Spider Dragline Research Articles

Persistence and variation in microstructural design during the evolution of spider silk.

The extraordinary mechanical performance of spider dragline silk is explained by its highly ordered microstructure and results from the sequences of its constituent proteins. This optimized microstructural organization simultaneously achieves high tensile strength and strain at breaking by taking advantage of weak molecular interactions. However, elucidating how the original design evolved over the 400 million year history of spider silk, and identifying the basic relationships between microstructural details and performance have proven difficult tasks. Here we show that the analysis of maximum supercontracted single spider silk fibers using X ray diffraction shows a complex picture of silk evolution where some key microstructural features are conserved phylogenetically while others show substantial variation even among closely related species. This new understanding helps elucidate which microstructural features need to be copied in order to produce the next generation of biomimetic silk fibers.

Self-assembled semi-crystallinity at parallel β-sheet nanocrystal interfaces in clustered MaSp1 (spider silk) proteins.

In this communication, we use molecular dynamics methods to model the self-assembly of semi-crystalline domains at β-sheet nanocrystal interfaces in clusters of spider silk (MaSp1) proteins. Our research elucidates that the energetics at interfaces between crystalline and amorphous domains control effectively, the extent to which semi-crystalline domains can form at interfaces. Stability at nanocrystal interfaces is not linearly related to the internal (bulk) stability of the β-sheet nanocrystal. Rather, interfacial stability is found to be highly sensitive to the number of alanine repeat units that make up each sheet. Intriguingly, the most stable interface for the development of semi-crystallinity is built up of polyalanine β-sheets of a length similar to that which is spun naturally in spider dragline silk.

Protein secondary structure of Green Lynx spider dragline silk investigated by solid-state NMR and X-ray diffraction

In this study, the secondary structure of the major ampullate silk from Peucetia viridans (Green Lynx) spiders is characterized by X-ray diffraction and solid-state NMR spectroscopy. From X-ray diffraction measurement, β-sheet nanocrystallites were observed and found to be highly oriented along the fiber axis, with an orientational order, fc≈0.98. The size of the nanocrystallites was determined to be on average 2.5nm×3.3nm×3.8nm. Besides a prominent nanocrystalline region, a partially oriented amorphous region was also observed with an fa≈0.89. Two-dimensional 13C–13C through-space and through-bond solid-state NMR experiments were employed to elucidate structure details of P. viridans silk proteins. It reveals that β-sheet nanocrystallites constitutes 40.0±1.2% of the protein and are dominated by alanine-rich repetitive motifs. Furthermore, based upon the NMR data, 18±1% of alanine, 60±2% glycine and 54±2% serine are incorporated into helical conformations.

Self-Assembly of Recombinant Hagfish Thread Keratins Amenable to a Strain-Induced α-Helix to β-Sheet Transition.

Hagfish slime threads are assembled from protein-based bundles of intermediate filaments (IFs) that undergo a strain-induced α-helical coiled-coil to β-sheet transition. Draw processing of native fibers enables the creation of mechanically tuned materials, and under optimized conditions this process results in mechanical properties similar to spider dragline silk. In this study, we develop the foundation for the engineering of biomimetic recombinant hagfish thread keratin (TK)-based materials. The two protein constituents from the hagfish Eptatretus stoutii thread, named EsTKα and EsTKγ, were expressed in Escherichia coli and purified. Individual (rec)EsTKs and mixtures thereof were subjected to stepwise dialysis to evaluate their protein solubility, folding, and self-assembly propensities. Conditions were identified that resulted in the self-assembly of coiled-coil rich IF-like filaments, as determined by circular dichroism (CD) and transmission electron microscopy (TEM). Rheology experiments indicated that the concentrated filaments assembled into gel-like networks exhibiting a rheological response reminiscent to that of IFs. Notably, the self-assembled filaments underwent an α-helical coiled-coil to β-sheet transition when subjected to oscillatory shear, thus mimicking the critical characteristic responsible for mechanical strengthening of native hagfish threads. We propose that our data establish the foundation to create robust and tunable recombinant TK-based materials whose mechanical properties are controlled by a strain-induced α-helical coiled-coil to β-sheet transition.

Dimerization of Terminal Domains in Spiders Silk Proteins Is Controlled by Electrostatic Anisotropy and Modulated by Hydrophobic Patches.

