Inducing β-sheets formation in synthetic spider silk fibers by aqueous post-spin stretching

As a promising biomaterial with numerous potential applications, various types of synthetic spider silk fibers have been produced and studied in an effort to produce man-made fibers with mechanical and physical properties comparable to those of native spider silk. In this study, two recombinant proteins based on Nephila clavipes Major ampullate Spidroin 1 (MaSp1) consensus repeat sequence were expressed and spun into fibers. Mechanical test results showed that fiber spun from the higher molecular weight protein had better overall mechanical properties (70 KD versus 46 KD), whereas postspin stretch treatment in water helped increase fiber tensile strength significantly. Carbon-13 solid-state NMR studies of those fibers further revealed that the postspin stretch in water promoted protein molecule rearrangement and the formation of β-sheets in the polyalanine region of the silk. The rearrangement correlated with improved fiber mechanical properties and indicated that postspin stretch is key to helping the spider silk proteins in the fiber form correct secondary structures, leading to better quality fibers.     

Molecular and mechanical properties of major ampullate silk of the black widow spider, Latrodectus hesperus

Molecular and material properties of major ampullate silk were studied for the cobweb-building black widow spider Latrodectus hesperus. Material properties were measured by stretching the silk to breaking. The strength was 1.0 +/- 0.2 GPa, and the extensibility was 34 +/- 8%. The secondary structure of the major ampullate silk protein was studied using carbon-13 NMR spectroscopy. Alanine undergoes a transition from a coiled structure in pre-spun silk to a beta sheet structure in post-spun silk. We have also isolated two distinct cDNAs (both about 500 bp) which encode proteins similar to major ampullate spidroin 1 and 2 (MaSp1 and MaSp2). The MaSp1-like silk contains polyalanine runs of 5-10 residues as well as GA and GGX motifs. The MaSp2-like silk contains polyalanine runs of varying length as well as GPG(X)(n) motifs. L. hesperus major ampullate silk is more like major ampullate silk from other species than other L. hesperus silks.     

An investigation of the divergence of major ampullate silk fibers from Nephila clavipes and Argiope aurantia

The major ampullate fiber of both Nephila clavipes and Argiope aurantia is composed of two different proteins, MaSp1 and MaSp2. Each of these proteins has a highly conserved pattern of silk-associated amino acid motifs. The GPGXX motif is the only source of proline and is unique to MaSp2. On the basis of the percent of proline, Nephila clavipes major ampullate silk was calculated to consist of 19% MaSp2 and 81% MaSp1, while Argiope aurantia was calculated to have a significantly higher MaSp2 content of 59% with MaSp1 comprising the remaining 41%. To investigate the functional implications of the difference in protein composition, major ampullate silk fibers from Nephila clavipes and Argiope aurantia were mechanically tested and compared. Stress-strain curves produced from polynomial regression show that the two significant differences between major ampullate silk fibers from Nephila clavipes and Argiope aurantia are the average peak load stress and Young's modulus, with Argiope higher for both.     

A MaSp2-like gene found in the Amazon mygalomorph spider Avicularia juruensis

Two unique spidroins are present in the silk of the Amazon mygalomorph spider — Avicularia juruensis (Theraphosidae), and for the first time the presence and expression of a major ampullate spidroin 2-like in Mygalomorphae are demonstrated. Molecular analysis showed the presence of (GA)_n, poly-A and GPGXX motifs in the amino acid sequence of Spidroin 2, the last being a motif described so far only in MaSp2 and Flag spidroins. Phylogenetic analysis confirmed the previously known orthologous silk gene clusters, and placed this gene firmly within the orbicularian MaSp2 clade. Gene tree–species tree reconciliations show a pattern of multiple gene duplication throughout spider silk evolution, and pinpoint the oldest speciation in which MaSps must have been present in spiders on the mygalomorph–araneomorph split, 240 MYA. Therefore, while not refuting orb weaver monophyly, MaSp2s, and major ampullate silks in general cannot be classified as orbicularian synapomorphies, but have to be considered plesiomorphic for Opisthothelae. The evidence presented here challenges the simplified notion that mygalomorphs spin only one kind of silk, and adds to the suite of information suggesting a pattern of early niche diversification between Araneomorphae and Mygalomorphae. Additionally, mygalomorph MaSp2-like might accommodate mechanical demands arising from the arboreal habitat preference of Avicularia.     

