Fine structure of cribellate spider silk

Sticky silk from webs of the spiders, Uloborus diversus andFilistata arizoniciis,were examined by election microscopy.The silk of U. diversus contains long fibrils, 200 –300A{ring} in diameter, consisting of an electron-dense centralfilament, 30 –60 A{ring} across, embedded in a lightermatrix. Transverse banding is distinguished in the matrix atintervals from over 200 to less than 50 A{ring}. Similar featuresare observed in the silk of F. arizonicus. Extended fibrilshave an altered structure.     

The formation of a quaternary structure by recombinant analogs of spider silk proteins

The morphology of the fibers formed by recombinant analogs of dragline spider silk proteins, spidroins 1 and 2, was studied. It has been shown that the extension of the initial fiber, the so-called as-spun fiber, leads to remodeling of the spongy matrix with the formation of microfibers, which is accompanied by a decrease in the fiber diameter. The breaking strength of the fiber depends not only on the primary structure of the constituent protein, but also on the way it was formed. Simulation of the assembly of microfibers and the fibers formed of them can clarify the natural spider web spinning and enhance the development of technology for producing biomaterials with unique properties.     

Molecular biology of spider silk

Spider silks are an intriguing family of fibrous proteins due to their highly repetitive primary sequence, their solution properties and their assembly and processing into fibers with remarkable mechanical properties. Current research efforts aimed at understanding and manipulating genes encoding these proteins are helping to gain insight into the relationships between protein sequence, protein assembly and macromolecular properties.     

Biology of spider silk.

Studies are beginning to show that spider silk can be highly variable in chemical composition and mechanical properties. Clearly, both external and internal conditions affect silk production and thus the mechanical properties of the finished thread. An argument can be made that silk is optimised for a wide range of conditions rather than maximised for strength or toughness. Moreover, it seems that the spider is able to induce rapid and temporary adaptations of silk properties.     

Molecular biology of spider silk.

Spider silks are an intriguing family of fibrous proteins due to their highly repetitive primary sequence, their solution properties and their assembly and processing into fibers with remarkable mechanical properties. Current research efforts aimed at understanding and manipulating genes encoding these proteins are helping to gain insight into the relationships between protein sequence, protein assembly and macromolecular properties.     

Amyloidogenic nature of spider silk.

In spiders soluble proteins are converted to form insoluble silk fibres, stronger than steel. The final fibre product has long been the subject of study; however, little is known about the conversion process in the silk-producing gland of the spider. Here we describe a study of the conversion of the soluble form of the major spider-silk protein, spidroin, directly extracted from the silk gland, to a beta-sheet enriched state using circular dichroism (CD) spectroscopy. Combined with electron microscopy (EM) data showing fibril formation in the beta-sheet rich region of the gland and amino-acid sequence analyses linking spidroin and amyloids, these results lead us to suggest that the refolding conversion is amyloid like. We also propose that spider silk could be a valuable model system for testing hypotheses concerning beta-sheet formation in other fibrilogenic systems, including amyloids.     

Microbial production of spider silk proteins

The remarkable properties of spider dragline silk and related protein polymers will find many applications if the materials can be produced economically. We have demonstrated the production of high molecular weight spider dragline silk analog proteins encoded by synthetic genes in several microbial systems, including Escherichia coli and Pichia pastoris. In E. coli, proteins of up to 1000 amino acids in length could be produced efficiently, but the yield and homogeneity of higher molecular weight silk proteins were found to be limited by truncated synthesis, probably as a result of ribosome termination errors. No such phenomenon was observed in the yeast P. pastoris, where higher molecular weight silk proteins could be produced without heterogeneity due to truncated synthesis. Spider dragline silk analog proteins could be secreted by P. pastoris when fused to both the signal sequence and N-terminal pro-sequence of the Saccharomyces cerevisiae alpha-mating factor gene.     

New internal structure of spider dragline silk revealed by atomic force microscopy.