The well-tuned spinning technology from spiders has attracted many researchers with the promise of producing high-performance, biocompatible, and yet biodegradable fibers. So far, the intricate chemistry and rheology of spinning have eluded us. A breakthrough was achieved recently, when the 3D structures of the N and C terminal domains of spider dragline silk were resolved and their pH-induced dimerization was revealed. To understand the terminal domains' dimerization mechanisms, we developed a protein model based on the experimental structures that reproduces charge and hydrophobic anisotropy of the complex protein surfaces. Monte Carlo simulations were used to study the thermodynamic dimerization of the N-terminal domain as a function of pH and ionic strength. We show that the hydrophobic and electrostatic anisotropies of the N-terminal domain cooperate constructively in the association process. The dipolar attractions at pH 6 lead to weakly bound dimers by forcing an antiparallel monomer orientation, stabilized by hydrophobic locking at close separations. Elevated salt concentrations reduce the thermodynamic dimerization constant due to screened electrostatic dipolar attraction. Moreover, the mutations on ionizable residues reveal a free energy of binding, proportional to the dipole moment of the mutants. It has previously been shown that dimers, formed at pH 6, completely dissociate at pH 7, which is thought to be due to altered protein charges. In contrast, our study indicates that the pH increase has no influence on the charge distribution of the N-terminal domain. Instead, the pH-induced dissociation is due to an adapted, loose conformation at pH 7, which significantly hampers both electrostatic and hydrophobic attractive interactions.

Nanofibrous Adhesion: The Twin of Gecko Adhesion

Inspired by dusty spider dragline silk, we studied the adhesive interaction between artificial nanofibers and their aerosol surroundings. The nanofibers are found to be able to actively capture particulate matters from the environment, exactly as the spider dragline silk does. Examinations prove that such nanofibrous adhesion is insensitive to the chemical nature of the fibers and the physical states of the particulate matter and depends only on the fiber diameters. Such facts indicate that nanofibrous adhesion is a case of dry adhesion, mainly governed by van der Waals force, sharing the same mechanism to gecko adhesion. Nanofibrous adhesion is of great importance and has promising potential. For instance, in this work, nanofibers are fabricated into a thin and translucent filter, which has a filtration performance, as high as 95%, that easily outperformed ordinary ones. We believe that this adhesive property of nanofibers will open up broader applications in both scientific and industrial fields.

Molecular dynamics of spider dragline silk fiber investigated by 2H MAS NMR.

The molecular dynamics of the proteins that comprise spider dragline silk were investigated with solid-state (2)H magic angle spinning (MAS) NMR line shape and spin-lattice relaxation time (T1) analysis. The experiments were performed on (2)H/(13)C/(15)N-enriched N. clavipes dragline silk fibers. The silk protein side-chain and backbone dynamics were probed for Ala-rich regions (β-sheet and 31-helical domains) in both native (dry) and supercontracted (wet) spider silk. In native (dry) silk fibers, the side chains in all Ala containing regions undergo similar fast methyl rotations (>10(9) s(-1)), while the backbone remains essentially static (<10(2) s(-1)). When the silk is wet and supercontracted, the presence of water initiates fast side-chain and backbone motions for a fraction of the β-sheet region and 31-helicies. β-Sheet subregion 1 ascribed to the poly(Ala) core exhibits slower dynamics, while β-sheet subregion 2 present in the interfacial, primarily poly(Gly-Ala) region that links the β-sheets to disordered 31-helical motifs, exhibits faster motions when the silk is supercontracted. Particularly notable is the observation of microsecond backbone motions for β-sheet subregion 2 and 31-helicies. It is proposed that these microsecond backbone motions lead to hydrogen-bond disruption in β-sheet subregion 2 and helps to explain the decrease in silk stiffness when the silk is wet and supercontracted. In addition, water mobilizes and softens 31-helical motifs, contributing to the increased extensibility observed when the silk is in a supercontracted state. The present study provides critical insight into the supercontraction mechanism and corresponding changes in mechanical properties observed for spider dragline silks.

Spiders spinning electrically charged nano-fibres.

Most spider threads are on the micrometre and sub-micrometre scale. Yet, there are some spiders that spin true nano-scale fibres such as the cribellate orb spider, Uloborus plumipes. Here, we analyse the highly specialized capture silk-spinning system of this spider and compare it with the silk extrusion systems of the more standard spider dragline threads. The cribellar silk extrusion system consists of tiny, morphologically basic glands each terminating through exceptionally long and narrow ducts in uniquely shaped silk outlets. Depending on spider size, hundreds to thousands of these outlet spigots cover the cribellum, a phylogenetically ancient spinning plate. We present details on the unique functional design of the cribellate gland-duct-spigot system and discuss design requirements for its specialist fibrils. The spinning of fibres on the nano-scale seems to have been facilitated by the evolution of a highly specialist way of direct spinning, which differs from the aqua-melt silk extrusion set-up more typical for other spiders.

