Comparison of fibroin cDNAs from webspinning insects: insight into silk formation and function

Embiopterans (webspinning insects) are renowned for their prolific use of silk. These organisms spin silk to construct elaborate networks of tubes in which they live, forage, and reproduce. The silken galleries are essential for protecting these soft-bodied insects from predators and other environmental hazards. Despite the ecological importance of embiopteran silk, very little is known about its constituent proteins. Here, we characterize the silk protein cDNAs from four embiopteran species to better understand the function and evolution of these adaptive molecules. We show that webspinner fibroins (silk proteins) are highly repetitive in sequence and possess several conserved characteristics, despite differences in habitat preferences across species. The most striking similarities are in the codon usage biases of the fibroin genes, particularly in the repetitive regions, as well as sequence conservation of the carboxyl-terminal regions of the fibroins. Based on analyses of the silk genes, we propose hypotheses regarding codon bias and its effect on the translation and replication of these unusual genes. Furthermore, we discuss the significance of specific fibroin motifs to the mechanical and structural characteristics of silk fibers. Lastly, we report that the conservation of webspinner fibroin carboxyl-terminal regions suggests that fiber formation may occur through a mechanism analogous to that found in Lepidoptera. From these results, insight is gained into the tempo and mode of evolution that has shaped embiopteran fibroins.     

Structures of Bombyx mori and Samia cynthia ricini silk fibroins studied with solid-state NMR

There are many kinds of silks spun by silkworms and spiders, which are suitable to study the structure-property relationship for molecular design of fibers with high strength and high elasticity. In this review, we mainly focus on the structural determination of two well-known silk fibroin proteins that are from the domesticated silkworm, Bombyx mori, and the wild silkworm, Samia cynthia ricini, respectively. The structures of B. mori silk fibroin before and after spinning were determined by using an appropriate model peptide, (AG)(15), with several solid-state NMR methods; (13)C two-dimensional spin-diffusion solid-state NMR and rotational echo double resonance (REDOR) NMR techniques along with the quantitative use of the conformation-dependent (13)C CP/MAS chemical shifts. The structure of S. c. ricini silk fibroin before spinning was also determined by using a model peptide, GGAGGGYGGDGG(A)(12)GGAGDGYGAG, which is a typical repeated sequence of the silk fibroin, with the solid-state NMR methods. The transition from the structure of B. mori silk fibroin before spinning to the structure after spinning was studied with molecular dynamics calculation by taking into account several external forces applied to the silk fibroin in the silkworm.     

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.     

Structural disorder in silk proteins reveals the emergence of elastomericity

Spider silks combine basic amino acids into strong and versatile fibers where the quality of the elastomer is attributed to the interaction of highly adapted protein motifs with a complex spinning process. The evaluation, however, of the interaction has remained elusive. Here, we present a novel analysis to study silk formation by examining the secondary structures of silk proteins in solution. Using the seven different silks of Nephila edulis as a benchmark system, we define a structural disorder parameter (the folding index, gamma). We found that gamma is highly correlated with the ratio of glycine present. Testing the correlation between glycine content and the folding index (gamma) against a selected range of silks, we find quantitatively that, in order to achieve specialization with changes in mechanical performance, the spider's silks require higher structural flexibility at the expense of reduced stability and consequently an increased conversion-energy cost. Taken together, our biophysical and evolutionary findings reveal that silk elastomericity evolved in tandem with specializations in the process of silk spinning.     

Spider flagelliform silk: lessons in protein design, gene structure, and molecular evolution

Spiders spin multiple types of silks that are renowned for their superb mechanical properties. Flagelliform silk, used in the capture spiral of an orb-web, is one of the few silks characterized by both cDNA and genomic DNA data. This fibroin is composed of repeating ensembles of three types of amino acid sequence motifs. The predominant subrepeat, GPGGX, likely forms a beta-turn, and tandem arrays of these turns are thought to create beta-spirals. These spring-like helices may be critical for the exceptional ability of capture silk to stretch and recoil. Each ensemble of motifs was found to correspond to a different exon within the flagelliform gene. The pattern of sequence similarity among exons indicates intragenic concerted evolution. Surprisingly, the introns between the iterated exons are also homogenized with each other. This unusual molecular architecture in the flagelliform silk gene has implications for the evolution and maintenance of spider silk proteins.     

悦目金蛛拖牵丝力学性能的变异

蜘蛛丝作为一种具有优良机械性能的天然动物蛋白纤维,其特有的结构和机械性能与其生物学功能密切相关。由大壶状腺纺出的拖牵丝在蜘蛛的行走、建网、捕食、逃生、繁殖等多种生命活动中均发挥了重要的功能,其机械性能会受到多种内外因素相互作用的影响。本文对在不同体重、不同猎物饲养和不同营养状态3种条件下人工抽出的悦目金蛛(Argiope amoena)拖牵丝与其不同单丝间的力学性能进行了比较研究。结果表明,悦目金蛛拖牵丝的力学性能在组间、组内不同个体,以及同一个体不同丝纤维间变异都较大。随着蜘蛛个体的增大,蛛丝横截面直径逐渐增大,这会使得蛛丝的力学性能更好,便于作为救命索的拖牵丝在遇到危险时承受蜘蛛体重;蜘蛛在经过1个月的饥饿后,蛛丝在屈服点附近的力学性能并未发生显著变化,而断裂点应变和断裂能均显著减小,同时也表明无论对于作为救命索还是网丝,拖牵丝的弹性形变性能在与蛛丝相关的微观进化中要优先于塑性形变。这是蜘蛛在能量摄入受到限制时对拖牵丝的投入权衡的结果。     

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.     