Multiple recombining loci encode MaSp1, the primary constituent of dragline silk, in widow spiders (Latrodectus: Theridiidae)

Spiders spin a functionally diverse array of silk fibers, each composed of one or more unique proteins. Most of these proteins, in turn, are encoded by members of a single gene family thought to have arisen through duplication and divergence of an ancestral silk gene. Because of its remarkable mechanical properties, orb weaver dragline silk, a composite of 2 proteins (MaSp1 and MaSp2), is the best studied. Here, we demonstrate that multiple loci encode MaSp1 in widow spiders (Latrodectus). Because these copies may be the result of more recent duplication events than those leading to the currently recognized silk gene paralogs, they offer insight into the early evolutionary fate of silk gene duplicates. In addition to 3 presumed functional MaSp1 loci in Latrodectus hesperus (Western black widow) and Latrodectus geometricus (brown widow) genomes, we find a MaSp1 pseudogene in L. hesperus, demonstrating the potential for unrecognized extinction of silk gene paralogs. We also document recombination events among L. hesperus MaSp1 loci and between Latrodectus MaSp1 loci and MaSp2. This result supports the hypothesis that concerted evolution occurs not only within an individual silk gene but also among silk gene paralogs. One of the L. geometricus MaSp1 copies encodes a protein that has diverged in amino acid composition and potentially converged on the secondary structure of MaSp2. Based on the presence of multiple MaSp1 loci and the phylogenetic distribution of MaSp1 versus MaSp2, we propose that MaSp2 is derived from an ancestral MaSp1 duplicate. Finally, divergence has occurred in the upstream flanking sequences of the L. hesperus MaSp1 loci, the region most likely to contain regulatory motifs, providing ample opportunity for differential expression. However, the benefits associated with increased protein production may be the primary mechanism maintaining multiple functional MaSp1 copies in widow genomes.     

Elucidating metabolic pathways for amino acid incorporation into dragline spider silk using 13C enrichment and solid state NMR

Spider silk has been evolutionarily optimized for contextual mechanical performance over the last 400 Ma. Despite precisely balanced mechanical properties, which have yet to be reproduced, the underlying molecular architecture of major ampullate spider silk can be simplified being viewed as a versatile block copolymer. Four primary amino acid motifs: polyalanine, (GA)(n), GPGXX, and GGX (X = G,A,S,Q,L,Y) will be considered in this study. Although synthetic mimetics of many of these amino acid motifs have been produced in several biological systems, the source of spider silk's mechanical integrity remains elusive. Mechanical robustness may be a product not only of the amino acid structure but also of the tertiary structure of the silk. Historically, solid state nuclear magnetic resonance (ssNMR) has been used to reveal the crystalline structure of the polyalanine motif; however, limitations in amino acid labeling techniques have obscured the structures of the GGX and GPGXX motifs thought to be responsible for the structural mobility of spider silk. We describe the use of metabolic pathways to label tyrosine for the first time as well as to improve the labeling efficiency of proline. These improved labeling techniques will allow the previously unknown tertiary structures of major ampullate silk to be probed.     

The molecular structures of major ampullate silk proteins of the wasp spider,Argiope bruennichi: A second blueprint for synthesizing de novo silk

The dragline silk of orb-weaving spiders possesses extremely high tensile strength and elasticity. To date, full-length sequences of only two genes encoding major ampullate silk protein (MaSp) in Latrodectus hesperus have been determined. In order to further understand this gene family, we utilized in this study a variety of strategies to isolate full-length MaSp1 and MaSp2 cDNAs in the wasp spider Argiope bruennichi. A. bruennichi MaSp1 and MaSp2 are primarily composed of remarkably homogeneous ensemble repeats containing several complex motifs, and both have highly conserved C-termini and N-termini. Two novel amino acid motifs, GGF and SGR, were found in MaSp1 and MaSp2, respectively. Amino acid composition analysis of silk, luminal contents and predicted sequences indicates that MaSp1 and MaSp2 are two major components of major ampullate glands and that the ratio of MaSp1 to MaSp2 is approximately 3:2 in dragline silk. Furthermore, both the MaSp1:MaSp2 ratio and the conserved termini are closely linked with the production of high quality synthetic fibers. Our results make an important contribution to our understanding of major ampullate silk protein structure and provide a second blueprint for creating new composite silk which mimics natural spider dragline silk.     