Structural hysteresis in dragline spider silks induced by supercontraction: An x-ray fiber micro-diffraction study.

Interaction with water causes shrinkage and significant changes in the structure of spider dragline silks, which has been referred to as supercontraction in the literature. Preferred orientation or alignment of protein chains with respect to the fiber axis is extensively changed during this supercontraction process. Synchrotron x-ray micro-fiber diffraction experiments have been performed on Nephila clavipes and Argiope aurantia major and minor ampullate dragline spider fibers in the native dry, contracted (by immersion in water) and restretched (from contracted) states. Changes in the orientation of β-sheet nanocrystallites and the oriented component of the amorphous network have been determined from wide-angle x-ray diffraction patterns. While both the crystalline and amorphous components lose preferred orientation on wetting with water, the nano-crystallites regain their orientation on wet-restretching, whereas the oriented amorphous components only partially regain their orientation. Dragline major ampullate silks in both the species contract more than their minor ampullate silks.

Stress-Induced Lamellar Order in Spider Silk Fibers

Spider dragline silk, solely made from protein, outperforms synthetic fibers in terms of toughness and extensibility, which is thought to originate from its refined nano-scale hierarchical structure. The key for understanding the outstanding mechanical performance of dragline silk lies in a detailed description of the underlying molecular structure and dynamics under load conditions. Here, we aim at determining the ingredients for silk's outstanding toughness using multi-scale computational simulations. First, we present large-scale molecular-dynamics simulations of the structure and dynamics of silk protein. We introduce the most comprehensive, to date, spider silk models comprising of hundred 500-residue chains of two spider proteins, namely MaSp1 and MaSp2, using a collapsing-annealing protocol [1]. Our systems are composed of crystalline beta-sheet segments completely embedded in and connected with an amorphous phase. On this basis, we developed a three-dimensional continuum model of dragline silk for finite element analysis. Such model takes into account the plasticity of the beta-sheets, the rate-dependent dynamics of the amorphous phase, and the viscous friction between them [2]. Tensile properties, velocity-dependent effects and hysteresis are in good agreement with experimental data. Intriguingly, the simulations revealed that under load conditions, crystals rearrange into lamellar bands. We could confirm this trend by small-angle neutron scattering of silk fibers under stress, with simulations and experiments showing quantitatively the same shift and intensity in the signal from the periodically forming bands. The increased order results in a more homogenous stress distribution and higher fiber toughness. Our combined atomistic and fiber-level simulations and experiments suggest that the test stress-induced order is a common feature in materials combining crystalline and amorphous phases on the nano-scale.[1] Cruz-Chu, E. R et al. Faraday Discussions (2009) 143, 47-62[2] Sandeep Patil et al. PLOS One (2014) 9, e104832

E-spun composite fibers of collagen and dragline silk protein: fiber mechanics, biocompatibility, and application in stem cell differentiation.

Biocomposite matrices with high mechanical strength, high stability, and the ability to direct matrix-specific stem cell differentiation are essential for the reconstruction of lesioned tissues in tissue engineering and cell therapeutics. Toward this end, we used the electrospinning technique to fabricate well-aligned composite fibers from collagen and spider dragline silk protein, obtained from the milk of transgenic goats, mimicking the native extracellular matrix (ECM) on a similar scale. Collagen and the dragline silk proteins were found to mix homogeneously at all ratios in the electrospun (E-spun) fibers. As a result, the ultimate tensile strength and elasticity of the fibers increased monotonically with silk percentage, whereas the stretchability was slightly reduced. Strikingly, we found that the incorporation of silk proteins to collagen dramatically increased the matrix stability against excessive fiber swelling and shape deformation in cell culture medium. When human decidua parietalis placental stem cells (hdpPSCs) were seeded on the collagen–silk matrices, the matrices were found to support cell proliferation at a similar rate as that of the pure collagen matrix, but they provided cell adhesion with reduced strengths and induced cell polarization at varied levels. Matrices containing 15 and 30 wt % silk in collagen (CS15, CS30) were found to induce a level of neural differentiation comparable to that of pure collagen. In particular, CS15 matrix induced the highest extent of cell polarization and promoted the development of extended 1D neural filaments strictly in-line with the aligned fibers. Taking the increased mechanical strength and fiber stability into consideration, CS15 and CS30 E-spun fibers offer better alternatives to pure collagen fibers as scaffolds that can be potentially utilized in neural tissue repair and the development of future nanobiodevices.

