinforcing and recyclable multifunctional lignin-based polyurethanes based on multi-level dynamic cross-linking

The preparation of lignin-based polyurethanes with excellent mechanical properties and recyclability remains extremely challenging because of the poor compatibility of lignin and matrix. Inspired by the multiple structures of spider silk and pearl layers, high-strength, heat-resistant and recyclable lignin-based polyurethanes (LPUs) were prepared by introducing weak hydrogen bonds, strong hydrogen bonds and dynamic silyl ether covalent bonds. Multiple hydrogen bonds provided a large energy dissipation mechanism. The covalent linkage between lignin and matrix promoted their compatibility and formed a robust cross-linking network. Depending on the multi-level dynamic interactions, the prepared elastomer exhibited a mechanical strength of 73 MPa, which was 1.9 times higher than that of the blank sample, with an initial thermal decomposition temperature of 310.5 °C and a good recyclability. Especially, the mechanical strength of LPUs was up to 99 MPa after mechanical training. In addition, the abundant conjugated structure of lignin endowed LPUs with an excellent photothermal conversion ability. Under the irradiation of NIR light with 4000 w m−2, the surface temperature increased to 95 °C within two minutes, exhibiting a stable photothermal performance even after 6 cycles. This work showed the application prospects of LPUs in the fields of high strength engineering materials and photo-induced actuators.

Pulling and analyzing silk fibers from aqueous solution using a robotic device

Spiders, silkworms, and many other animals can spin silk with exceptional properties. However, artificially spun fibers often fall short of their natural counterparts partly due sub-optimal production methods. A variety of methods, such as wet-, dry-, and biomimetic spinning have been used. The methods are based on extrusion, whereas natural spinning also involves pulling. Another shortcoming is that there is a lack feedback control during extension. Here we demonstrate a robotic fiber pulling device that enables controlled pulling of silk fibers and in situ measurement of extensional forces during the pulling and tensile testing of the pulled fibers. The pulling device was used to study two types of silk—one recombinant spider silk (a structural variant of ADF3) and one regenerated silk fibroin. Also, dextran—a branched polysaccharide—was used as a reference material for the procedure due to its straightforward preparation and storage. No post-treatments were applied. The pulled regenerated silk fibroin fibers achieved high tensile strength in comparison to similar extrusion-based methods. The mechanical properties of the recombinant spider silk fibers seemed to be affected by the liquid-liquid phase separation of the silk proteins.

Special fog harvesting mode on bioinspired hydrophilic dual-thread spider silk fiber

Normal bioinspired spider silk fibers (BSSFs) show effective fog collection abilities but are weak in the rapid transport of captured droplets, which could potentially hinder the further improvement of fog harvesting efficiency. Factually, previous works have shown that the main axis of natural spider silk consists of two separate, parallel threads, similar to the microchannel structure of Sarracenia trichome. This structural feature may be an important factor contributing to the highly efficient fog harvesting ability of spider silks. However, to the best of our knowledge, previous studies on BSSFs have ignored this feature. In this work, inspired by spider silk and Sarracenia trichome, a novel bioinspired hydrophilic dual-thread spider silk fiber (HDSSF) is obtained via a simple dipping coating method to introduce capillary force. On the HDSSF, compared to previous works, a totally different fog harvesting mode was observed, i.e., captured droplets could be rapidly transported to spindle knots under the effect of capillary force and internal Laplace pressure difference via the connectivity of liquid film and no obvious droplets could be observed on the threads. The experimental observations and underlying mechanism analyses have verified that this special fog harvesting mode allows rapid transport and directional collection of droplets to accelerate the surface reconstruction rate, coupled with the high droplet capture ability of fiber, resulting in a significant improvement of fog harvesting efficiency (9.03 g cm−2·h−1, a 590% increase compared with normal BSSF). Such HDSSF is potentially applicable in high-efficiency fog harvesting systems, droplet transport devices and other engineering applications related to fog harvesting.