Atomic force microscopy was used to study the three-dimensional nanometer-scale structure of the dragline silk of Nephila clavipes from microtomed sections of the silk. Contrary to a previously proposed model of randomly distributed protein crystallites interspersed in amorphous regions, a highly organized skin-core structure of the fiber was observed. The skin appeared to be thin with no discernible distinct features. The core consists of pleated fibril-like structures, which are arranged in two concentric cylinders. Upon stretching, the pleats were smoothed out substantially. The mechanical properties of spider silk can quite straightforwardly be related to the newly observed structures.     

Polarized light microscopy, variability in spider silk diameters, and the mechanical characterization of spider silk

Abstract. Spider silks possess a remarkable combination of high tensile strength and extensibility that makes them among the toughest materials known. Despite the potential exploitation of these properties in biotechnology, very few silks have ever been characterized mechanically. This is due in part to the difficulty of measuring the thin diameters of silk fibers. The largest silk fibers are only 5–10 μm in diameter and some can be as fine as 50 nm in diameter. Such narrow diameters, coupled with the refraction of light due to the anisotropic nature of crystalline regions within silk fibers, make it difficult to determine the size of silk fibers. Here, we report upon a technique that uses polarized light microscopy (PLM) to accurately and precisely characterize the diameters of spider silk fibers. We found that polarized light microscopy is as precise as scanning electron microscopy (SEM) across repeated measurements of individual samples of silk and resulted in mean diameters that were ～0.10 μm larger than those from SEM. Furthermore, we demonstrate that thread diameters within webs of individual spiders can vary by as much as 600%. Therefore, the ability of PLM to non‐invasively characterize the diameters of each individual silk fiber used in mechanical tests can provide a crucial control for natural variation in silk diameters, both within webs and among spiders.     

RGD-functionalized bioengineered spider dragline silk biomaterial

Spider silk fibers have remarkable mechanical properties that suggest the component proteins could be useful biopolymers for fabricating biomaterial scaffolds for tissue formation. Two bioengineered protein variants from the consensus sequence of the major component of dragline silk from Nephila clavipes were cloned and expressed to include RGD cell-binding domains. The engineered silks were characterized by CD and FTIR and showed structural transitions from random coil to insoluble beta-sheet upon treatment with methanol. The recombinant proteins were processed into films and fibers and successfully used as biomaterial matrixes to culture human bone marrow stromal cells induced to differentiate into bone-like tissue upon addition of osteogenic stimulants. The recombinant spider silk and the recombinant spider silk with RGD encoded into the protein both supported enhanced the differentiation of human bone marrow derived mesenchymal stem cells (hMSCs) to osteogenic outcomes when compared to tissue culture plastic. The recombinant spider silk protein without the RGD displayed enhanced bone related outcomes, measured by calcium deposition, when compared to the same protein with RGD. Based on comparisons to our prior studies with silkworm silks and RGD modifications, the current results illustrate the potential to bioengineer spider silk proteins into new biomaterial matrixes, while also highlighting the importance of subtle differences in silk sources and modes of presentation of RGD to cells in terms of tissue-specific outcomes.     

The evolution of cryptic spider silk: a behavioral test

Phylogenetic patterns of change in spider silk coloration provideinsight into the selective pressures directing evolution ofsilks. Trends toward evolution of silks with low reflectanceof ultraviolet (UV) light suggest that reduced UV reflectancemay be an adaptation to reduce visibility of webs to insectprey. However, a test of the visibility of primitive and derivedspider silks is lacking. Several genera of orb-weaving spidersinclude conspicuous designs of silk, called "stabilimenta,"at the center of their webs. Due to their large size, stabilimentapresent signals that insects can use to avoid webs. Unlikeother silks in the orb web, which reflect little UV light,evolutionarily derived stabilimentum silk retains a bright UV reflectance. But, unlike primitive silks, stabilimentum silkalso reflects large amounts of blue and green light. We comparedthe visibility of primitive tarantula silks and derived stabilimentumsilks to insects by using the ability of honey bees to learnto forage at targets of spider silk. We found that the uniquespectral properties of stabilimentum silk render it crypticto insects and that primitive silks are more visible to bees.Our findings support a hypothesis that the coloration of stabilimentumsilk is an adaptation to reduce the ability of insects to avoidwebs and that ancient biases in the color vision of insectshave acted upon the evolution of spider silk coloration throughsensory drive. But our findings question the emphasis on UVreflectance alone for visibility of spider silks to insects.     