Diverse Formulas for Spider Dragline Fibers Demonstrated by Molecular and Mechanical Characterization of Spitting Spider Silk

Spider silks have outstanding mechanical properties. Most research has focused on dragline silk proteins (major ampullate spidroins, MaSps) from orb-weaving spiders. Using silk gland expression libraries from the haplogyne spider Scytodes thoracica, we discovered two novel spidroins (S. thoracica fibroin 1 and 2). The amino acid composition of S. thoracica silk glands and dragline fibers suggest that fibroin 1 is the major component of S. thoracica dragline silk. Fibroin 1 is dominated by glycine-alanine motifs, and lacks sequence motifs associated with orb-weaver MaSps. We hypothesize fibroin 2 is a piriform or aciniform silk protein, based on amino acid composition, spigot morphology, and phylogenetic analyses. S. thoracica's dragline silk is less tough than previously reported, but is still comparable to other dragline silks. Our analyses suggest that dragline silk proteins evolved multiple times. This demonstrates that spider dragline silk is more diverse than previously understood, providing alternative high performance silk designs.

Synthetic biology increases efficiency of Escherichia coli to produce Parawixia bistriata spider silk protein

Background Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. With synthetic biology it is possible to express recombinant spider silk proteins, which are characterized by a highly repetitive rich glycine and alanine sequence [1]. However, production of high molecular weight spider silk protein can be difficult due to DNA instability, transcription and translation errors. Here we show, for the first time, Masp2 (105 kDa) spidroin silk protein production from the Brazilian spider Parawixia bistriata in different metabolically engineered E.coli strains.

In situ Raman spectroscopic study of Al-infiltrated spider dragline silk under tensile deformation.

Natural materials consisting of protein structures impregnated with a tiny amount of metals often exhibit impressive mechanical behavior, which represents a new design paradigm for the development of biomimetic materials. Here, we produced Al-infiltrated silks by applying a modified Al2O3 atomic layer deposition process to the dragline silk of the Nephila pilipes spider, which showed unusual mechanical properties. The deformation behavior of the molecular structure of the Al-infiltrated silk was investigated by performing in situ Raman spectroscopy, where Raman shifts were measured concurrently with macroscopic mechanical deformations. For identifying the role of the infiltrated Al atoms, the study was performed in parallel with untreated silk, and the results were compared. Our experimental results revealed that superior mechanical properties of the Al-infiltrated silk are likely to be caused by the alterations of the sizes of the β-sheet crystals and their distribution.

INVESTIGATION OF NANO- AND MICROSTRUCTURE OF BIOMATERIALS FOR REGENERATIVE MEDICINE BY METHOD OF SCANNING PROBE NANOTOMOGRAPHY

Aim. To perform a study of three-dimensional micro- and nanostructure of porous biocompatible scaffolds and quantitative analysis of nanoscale porosity parameters. Materials and methods . Three-dimensional porous scaffolds made from spidroin rS1/9 (recombinant analog of spider dragline protein) were produced by salt leaching technique. Dimensions of macropores in produced three-imensional scaffolds were in range from 200 to 400 microns. The study of three-dimensional structure of scaffolds was carried out by scanning probe nanotomography technique with the use of experimental setup combining ultramicrotome and scanning probe microscope. Results. Three-dimensional nanotomographical reconstruction of scaffold macropore wall structure is obtained. The formation of three-dimensional network of interconnected pores and channels with characteristic dimensions in range from 20 to 700 nm in the volume of macropore walls of studied scaffolds is observed. Mean pore diameter is 150 nm. Volume porosity of macropore walls is 22% while volume fraction of pores interconnected in large pore clusters is about 20% of all pore volume. Conclusion. Obtained as a result of the study quantitative characteristics of porous micro- and nanostructure of scaffolds show signifi cant degree of nanoscale porosity and percolation of macropore walls what correlates with reported high effi ciency of tissue regeneration on such scaffolds implanted in vivo. Use of scanning probe nanotomography technique for analysis of characteristics and topology of micro- and nanopore systems enables to improve effi ciency of development of novel biocompatible and biodegradable materials with predicted morphological, physical, chemical and biological characteristics.

Spider-silk-like shape memory polymer fiber for vibration damping

In this study, the static and dynamic properties of shape memory polyurethane (SMPU) fiber are reported and compared to those of spider dragline silk. Although the polymeric fiber has a lower strength compared to spider dragline silks (0.2–0.3 GPa versus 1.1 GPa), it possesses much higher toughness (276–289 MJ m−3 versus 160 MJ m−3), due to its excellent extensibility. The dynamic mechanical tests reveal that SMPU fiber has a high damping capacity (tan δ = 0.10–0.35) which is comparable to or even higher than that of spider silks (tan δ = 0.15). In addition, we found that, different programming methods change the shape memory and damping properties of the fiber in different ways and cold-drawing programming is more advocated in structural applications. These results suggest that the SMPU fiber has similar vibration damping and mechanical properties as spider silk, and may find applications in lightweight engineering structures.