Wet spinning is employed to produce spider silk with high elasticity

Spider silk exhibits exceptional strength and elasticity in its natural form. Over the course of several decades, researchers have been working on artificially spinning recombinant spider silk proteins (spidroin) in order to replicate the remarkable mechanical properties of natural spider silk. In this study, we utilized the wet spinning method to investigate the relationship between the concentration of the coagulation bath and fiber performance. We discovered that the concentration of methanol plays a crucial role in determining the continuity, diameter, and mechanical properties of the fibers. Lower concentrations of methanol favor the production of continuous, thinner fibers with higher strain. Additionally, secondary stretching during the spinning process contributes to the production of silk fibers with stable mechanical properties and thermal stability. By employing different concentrations of methanol and applying additional stretching, we successfully produced silk fibers with a high strain of 2.1652 ± 0.3871 mm/mm. Furthermore, these wet-spun fibers demonstrated the ability to promote the growth of Schwann cells, indicating their potential application in the field of biomedical engineering. Hence, the exceptional mechanical properties and the ability to promote cellular growth make the obtained spider silk fibers highly promising for various biomedical applications.

Insights into surface chemistry down to nanoscale: An accessible colour hyperspectral imaging approach for scanning electron microscopy

Chemical imaging (CI) is the spatial identification of molecular chemical composition and is critical to characterising the (in-) homogeneity of functional material surfaces. Nanoscale CI on bulk functional material surfaces is a longstanding challenge in materials science and is addressed here.We demonstrate the feasibility of surface sensitive CI in the scanning electron microscope (SEM) using colour enriched secondary electron hyperspectral imaging (CSEHI). CSEHI is a new concept in the SEM, where secondary electron emissions in up to three energy ranges are assigned to RGB (red, green, blue) image colour channels. The energy ranges are applied to a hyperspectral image volume which is collected in as little as 50 s. The energy ranges can be defined manually or automatically.Manual application requires additional information from the user as first explained and demonstrated for a lithium metal anode (LMA) material, followed by manual CSEHI for a range of materials from art history to zoology.We introduce automated CSEHI, eliminating the need for additional user information, by finding energy ranges using a non-negative matrix factorization (NNMF) based method. Automated CSEHI is evaluated threefold: (1) benchmarking to manual CSEHI on LMA; (2) tracking controlled changes to LMA surfaces; (3) comparing automated CSEHI and manual CI results published in the past to reveal nanostructures in peacock feather and spider silk. Based on the evaluation, CSEHI is well placed to differentiate/track several lithium compounds formed through a solution reaction mechanism on a LMA surface (eg. lithium carbonate, lithium hydroxide and lithium nitride). CSEHI was used to provide insights into the surface chemical distribution on the surface of samples from art history (mineral phases) to zoology (di-sulphide bridge localisation) that are hidden from existing surface analysis techniques. Hence, the CSEHI approach has the potential to impact the way materials are analysed for scientific and industrial purposes.

A Density Functional Theory (DFT) Perspective on Optical Absorption of Modified Graphene Interacting with the Main Amino Acids of Spider Silk.

We investigated the possible adsorption of each of the main building blocks of spider silk: alanine, glycine, leucine, and proline. This knowledge could help develop new biocompatible materials and favors the creation of new biosensors. We used ab initio density functional theory methods to study the variations in the optical absorption, reflectivity, and band structure of a modified graphene surface interacting with these four molecules. Four modification cases were considered: graphene with vacancies at 5.55% and fluorine, nitrogen, or oxygen doping, also at 5.55%. We found that, among the cases considered, graphene with vacancies is the best candidate to develop optical biosensors to detect C=O amide and differentiate glycine and leucine from alanine and proline in the visible spectrum region. Finally, from the projected density of states, the main changes occur at deep energies. Thus, all modified graphene's electronic energy band structure undergoes only tiny changes when interacting with amino acids.

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.

Disabling spidroin N-terminal homologs' reverse reaction unveils why its intermolecular disulfide bonds have not evolved for 380 million years

Spiders, ubiquitous predators known for their powerful silks, rely on spidroins that self-assemble from high-concentration solutions stored in silk glands, which are mediated by the NT and CT domains. CT homodimers containing intermolecular disulfide bonds enhance silk performance, promoting spider survival and reproduction. However, no NT capable of forming such disulfide bonds has been identified. Our study reveals that NT homodimers with sulfur substitution can form under alkaline conditions, shedding light on why spiders have not evolved intermolecular disulfide bonds in the NT module during their 380 million years of evolution. This discovery significantly advances our comprehension of spider evolution and silk spinning mechanisms, while also providing novel insights into protein storage, assembly, as well as the mechanisms and therapeutic strategies for neurodegenerative diseases associated with protein aggregation.