Expression and Purification of a Spider Silk Protein: A New Strategy for Producing Repetitive Proteins

Synthetic genes were constructed based on the known sequence of the spider dragline silk protein MaSp 2. The genes had 8, 16, or 32 contiguous units of the consensus repeat sequence of the protein. These artificial genes were constructed using a strategy involving compatible but nonregenerable restriction sites, which allowed construction of very large inserts in a precisely controlled manner. This strategy should have general utility in the controlled construction of repetitive proteins composed of identical or different repeat units. The protein from the 16-unit repeat was produced inEscherichia coliat levels up to 10 mg/g wet wt of cells although yields of 1–2 mg/g were more typical. The protein was easily purified with high recovery using an affinity column. The purified protein had the predicted amino acid composition and N-terminal sequence after cleavage of a leader sequence. The methodology described will allow production of sufficient quantities of protein for basic structure/function studies including production of synthetic fibers.     

Variation in protein intake induces variation in spider silk expression

Background

It is energetically expensive to synthesize certain amino acids. The proteins (spidroins) of spider major ampullate (MA) silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved.

Methodology/Principal Findings

We fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species'' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake.

Conclusions

Our results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact mechanics.     

Synthesis and characterization of multiblock copolymers based on spider dragline silk proteins

Spider dragline silk with its superlative tensile properties provides an ideal system to study the relationship between morphology and mechanical properties of a structural protein. Accordingly, we synthesized two hybrid multiblock copolymers by condensing poly(alanine) [(Ala)(5)] blocks of the structural proteins (spidroin MaSp1 and MaSp2) of spider dragline silk with different oligomers of isoprene (2200 and 5000 Da) having reactive end groups. The synthetic multiblock polymer displayed similar secondary structure to that of natural spidroin, the peptide segment forming a beta-sheet structure. These multiblock polymers showed a significant solubility in the component solvents. Moreover, the copolymer which contains the short polyisoprene segment would aggregate into a micellar-like structure, as observed by TEM.     

Web building and silk properties functionally covary among species of wolf spider

Although phylogenetic studies have shown covariation between the properties of spider major ampullate (MA) silk and web building, both spider webs and silks are highly plastic so we cannot be sure whether these traits functionally covary or just vary across environments that the spiders occupy. As MaSp2‐like proteins provide MA silk with greater extensibility, their presence is considered necessary for spider webs to effectively capture prey. Wolf spiders (Lycosidae) are predominantly non‐web building, but a select few species build webs. We accordingly collected MA silk from two web‐building and six non‐web‐building species found in semirural ecosystems in Uruguay to test whether the presence of MaSp2‐like proteins (indicated by amino acid composition, silk mechanical properties and silk nanostructures) was associated with web building across the group. The web‐building and non‐web‐building species were from disparate subfamilies so we estimated a genetic phylogeny to perform appropriate comparisons. For all of the properties measured, we found differences between web‐building and non‐web‐building species. A phylogenetic regression model confirmed that web building and not phylogenetic inertia influences silk properties. Our study definitively showed an ecological influence over spider silk properties. We expect that the presence of the MaSp2‐like proteins and the subsequent nanostructures improves the mechanical performance of silks within the webs. Our study furthers our understanding of spider web and silk co‐evolution and the ecological implications of spider silk properties.     

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.     

