Protein and amino acid composition of silks from the cob weaver, Latrodectus hesperus (black widow)

The silks from the cob weaving spider, Latrodectus hesperus (black widow), have been examined with the goal of expanding our understanding of the relationship between the protein structure and mechanical performance of these unique biomaterials. The scaffolding, dragline and inner egg case silks each appear to be distinct fibers based on mole percent amino acid composition and polypeptide composition. Further, we find that the amino acid composition of dragline and egg case silk are similar to the analogous silks produced by orb weaving spiders, while scaffolding silk may represent a novel silk. The black widow silks are comprised of multiple high molecular weight polypeptides, however, the egg case and scaffolding silks also contain some smaller polypeptides.     

Characterization and expression of a cDNA encoding a tubuliform silk protein of the golden web spider Nephila antipodiana

Spider silks are renowned for their excellent mechanical properties. Although several spider fibroin genes, mainly from dragline and capture silks, have been identified, there are still many members in the spider fibroin gene family remain uncharacterized. In this study, a novel silk cDNA clone from the golden web spider Nephila antipodiana was isolated. It is serine rich and contains two almost identical fragments with one varied gap region and one conserved spider fibroin-like C-terminal domain. Both in situ hybridization and immunoblot analyses have shown that it is specifically expressed in the tubuliform gland. Thus, it likely encodes the silk fibroin from the tubuliform gland, which supplies the main component of the inner egg case. Unlike other silk proteins, the protein encoded by the novel cDNA in water solution exhibits the characteristic of an alpha-helical protein, which implies the distinct property of the egg case silk, though the fiber of tubuliform silk is mainly composed of beta-sheet structure. Its sequence information facilitates elucidation of the evolutionary history of the araneoid fibroin genes.     

悦目金蛛和棒络新妇卵袋丝物理化学结构表征及其力学性能研究

蜘蛛丝是一种具有优良机械性能的天然动物蛋白纤维,它特有的结构和性能与其生物学功能密切相关。作者采用氨基酸自动分析仪、傅立叶转换红外光谱仪、扫描电镜和电子单纤强力仪对悦目金蛛（Argiope amoena）和棒络新妇（Nephila clavata）的卵袋丝进行了物理化学结构表征与力学性能的研究,结果表明两种蜘蛛卵袋均由微米级柱状腺丝、大壶状腺丝、亚微米级或纳米级葡萄状腺丝构成。卵袋丝的表面形貌特征、极性氨基酸含量、大侧链与小侧链氨基酸的比值、无定型区、β-折叠结构与结晶结构的含量等氨基酸组成种类与蛋白质二级结构特征,均满足各自生物学功能对断裂强度、延展性、初始模量等力学性能的要求。     

Variability in the mechanical properties of spider silks on three levels: interspecific, intraspecific and intraindividual.

The mechanical characteristics of dragline silks collected from a range of spiders drawn from the Argiopidae, Tetragnathidae, Theridiidae and Pisauridae displayed significant inter- and intraspecific differences. Dragline silks of the same species could show considerable variability probably dependent upon spider condition: starvation, for example, lead to decreased breaking elongation in Nephila edulis. Environmental conditions such as reeling speed affected silk properties such that (i) breaking elongation decreased, (ii) breaking stress increased and (iii) Young's modulus increased with increasing reeling speed. However, N. edulis and Araneus diadematus responded differently to the reeling speed treatments suggesting differences in basic silk properties.     

悦目金蛛3种蛛丝超微结构和理化特性的初步研究

利用扫描电镜、氨基酸分析仪、X-衍射仪和单纤维电子强力仪分别对悦目金蛛Argiopeamoena拖丝、网框丝和卵袋丝的超微结构和理化特性进行了测试和观察。结果表明,悦目金蛛卵袋不是由一种结构均一的丝纤维构成,而是由直径相差悬殊的Ⅰ型卵袋丝和Ⅱ型卵袋丝2种丝纤维共同组成,该结果对卵袋丝仅由管状腺产生的观点提出了疑问。在氨基酸组成上悦目金蛛拖丝和网框丝相似,但其卵袋丝的氨基酸组成与拖丝和网框丝相比差别明显。另外还发现卵袋丝的强度、结晶度大于拖丝和网框丝,而它的延伸性能却不及拖丝和网框丝。     

