Structural studies of spider silk proteins in the fiber

Although spider silk has been studied for a number of years the structures of the proteins involved have yet to be definitely determined. X-ray diffraction and solid-state nuclear magnetic resonance (NMR) were used to study major ampullate (dragline) silk from Nephila clavipes. The silk was studied in its natural state, in the supercontacted state and in the restretched state following supercontraction. The natural silk structure is dominated by β-sheets aligned parallel to the fiber axis. Supercontraction is characterized by randomizing of the orientation of the β-sheet. When the fiber is restretched alignment is regained. However, the same reorientation was observed for wetting of minor ampullate silk which does not supercontract. Thus, the reorientation of β-sheets alone cannot explain the supercontraction in dragline silk. Cocoon silk showed very little β-sheet orientation in the natural state and there were no changes upon wetting. NMR and X-ray diffraction data are consistent with the β-sheets arising from the poly-alanine sequences known to be present in the proteins of major ampullate silk as has been proposed previously. © 1997 John Wiley & Sons, Ltd.     

Chimeric spider silk proteins mediated by intein result in artificial hybrid silks

Hybrid silks hold a great potential as specific biomaterials due to its controlled mechanical properties. To produce fibers with tunable properties, here we firstly made chimeric proteins in vitro, called W2C4CT and W2C8CT, with ligation of MaSp repetitive modules (C) with AcSp modules (W) by intein trans splicing technology from smaller precursors without final yield reduction. Intein mediated chimeric proteins form fibers at a low concentration of 0.4 mg/mL in 50 mM K₃PO₄ pH 7.5 just drawn by hand. Hybrid fibers show smoother surface, and also have stronger chemical resistance as compared with fibers from W2CT (W fibers) and mixture of W2CT/C8CT (MHF8 fibers). Fibers from chimeric protein W2C4CT (HFH4) have improved mechanical properties than W fibers; however, with more C modules W2C8CT fibers (HFH8) properties decreased, indicates the length proportion of various modules is very important and should be optimized for fibers with specific properties. Generally, hybrid silks generated via chimeric proteins, which can be simplified by intein trans splicing, has greater potential to produce fibers with tunable properties. Our research shows that intein mediated directional protein ligation is a novel way to make large chimeric spider silk proteins and hybrid silks. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 385–392, 2016.     

13C CP/MAS NMR study on structural heterogeneity in Bombyx mori silk fiber and their generation by stretching

It is important to resolve the structure of Bombyx mori silk fibroin before spinning (silk I) and after spinning (silk II), and the mechanism of the structural transition during fiber formation in developing new silk-like fiber. The silk I structure has been recently resolved by (13)C solid-state NMR as a "repeated beta-turn type II structure." Here, we used (13)C solid-state NMR to clarify the heterogeneous structure of the natural fiber from Bombyx mori silk fibroin in the silk II form. Interestingly, the (13)C CP/MAS NMR revealed a broad and asymmetric peak for the Ala Cbeta carbon. The relative proportions of the various heterogeneous components were determined from their relative peak intensities after line shape deconvolution. Namely, for 56% crystalline fraction (mainly repeated Ala-Gly-Ser-Gly-Ala-Gly sequences), 18% distorted beta-turn, 13% beta-sheet (parallel Ala residues), and 25% beta-sheet (alternating Ala residues). The remaining fraction of 44% amorphous Tyr-rich region, 22% in both distorted beta-turn and distorted beta-sheet. Such a heterogeneous structure including distorted beta-turn can be observed for the peptides (AG)(n) (n > 9 ). The structural change from silk I to silk II occurs exclusively for the sequence (Ala-Gly-Ser-Gly-Ala-Gly)(n) in B. mori silk fibroin. The generation of the heterogeneous structure can be studied by change in the Ala Cbeta peak of (13)C CP/MAS NMR spectra of the silk fibroin samples with different stretching ratios.     