Expression and characterization of chimeric spidroins from flagelliform-aciniform repetitive modules

Spiders can produce up to seven different types of silks or glues with different mechanical properties. Of these, flagelliform (Flag) silk is the most elastic, and aciniform (AcSp1) silk is the toughest. To produce a chimeric spider silk (spidroin) Flag_R-AcSp1_R, we fused one repetitive module of flagelliform silk from Araneus ventricosus and one repetitive module of aciniform silk from Argiope trifasciata. The recombinant protein expressed in E. coli formed silk-like fibers by manual-drawing. CD analysis showed that the secondary structure of Flag_R-AcSp1_R spidroin remained stable during the gradual reduction of pH from 7.0 to 5.5. The spectrum of FTIR indicated that the secondary structure of Flag_R-AcSp1_R changed from α-helix to β-sheet. The conformation change of Flag_R-AcSp1_R was similar to other spidroins in the fiber formation process. SEM analysis revealed that the mean diameter of the fibers was around 1 ~ 2 μm, and the surface was smooth and uniform. The chimeric fibers exhibited superior toughness (~33.1 MJ/m³) and tensile strength (~261.4 MPa). This study provides new insight into design of chimeric spider silks with high mechanical properties.     

Environmentally induced post‐spin property changes in spider silks: influences of web type,spidroin composition and ecology

Many spiders use silk to construct webs that must function for days at a time, whereas many other species renew their webs daily. The mechanical properties of spider silk can change after spinning under environmental stress, which could influence web function. We hypothesize that spiders spinning longer‐lasting webs produce silks composed of proteins that are more resistant to environmental stresses. The major ampullate (MA) silks of orb web spiders are principally composed of a combination of two proteins (spidroins) called MaSp1 and MaSp2. We expected spider MA silks dominated by MaSp1 to have the greatest resistance to post‐spin property change because they have high concentrations of stable crystalline β‐sheets. Some orb web spiders that spin three‐dimensional orb webs, such as Cyrtophora, have MA silks that are predominantly composed of MaSp1. Hence, we expected that the construction of three‐dimensional orb webs might also coincide with MA silk resistance to post‐spin property change. Alternatively, the degree of post‐spin mechanical property changes in different spider silks may be explained by factors within the spider's ecosystem, such as exposure to solar radiation. We exposed the MA silks of ten spider species from five genera (Nephila, Cyclosa, Leucauge, Cyrtophora, and Argiope) to ecologically high temperatures and low humidity for 4 weeks, and compared the mechanical properties of these silks with unexposed silks. Using species pairs enabled us to assess the influence of web dimensionality and MaSp composition both with and without phylogenetic influences being accounted for. We found neither the MaSp composition nor the three‐dimensionality of the orb web to be associated with the degree of post‐spin mechanical property changes in MA silk. The MA silks in Leucauge spp. are dominated by MaSp2, which we found to have the least resistance to post‐spin property change. The MA silk in Argiope spp. is also dominated by MaSp2, but has high resistance to post‐spin property change. The ancestry of Argiope is unresolved, but it is largely a tropical genus inhabiting hot, open regions that present similar stressors to silk as those of our experiment. Ecological factors thus appear to influence the vulnerability of orb web spider MA silks to post‐spin property change. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 580–588.     

An Australian webspinner species makes the finest known insect silk fibers

Aposthonia gurneyi, an Australian webspinner species, is a primitive insect that constructs and lives in a silken tunnel which screens it from the attentions of predators. The insect spins silk threads from many tiny spines on its forelegs to weave a filmy sheet. We found that the webspinner silk fibers have a mean diameter of only 65nm, an order of magnitude smaller than any previously reported insect silk. The purpose of such fine silk may be to reduce the metabolic cost of building the extensive tunnels. At the molecular level, the A. gurneyi silk has a predominantly beta-sheet protein structure. The most abundant clone in a cDNA library produced from the webspinner silk glands encoded a protein with extensive glycine-serine repeat regions. The GSGSGS repeat motif of the A. gurneyi silk protein is similar to the well-known GAGAGS repeat motif found in the heavy fibroin of silkworm silk, which also has beta-sheet structure. As the webspinner silk gene is unrelated to the silk gene of the phylogenetically distant silkworm, this is a striking example of convergent evolution.     