Novel In Silico Analyses of Repetitive Spider Silk Sequences to Understand the Evolution and Mechanical Properties of Fibrous Protein Materials

The mechanical properties of spider silks have diverged as spiders have diversely speciated. Because the main components of silks are proteins, it is valuable to investigate their sequences. However, silk sequences have been regarded as difficult information to analyze due to their imbalance and imperfect tandem repeats (ITR). Here, an in silico approach is applied to systemically analyze a group of silk sequences. It is found that every time new spider groups emerge, unique trimer motifs appear. These trimer motifs are used to find additional clues of evolution and to determine relationships with mechanical properties. For the first time, crucial evidence is provided that shows silk sequences coevolved with spider species and the mechanical properties of their fibers to adapt to new living environments. This novel approach can be used as a platform for analyzing other groups of ITR‐harboring proteins and to obtain information for the design of tailor‐made fibrous protein materials.     

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.     

Analyis of structure/property relationships in silkworm (Bombyx mori) and spider dragline (Nephila edulis) silks using Raman spectroscopy

The molecular deformation of both silkworm (Bombyx mori) and spider dragline (Nephila edulis) silks has been studied using a combination of mechanical deformation and Raman spectroscopy. The stress/strain curves for both kinds of silk showed elastic behavior followed by plastic deformation. It was found that both materials have well-defined Raman spectra and that some of the bands in the spectra shift to lower frequency under the action of tensile stress or strain. The band shift was linearly dependent upon stress for both types of silk fiber. This observation provides a unique insight into the effect of tensile deformation upon molecular structure and the relationship between structure and mechanical properties. Two similar bands in the Raman spectra of both types of silk in the region of 1000-1300 cm(-1) had significant identical rates of Raman band shift of about 7 cm(-1)/GPa and 14 cm(-1)/GPa demonstrating the similarity between the silk fibers from two different animals.     

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.     

Conserved C-terminal domain of spider tubuliform spidroin 1 contributes to extensibility in synthetic fibers

Spider silk is renowned for its extraordinary mechanical properties, having a balance of high tensile strength and extensibility. To date, the majority of studies have focused on the production of dragline silks from synthetic spider silk gene products. Here we report the first mechanical analysis of synthetic egg case silk fibers spun from the Latrodectus hesperus tubuliform silk proteins, TuSp1 and ECP-2. We provide evidence that recombinant ECP-2 proteins can be spun into fibers that display mechanical properties similar to other synthetic spider silks. We also demonstrate that silks spun from recombinant thioredoxin-TuSp1 fusion proteins that contain the conserved C-terminal domain exhibit increased extensibility and toughness when compared to the identical fibers spun from fusion proteins lacking the C-terminus. Mechanical analyses reveal that the properties of synthetic tubuliform silks can be modulated by altering the postspin draw ratios of the fibers. Fibers subject to increased draw ratios showed elevated tensile strength and decreased extensibility but maintained constant toughness. Wide-angle X-ray diffraction studies indicate that postdrawn fibers containing the C-terminal domain of TuSp1 have more amorphous content when compared to fibers lacking the C-terminus. Taken together, these studies demonstrate that recombinant tubuliform spidroins that contain the conserved C-terminal domain with embedded protein tags can be effectively spun into fibers, resulting in similar tensile strength but increased extensibility relative to nontagged recombinant dragline silk proteins spun from equivalently sized proteins.     

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.     

Proline and processing of spider silks

Major ampullate (MAA) silks from a variety of spider species were collected by artificial silking that adjusted the samples to have similar breaking strains. Those silks are highly comparable in post-yield mechanical properties, but their supercontraction behaviors and initial moduli vary in large ranges and both correlate with the content of one amino acid, proline. These relationships, in combination with protein sequence data, support the hypothesis that the proline-related motif, that is, GPGXX, may play a key role in silk. This also explains the interspecific variability of spider dragline silk. Moreover, MAA silks from three representative species were prepared in a range of processing conditions and their mechanical properties were compared. Our results indicate how chemical compositions, coupled with processing conditions, shape the mechanical properties of the spider silk.     