A proposed model for dragline spider silk self‐assembly: Insights from the effect of the repetitive domain size on fiber properties

Dragline spider silk has been intensively studied for its superior qualities as a biomaterial. In previous studies, we made use of the baculovirus mediated expression system for the production of a recombinant Araneus diadematus spider silk dragline ADF4 protein and its self‐assembly into intricate fibers in host insect cells. In this study, our aim was to explore the function of the major repetitive domain of the dragline spider silk. Thus, we generated an array of synthetic proteins, each containing a different number of identical repeats up to the largest recombinantly expressed spider silk to date. Study of the self‐assembly properties of these proteins showed that depending on the increasing number of repeats they give rise to different assembly phenotypes, from a fully soluble protein to bona fide fibers with superior qualities. The different assembly forms, the corresponding chemical resistance properties obtained as well as ultrastructural studies, revealed novel insights concerning the structure and intermolecular interactions of the repetitive and nonrepetitive domains. Based on these observations and current knowledge in the field, we hereby present a comprehensive hypothetical model for the mechanism of dragline silk self‐assembly and fiber formation. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 458–468, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The dimerization mechanism of the N-terminal domain of spider silk proteins is conserved despite extensive sequence divergence

The N-terminal (NT) domain of spider silk proteins (spidroins) is crucial for their storage at high concentrations and also regulates silk assembly. NTs from the major ampullate spidroin (MaSp) and the minor ampullate spidroin are monomeric at neutral pH and confer solubility to spidroins, whereas at lower pH, they dimerize to interconnect spidroins in a fiber. This dimerization is known to result from modulation of electrostatic interactions by protonation of well-conserved glutamates, although it is undetermined if this mechanism applies to other spidroin types as well. Here, we determine the solution and crystal structures of the flagelliform spidroin NT, which shares only 35% identity with MaSp NT, and investigate the mechanisms of its dimerization. We show that flagelliform spidroin NT is structurally similar to MaSp NT and that the electrostatic intermolecular interaction between Asp 40 and Lys 65 residues is conserved. However, the protonation events involve a different set of residues than in MaSp, indicating that an overall mechanism of pH-dependent dimerization is conserved but can be mediated by different pathways in different silk types.     

Evolution of aerial spider webs coincided with repeated structural optimization of silk anchorages

Physical structures built by animals challenge our understanding of biological processes and inspire the development of smart materials and green architecture. It is thus indispensable to understand the drivers, constraints, and dynamics that lead to the emergence and modification of building behavior. Here, we demonstrate that spider web diversification repeatedly followed strikingly similar evolutionary trajectories, guided by physical constraints. We found that the evolution of suspended webs that intercept flying prey coincided with small changes in silk anchoring behavior with considerable effects on the robustness of web attachment. The use of nanofiber based capture threads (cribellate silk) conflicts with the behavioral enhancement of web attachment, and the repeated loss of this trait was frequently followed by physical improvements of web anchor structure. These findings suggest that the evolution of building behavior may be constrained by major physical traits limiting its role in rapid adaptation to a changing environment.     

Transglutamination allows production and characterization of native‐sized ELPylated spider silk proteins from transgenic plants

In the last two decades it was shown that plants have a great potential for production of specific heterologous proteins. But high cost and inefficient downstream processing are a main technical bottleneck for the broader use of plant‐based production technology especially for protein‐based products, for technical use as fibres or biodegradable plastics and also for medical applications. High‐performance fibres from recombinant spider silks are, therefore, a prominent example. Spiders developed rather different silk materials that are based on proteins. These spider silks show excellent properties in terms of elasticity and toughness. Natural spider silk proteins have a very high molecular weight, and it is precisely this property which is thought to give them their strength. Transgenic plants were generated to produce ELPylated recombinant spider silk derivatives. These fusion proteins were purified by Inverse Transition Cycling (ITC) and enzymatically multimerized with transglutaminase in vitro. Layers produced by casting monomers and multimers were characterized using atomic force microscopy (AFM) and AFM‐based nanoindentation. The layered multimers formed by mixing lysine‐ and glutamine‐tagged monomers were associated with the highest elastic penetration modulus.     