Araneoid egg case silk: a fibroin with novel ensemble repeat units from the black widow spider, Latrodectus hesperus

Araneoid spiders use specialized abdominal glands to manufacture up to seven different protein-based silks/glues that have diverse physical properties. The fibroin sequences that encode egg case fibers (cover silk for the egg case sac) and the secondary structure of these threads have not been previously determined. In this study, MALDI tandem TOF mass spectrometry (MS/MS) and reverse genetics were used to isolate the first egg case fibroin, named tubuliform spidroin 1 (TuSp1), from the black widow spider, Latrodectus hesperus. Real-time quantitative PCR analysis demonstrates TuSp1 is selectively expressed in the tubuliform gland. Analysis of the amino acid composition of raw egg case silk closely aligns with the predicted amino acid composition from the primary sequence of TuSp1, which supports the assertion that TuSp1 represents a major component of egg case fibers. TuSp1 is composed of highly homogeneous repeats that are 184 amino acids in length. The long stretches of polyalanine and glycine-alanine subrepeats, which account for the crystalline regions of minor ampullate and major ampullate fibers, are very poorly represented in TuSp1. However, polyserine blocks and short polyalanine stretches were highly iterated within the primary sequence, and (13)C NMR spectroscopy demonstrated that the majority of alanine was found in a beta-sheet structure in post-spun egg case silk. The TuSp1 repeat unit does not display substantial sequence similarity to any previously described fibroin genes or proteins, suggesting that TuSp1 is a highly divergent member of the spider silk gene family.     

Isolation of a clone encoding a second dragline silk fibroin. Nephila clavipes dragline silk is a two-protein fiber.

Spider dragline silk is a unique protein fiber possessing both high tensile strength and high elasticity. A partial cDNA clone for one dragline silk protein (Spidroin 1) was previously isolated. However, the predicted amino acid sequence could not account for the amino acid composition of dragline silk. We have isolated a partial cDNA clone for another dragline silk protein (Spidroin 2), demonstrating that dragline silk is composed of multiple proteins. The amino acid sequence exhibits an entirely different repetitive motif than Spidroin 1. Spidroin 2 is predicted to consist of linked beta-turns in proline-rich regions which alternate with beta-sheet regions composed of polyalanine segments. This structure for Spidroin 2 provides a model for dragline silk structure and function.     

Spidroins from the Brazilian spider Nephilengys cruentata (Araneae: Nephilidae)

Spiders produce up to six different kinds of silk, each one for a specific biological function. Spider silks are also known for their unique mechanical properties. The possibility of producing new materials with similar properties motivated research on these silk proteins (spidroins). Using expression sequence tags, we identified four spidroins produced by major ampullate, minor ampullate, flagelliform and tubuliform silk glands from the Brazilian spider Nephilengys cruentata (Araneae: Nephilidae). The new protein sequences showed substantial similarity to other spidroins previously described, with high content of alanine and glycine due to the presence of the highly repetitive motifs (polyAla, (GA)_n, (GGX)_n, (GPGGX)_n). Similarities among sequences were also observed between the different spidroins with the exception of tubuliform spidroin, which presents a unique complex amino acid sequence with high amounts of serine and low amounts of glycine.     

A CRAC-like motif in BAX sequence: relationship with protein insertion and pore activity in liposomes

Several proteins that interact with cholesterol have a highly conserved sequence, corresponding to the cholesterol recognition/interaction amino acid consensus. Since cholesterol has been proposed to modulate both oligomerization and insertion of the pro-apoptotic protein BAX, we investigated the existence of such a motif in the BAX sequence. Residues 113 to 119 of the recombinant BAX α5-helix, LFYFASK, correspond with the sequence motif described for the consensus pattern, -L/V-(X)(1-5)-Y-(X)(1-5)-R/K. Functional characterization of the point mutations, K119A, Y115F, and L113A in BAX, was performed in liposomes supplemented with cholesterol, comparing binding, integration, and pore forming activities. Our results show that the mutations Y115F and L113A changed the cholesterol-dependent insertion observed in the wild type protein. In addition, substitutions in the BAX sequence modified the concentration dependency of carboxyfluorescein release in liposomes, although neither pore activity of the wild type or of any of the mutants significantly increased in cholesterol-enriched liposomes. Thus, while it is likely that the putative CRAC motif in BAX accounts for its enhanced insertion in cholesterol-enriched liposomes; the pore forming properties of BAX did not depend on cholesterol content in the membranes, albeit those mutations changed the pore channeling activity of the protein.