Novel molecular and mechanical properties of egg case silk from wasp spider, Argiope bruennichi

Araneoid spiders use specialized abdominal glands to produce up to seven different protein-based silks/glues that have various mechanical properties. To date, the fibroin sequences encoding egg case fibers have not been fully determined. To gain further understanding of a recently reported spider silk protein gene family, several novel strategies were utilized in this study to isolate two full-length cDNAs of egg case silk proteins, cylindrical silk protein 1 (CySp1, 9.1 kb) and cylindrical silk protein 2 (CySp2, 9.8 kb), from the wasp spider, Argiope bruennichi. Northern blotting analysis demonstrated that CySp1 and CySp2 are selectively expressed in the cylindrical glands. The amino acid composition of raw egg case silk was closely consistent with the deduced amino acid composition based on the sequences of CySp1 and CySp2, which supports the assertion that CySp1 and CySp2 represent two major components of egg case silk. CySp1 and CySp2 are primarily composed of remarkable homogeneous assemble repeats that are 180 residues in length and consist of several complex subrepeats, and they contain highly homologous C-termini and markedly different N-termini. Our results suggest a possible link between CySp1 and CySp2. In addition, comparisons of stress/strain curves for dragline and egg case silk from Argiope bruennichi showed obvious differences in ultimate strength and extensibility, and similarities in toughness.     

Molecular studies of a novel dragline silk from a nursery web spider, Euprosthenops sp. (Pisauridae)

Various spider species produce dragline silks with different mechanical properties. The primary structure of silk proteins is thought to contribute to the elasticity and strength of the fibres. Previously published work has demonstrated that the dragline silk of Euprosthenops sp. is stiffer then comparable silk of Nephila edulis, Araneus diadematus and Latrodectus mactans. Our studies of Euprosthenops dragline silk at the molecular level have revealed that nursery web spider fibroin has the highest polyalanine content among previously characterised silks and this is likely to contribute to the superior qualities of pisaurid dragline.     

Microdissection of Black Widow Spider Silk-producing Glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring ^1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion.Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel ³. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences ⁴. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands.Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] ^5,6, tubuliform [synthesizes egg case silk] ^7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] ⁹ and pyriform [produces attachment disc silk] ¹⁰. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.     

NMR characterization of native liquid spider dragline silk from Nephila edulis

Solid spider dragline silk is well-known for its mechanical properties. Nonetheless a detailed picture of the spinning process is lacking. Here we report NMR studies on the liquid silk within the wide sac of the major ampullate (m.a.) gland from the spider Nephila edulis. The resolution in the NMR spectra is shown to be significantly improved by the application of magic-angle spinning (MAS). From the narrow width of the resonance lines and the chemical shifts observed, it is concluded that the silk protein within the wide sac of the m.a. gland is dynamically disordered throughout the molecule in the sense that each amino acid of a given type senses an identical environment, on average. The NMR data obtained are consistent with an isotropic liquid phase.     

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.     

Spinning activity of the spider Trogloneta granulum (Araneae, Mysmenidae): web, cocoon, cocoon handling behaviour, draglines and attachment discs

The spider species Trogloneta granulum, which in the wild lives inside scree slopes, builds three-dimensional orb webs. During egg-laying and egg sac building, the females stay with their dorsa down at the central part of the web. In this process, the hub is used as a platform. The threads of the hub are not incorporated into the silk cover of the egg sac. The silk wall of the egg sac is very thin, with all the silken threads constituting it having a uniform ultrastructure. The silk wall of the egg sac and the spinnerets are permanently linked by a dragline. Draglines produced by T. granulum affect the direction of movements of the female carrying its cocoon. Egg sacs are handled using draglines. The low number of piriform glands leads to the formation of very simple attachment discs, which fix the individual threads to the substratum. Thread bundles are attached to the substratum by means of accumulated attachment discs.