Water extraction by the major ampullate duct during silk formation in the spider, Argiope aurantia Lucas

The water, K⁺ and Na⁺ content of naturally produced major ampullate silk as well as silk mechanically drawn from the spider Argiope aurantia have been compared to that of the major ampullate gland. It is demonstrated that water is extracted by the major ampullate duct and that this process is accompanied by an exchange of K⁺ for Na⁺. The significance of these observations is discussed.     

A novel property of spider silk: chemical defence against ants

Spider webs are made of silk, the properties of which ensure remarkable efficiency at capturing prey. However, remaining on, or near, the web exposes the resident spiders to many potential predators, such as ants. Surprisingly, ants are rarely reported foraging on the webs of orb-weaving spiders, despite the formidable capacity of ants to subdue prey and repel enemies, the diversity and abundance of orb-web spiders, and the nutritional value of the web and resident spider. We explain this paradox by reporting a novel property of the silk produced by the orb-web spider Nephila antipodiana (Walckenaer). These spiders deposit on the silk a pyrrolidine alkaloid (2-pyrrolidinone) that provides protection from ant invasion. Furthermore, the ontogenetic change in the production of 2-pyrrolidinone suggests that this compound represents an adaptive response to the threat of natural enemies, rather than a simple by-product of silk synthesis: while 2-pyrrolidinone occurs on the silk threads produced by adult and large juvenile spiders, it is absent on threads produced by small juvenile spiders, whose threads are sufficiently thin to be inaccessible to ants.     

Microstructure of the silk apparatus in the coelotine spider Paracoelotes spinivulva (Araneae: Amaurobiidae)

The microstructural characteristics of the silk‐spinning apparatus and its ecological significance in the coelotine spider Paracoelotes spinivulva were examined by field emission scanning electron microscopy, with the goal of understanding the properties and the evolutionary origins of these silk constructs. The silk apparatuses of this spider were composed of four basic types of silk‐spinning spigot (ampullate, pyriform, aciniform and tubuliform), which connected with typical silk glands in the abdominal cavity. Of the three pairs of spinnerets, the posterior pairs were highly elongated along the body axis. Anterior spinnerets comprised two pairs of ampullate glands and approximately 70–80 pairs of pyriform glands in both sexes. Middle spinnerets had one to two pairs of ampullate spigots, three pairs of tubuliform spigots in females, and 50–60 (female) or 80–90 (male) pairs of aciniform spigots. An additional two pairs of tubuliform spigots in females and 70–80 (female) or 100–120 (male) pairs of aciniform spigots were counted on the spinning surfaces of the posterior spinnerets in both sexes. Although the coelotine spiders use their silk to catch prey, P. spinivulva characteristically do not have a typical “triad” spigot, including a flagelliform and two aggregate spigots, for capture thread production.     

Review the role of terminal domains during storage and assembly of spider silk proteins

Fibrous proteins in nature fulfill a wide variety of functions in different structures ranging from cellular scaffolds to very resilient structures like tendons and even extra-corporal fibers such as silks in spider webs or silkworm cocoons. Despite their different origins and sequence varieties many of these fibrous proteins share a common building principle: they consist of a large repetitive core domain flanked by relatively small non-repetitive terminal domains. Amongst protein fibers, spider dragline silk shows prominent mechanical properties that exceed those of man-made fibers like Kevlar. Spider silk fibers assemble in a spinning process allowing the transformation from an aqueous solution into a solid fiber within milliseconds. Here, we highlight the role of the non-repetitive terminal domains of spider dragline silk proteins during storage in the gland and initiation of the fiber assembly process.     