Suitable electrospinning approaches for recombinant spider silk proteins

Among the versatile opportunities of processing recombinant spider silk proteins into coatings, hydrogels, particles, fibrils and foams, electrospinning provides a further highly important morphology: submicron- and nanofibers. These fibers display a high surface-to-volume ratio, which, in combination with the silk's biocompatibility, flexibility and functionality allows various applications including drug-delivery, tissue engineering, wound dressings as well as particle filtration from liquid or gas. Based on the chosen application, individual spider silk proteins can be spun out of a choice of solvents using different spinning techniques in order to achieve the desired properties of the fibers. In this perspective article so far used electrospinning solvents, techniques and collectors are displayed and brought into context with their possible application.

Global analysis of kinetics reveals the role of secondary nucleation in recombinant spider silk self-assembly.

Recombinant spider silk proteins can be prepared in scalable fermentation processes and have been proven as sources of biomaterials for biomedical and technical applications. Nanofibrils, formed through the self-assembly of these proteins, possess unique structural and mechanical properties, serving as fundamental building blocks for the fabrication of micro- and nanostructured scaffolds. Despite significant progress in utilizing nanofibrils-based morphologies of recombinant spider silk proteins, a comprehensive understanding of the molecular mechanisms of nanofibrils self-assembly remains a challenge. Here, a detailed kinetic study of nanofibril formation from a recombinant spider silk protein eADF4(C16) in dependence on the protein concentration, seeding and temperature is provided. For the global fitting of kinetic data obtained during the fibril formation, we utilized the online platform AmyloFit. Evaluation of the data revealed that the self-assembly mechanism of recombinant spider silk is dominated by secondary nucleation. Thermodynamic analyses show that both primary and secondary nucleations, as well as the elongation step of the eADF4(C16), are endothermic processes. This article is protected by copyright. All rights reserved.

A high-strength bonding, water-resistance, flame-retardant magnesium oxychloride cement based inorganic adhesive via the construction of supramolecular system

Magnesium oxychloride cement (MOC) adhesive has the advantages of being non-formaldehyde and flame retardant and having low energy consumption; however, its poor compatibility with the wood interface results in low bonding strength. Inspired by the H-bonded β-sheets nanoconfinement phase of spider silk, a high-performance inorganic adhesive was developed based on the supramolecular network system of poly (vinyl alcohol)/phytic acid (PVA/PA) with dissolved ions and the synchronous hydration process of MOC. Benefiting from the H-bond crosslinking and chelation confinement effects, the addition of PVA/PA enables MOC to penetrate steadily into the wood. Under this strategy, the compressive strength and softening coefficient of the modified MOC adhesive were 65.07 MPa and 0.84, which were 17.03% and 425% higher than the unmodified MOC adhesive, respectively. In addition, the MOC/PVA/PA adhesive achieved a wet shear strength of 2.02 MPa, 68.3% higher than that of the MOC adhesive. These results demonstrate that the MOC/PVA/PA adhesive would be a promising inorganic adhesive in wood-based building materials.

Microstructure similarity analysis between synthetic phase-separated block copolymers and natural spider silk

This papers primary purpose is to explore why the microstructure of synthetic block copolymers and natural spider silk is similar at the nanoscale. This paper analyses the main chain segments and secondary structures of natural spider silk, clarifies the aggregation order, and introduces the contribution of secondary systems to the properties of natural block copolymers. Secondly, combined with the synthesis mechanism of natural spider silk, this paper summarizes and analyzes the general process of the synthesized block copolymer. Finally, the conditions of self-assembly of artificial fragments to form the structure are analyzed by employing mean-field theory and a phase diagram. Natural and ideal synthetic spider silk block copolymers have high similarity in performance. To achieve this, scientists can only start from the primary segment to understand their arrangement order in the secondary structure. Then the influence of each block on the properties of the silk fiber is analyzed. At the same time, the size and shape of self-assembled block copolymers are controlled at the micro-scale, thereby changing their properties. The mechanism above shows that the synthesized spider silk block copolymer is similar to natural spider silk in microscopic morphology.