High-toughness silk produced by a transgenic silkworm expressing spider (Araneus ventricosus) dragline silk protein.

Spider dragline silk is a natural fiber that has excellent tensile properties; however, it is difficult to produce artificially as a long, strong fiber. Here, the spider (Araneus ventricosus) dragline protein gene was cloned and a transgenic silkworm was generated, that expressed the fusion protein of the fibroin heavy chain and spider dragline protein in cocoon silk. The spider silk protein content ranged from 0.37 to 0.61% w/w (1.4–2.4 mol%) native silkworm fibroin. Using a good silk-producing strain, C515, as the transgenic silkworm can make the raw silk from its cocoons for the first time. The tensile characteristics (toughness) of the raw silk improved by 53% after the introduction of spider dragline silk protein; the improvement depended on the quantity of the expressed spider dragline protein. To demonstrate the commercial feasibility for machine reeling, weaving, and sewing, we used the transgenic spider silk to weave a vest and scarf; this was the first application of spider silk fibers from transgenic silkworms.

Viscous friction between crystalline and amorphous phase of dragline silk.

The hierarchical structure of spider dragline silk is composed of two major constituents, the amorphous phase and crystalline units, and its mechanical response has been attributed to these prime constituents. Silk mechanics, however, might also be influenced by the resistance against sliding of these two phases relative to each other under load. We here used atomistic molecular dynamics (MD) simulations to obtain friction forces for the relative sliding of the amorphous phase and crystalline units of Araneus diadematus spider silk. We computed the coefficient of viscosity of this interface to be in the order of 102 Ns/m2 by extrapolating our simulation data to the viscous limit. Interestingly, this value is two orders of magnitude smaller than the coefficient of viscosity within the amorphous phase. This suggests that sliding along a planar and homogeneous surface of straight polyalanine chains is much less hindered than within entangled disordered chains. Finally, in a simple finite element model, which is based on parameters determined from MD simulations including the newly deduced coefficient of viscosity, we assessed the frictional behavior between these two components for the experimental range of relative pulling velocities. We found that a perfectly relative horizontal motion has no significant resistance against sliding, however, slightly inclined loading causes measurable resistance. Our analysis paves the way towards a finite element model of silk fibers in which crystalline units can slide, move and rearrange themselves in the fiber during loading.

Nutrient and Wind Effects on Dragline Properties: Perspectives from WAXS &amp; SAXS

Spider dragline silk is one of the strongest nature fibers and some of their features are even better than those of the best synthetic fibers. Understanding the mechanisms inducing silk variability may have implications for biomimetics and the synthesis of environmentally responsive materials. Dragline silk contains both elasticity (amorphous) and crystalline regions. Our previous studies had demonstrated that spiders might vary the protein composition and thus physical properties of silks when experiencing food with different nutrient level. In this study we fed Nephila pilipes with high, low and no protein foods and collected their dragline silks for synchrotron Radiation (SR) wide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS) examinations. The WAXS data showed a significant difference in crystalline fractions of dragline silks produced by N. pilipes experiencing different food treatments. In addition, the orientation of crystallines also varied considerably among silks produced by spiders in three treatment groups. The SAXS data, obtained with the beam incident along and perpendicular to the fiber axis revealed a mesostructure comprising nano crystallites (beta sheets) stack spirally along the spider fibril axis. Such results indicate that spiders experiencing different nutrient stress level might produce dragline silks of different physical properties due to variations in crystalline density, orientation and the meso-phase structures in nano scale. Furthermore, varying environmental wind strength leads to changes in tensile mechanics of spider dragline silk hence produced. Exposing the spider Cyclosa mulmeinensis to controlled stress from constant airflow, we found correlated changes in (i) amino acid composition, (ii) tensile mechanics and (iii) crystallinity, of the dragline silk; which results suggest that protein variation and/or post secretion crystalline variations are associated with the mechanical properties of the spider silks.

Coatings and Films Made of Silk Proteins

Silks are a class of proteinaceous materials produced by arthropods for various purposes. Spider dragline silk is known for its outstanding mechanical properties, and it shows high biocompatibility, good biodegradability, and a lack of immunogenicity and allergenicity. The silk produced by the mulberry silkworm B. mori has been used as a textile fiber and in medical devices for a long time. Here, recent progress in the processing of different silk materials into highly tailored isotropic and anisotropic coatings for biomedical applications such as tissue engineering, cell adhesion, and implant coatings as well as for optics and biosensors is reviewed.