Spinning an elastic ribbon of spider silk

The Sicarid spider Loxosceles laeta spins broad but very thin ribbons of elastic silk that it uses to form a retreat and to capture prey. A structural investigation into this spider's silk and spinning apparatus shows that these ribbons are spun from a gland homologous to the major ampullate gland of orb web spiders. The Loxosceles gland is constructed from the same basic parts (separate transverse zones in the gland, a duct and spigot) as other spider silk glands but construction details are highly specialized. These differences are thought to relate to different ways of spinning silk in the two groups of spiders. Loxosceles uses conventional die extrusion, feeding a liquid dope (spinning solution) to the slit-like die to form a flat ribbon, while orb web spiders use an extrusion process in which the silk dope is processed in an elongated duct to produce a cylindrical thread. This is achieved by the combination of an initial internal draw down, well inside the duct, and a final draw down, after the silk has left the spigot. The spinning mechanism in Loxosceles may be more ancestral.     

Microbial production of spider silk proteins.

The remarkable properties of spider dragline silk and related protein polymers will find many applications if the materials can be produced economically. We have demonstrated the production of high molecular weight spider dragline silk analog proteins encoded by synthetic genes in several microbial systems, including Escherichia coli and Pichia pastoris. In E. coli, proteins of up to 1000 amino acids in length could be produced efficiently, but the yield and homogeneity of higher molecular weight silk proteins were found to be limited by truncated synthesis, probably as a result of ribosome termination errors. No such phenomenon was observed in the yeast P. pastoris, where higher molecular weight silk proteins could be produced without heterogeneity due to truncated synthesis. Spider dragline silk analog proteins could be secreted by P. pastoris when fused to both the signal sequence and N-terminal pro-sequence of the Saccharomyces cerevisiae alpha-mating factor gene.     

Recombinant DNA production of spider silk proteins

Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks.     

Transmission X-ray microscopy of spider dragline silk

We have investigated the structure of spider silk fibers from two different Nephila species and three different Araneus species by transmission X-ray microscopy (TXM). Single fibers and double fibers have been imaged. All images are in agreement with a homogenous density on length scales between the fiber diameter and the resolution of the instrument, which is about 25 nm.     

Supercontracted spider dragline silk: a solid-state NMR study of the local structure.

The local structure of supercontracted dragline silk from the spider Nephila madagascariensis was investigated by solid-state nuclear magnetic resonance. Two-dimensional (2D) spin-diffusion experiments did not show any significant conformational changes in short-range order (and the secondary structure of the protein) upon supercontraction. Our results are in accordance with the proposal by Vollrath et al. (Proc R Soc London B 1996;263:147-151) that urea-supercontraction does not alter the local structure of spider dragline silk fundamentally. However, significant differences in the dynamics of the polypeptide chain upon supercontraction are detected at room temperature. At low temperature, these dynamics are frozen out. In addition, the role of the solvent (water) in the silk is investigated in Nephila edulis. Mobile water is detected at temperatures significantly below the freezing point of bulk water.     

The evolution of complex biomaterial performance: The case of spider silk

Spider silk is a high-performance biomaterial with exceptional mechanical properties and over half a century of research into its mechanics, structure, and biology. Recent research demonstrates that it is a highly variable class of materials that differs across species and individuals in complex and interesting ways. Here, we review recent literature on mechanical variation and evolution in spider silk. We then present new data on material properties of silk from nine species of spiders in the Mesothelae and Mygalomorphae, the two basal clades of spiders. Silk from spiders in the Araneomorphae (true spiders where most previous research on silk has focused) is significantly stronger and therefore much tougher than the silk produced by spiders in the basal groups. These data support the hypothesis that the success and diversity seen in araneomorph spiders is associated with the evolution of this high-performance fiber. This comparative approach shows promise as a way to understand complex, high-performance biomaterials.