Genetically engineered syntheses of tandem repetitive polypeptides consisting of glycine-rich sequence of spider dragline silk

We described genetically engineered syntheses of tandem repetitive polypeptides consisting of glycine-rich sequence, GlyLeuGlyGlyGlnGlyGlyGlyAlaGlyGlnGlyGlyTyrGly, designated SCAP(1), in spidroin I of spider dragline silk from Nephila clavipes and the secondary conformational analyses in the solid state by Fourier transform ir measurements. The polypeptides composed of 4, 5, 6, 7, 11, 12, or 13 repeats of SCAP(1) were expressed in Escherichia coli, purified by nickel chelate affinity chromatography, and then cleaved with cyanogen bromide to release N- and C-terminal extensions. Typical yields were from 1.2 to 5.2 mg of lyophilized uncleaved polypeptides per liter of fermentation medium at an absorbance of 2.0 at 600 nm, and the production levels increased with decreasing the molecular weight of the expressed polypeptides. The lyophilized powder of cleaved SCAP(13) adopted the random coil, whereas the cast film from formic acid formed the β-sheet structure. The conformational results might indicate that the glycine-rich sequence formed β-sheet structure in spidroin I. Cleaved SCAP(13) started to decompose under nitrogen at ca. 230°C, which was in agreement with the decomposition temperature of the spider dragline silk from N. clavipes. © 1998 John Wiley & Sons, Inc. Biopoly 45: 269–279, 1998     

Solution structure of two insect-specific spider toxins and their pharmacological interaction with the insect voltage-gated Na+ channel

Delta-paluIT1 and delta-paluIT2 are toxins purified from the venom of the spider Paracoelotes luctuosus. Similar in sequence to mu-agatoxins from Agelenopsis aperta, their pharmacological target is the voltage-gated insect sodium channel, of which they alter the inactivation properties in a way similar to alpha-scorpion toxins, but they bind on site 4 in a way similar to beta-scorpion toxins. We determined the solution structure of the two toxins by use of two-dimensional nuclear magnetic resonance (NMR) techniques followed by distance geometry and molecular dynamics. The structures of delta-paluIT1 and delta-paluIT2 belong to the inhibitory cystine knot structural family, i.e. a compact disulfide-bonded core from which four loops emerge. Delta-paluIT1 and delta-paluIT2 contain respectively two- and three-stranded anti-parallel beta-sheets as unique secondary structure. We compare the structure and the electrostatic anisotropy of those peptides to other sodium and calcium channel toxins, analyze the topological juxtaposition of key functional residues, and conclude that the recognition of insect voltage-gated sodium channels by these toxins involves the beta-sheet, in addition to loops I and IV. Besides the position of culprit residues on the molecular surface, difference in dipolar moment orientation is another determinant of receptor binding and biological activity differences. We also demonstrate by electrophysiological experiments on the cloned insect voltage-gated sodium channel, para, heterologuously co-expressed with the tipE subunit in Xenopus laevis oocytes, that delta-paluIT1 and delta-paluIT2 procure an increase of Na+ current. delta-PaluIT1-OH seems to have less effect when the same concentrations are used.     

Biosorption of Cu(II) and Pb(II) ions from aqueous solution by natural spider silk

Aside from its excellent mechanical properties, spider silk (SS) would offer an active surface for heavy metal interaction due to its rich protein structure. The present study describes the potential use of natural (SS) as a sorbent of heavy metals from aqueous solutions. Single and multi-species biosorption experiments of heavy metals by natural SS were conducted using batch and column experiments. The biosorption kinetics, in general, was found to follow the second-order rate expression, and the experimental equilibrium biosorption data fitted reasonably well to Freundlich isotherm. From the Freundlich isotherm, the biosorption capacities of Cu(II) and Pb(II) ions onto SS were found as 0.20 and 0.007 mmol g?1, respectively. The results showed a decrease in the extent of metal ion uptake with lowering the pH.     

Fine structural aspects of silk secretion in a spider. II. Conduction in the pyriform glands

The excretory duct of pyriform glands in Araneus diadematus is connected to the secretory sac through an intermediary cell ring. Apices of these cells bear thick, long microvilli and cytoplasmic extensions containing microtubules in bundles, some of which are derived from normal basal bodies. These finger-like extensions lie between the cuticular intima and the secretory product; they are thought to protect the intima and to initiate moulding of the silk thread. Structural features of the duct cells suggest that the latter play a role in the control of the water content of the silk glue which is restricted to the last portion of the duct where numerous nerve endings are inserted between cells. It is evident that duct structure and chemical and physical characteristics of silk are correlated in all spider silk glands.