In vivo effects of metal ions on conformation and mechanical performance of silkworm silks

BackgroundThe mechanism of silk fiber formation is of particular interest. Although in vitro evidence has shown that metal ions affect conformational transitions of silks, the in vivo effects of metal ions on silk conformations and mechanical performance are still unclear.MethodsThis study explored the effects of metal ions on silk conformations and mechanical properties of silk fibers by adding K⁺ and Cu^2 + into the silk fibroin solutions or injecting them into the silkworms. Aimed by CD analysis, FTIR analysis, and mechanical testing, the conformational and mechanical changes of the silks were estimated. By using BION Web Server, the interactions of K⁺ and N-terminal of silk fibroin were also simulated.ResultsWe presented that K⁺ and Cu^2 + induced the conformational transitions of silk fibroin by forming β-sheet structures. Moreover, the mechanical parameters of silk fibers, such as strength, toughness and Young's modulus, were also improved after K⁺ or Cu^2 + injection. Using BION Web Server, we found that potassium ions may have strong electrostatic interactions with the negatively charged residues.ConclusionWe suggest that K⁺ and Cu^2 + play crucial roles in the conformation and mechanical performances of silks and they are involved in the silk fiber formation in vivo.General significanceOur results are helpful for clarifying the mechanism of silk fiber formation, and provide insights for modifying the mechanical properties of silk fibers.     

Spider minor ampullate silk proteins are constituents of prey wrapping silk in the cob weaver Latrodectus hesperus

Spiders spin high performance fibers with diverse biological functions and mechanical properties. Molecular and biochemical studies of spider prey wrapping silks have revealed the presence of the aciniform silk fibroin AcSp1-like. In our studies we demonstrate the presence of a second distinct polypeptide present within prey wrapping silk. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called MiSp1-like and demonstrate that its protein product is a constituent of prey wrap silks from the black widow spider, Latrodectus hesperus. BLAST searches of the NCBInr protein database using the amino acid sequence of MiSp1-like revealed similarity to the conserved C-terminal domain of silk family members. In particular, MiSp1-like showed the highest degree of sequence similarity to the nonrepetitive C-termini of published orb-weaver minor ampullate fibroin molecules. Analysis of the internal amino acid sequence of the black widow MiSp1-like revealed polyalanine stretches interrupted by glycine residues and glycine-alanine couplets within MiSp1-like as well as repeats of the heptameric sequence AGGYGQG. Real-time quantitative PCR analysis demonstrates that the MiSp1-like gene displays a minor ampullate gland-restricted pattern of expression. Furthermore, amino acid composition analysis, coupled with scanning electron microscopy of raw wrapping silk, supports the assertion that minor ampullate silks are important constituents of black widow spider prey wrap silk. Collectively, our findings provide direct molecular evidence for the involvement of minor ampullate fibroins in swathing silks and suggest composite materials play an important role in the wrap attack process for cob-weavers.     

Conservation of essential design features in coiled coil silks

Silks are strong protein fibers produced by a broad array of spiders and insects. The vast majority of known silks are large, repetitive proteins assembled into extended beta-sheet structures. Honeybees, however, have found a radically different evolutionary solution to the need for a building material. The 4 fibrous proteins of honeybee silk are small ( approximately 30 kDa each) and nonrepetitive and adopt a coiled coil structure. We examined silks from the 3 superfamilies of the Aculeata (Hymenoptera: Apocrita) by infrared spectroscopy and found coiled coil structure in bees (Apoidea) and in ants (Vespoidea) but not in parasitic wasps of the Chrysidoidea. We subsequently identified and sequenced the silk genes of bumblebees, bulldog ants, and weaver ants and compared these with honeybee silk genes. Each species produced orthologues of the 4 small fibroin proteins identified in honeybee silk. Each fibroin contained a continuous predicted coiled coil region of around 210 residues, flanked by 23-160 residue length N- and C-termini. The cores of the coiled coils were unusually rich in alanine. There was extensive sequence divergence among the bee and ant silk genes (<50% similarity between the alignable regions of bee and ant sequences), consistent with constant and equivalent divergence since the bee/ant split (estimated to be 155 Myr). Despite a high background level of sequence diversity, we have identified conserved design elements that we propose are essential to the assembly and function of coiled coil silks.     

Silks produced by insect labial glands

Insect silks are secreted from diverse gland types; this chapter deals with the silks produced by labial glands of Holometabola (insects with pupa in their life cycle). Labial silk glands are composed of a few tens or hundreds of large polyploid cells that secrete polymerizing proteins which are stored in the gland lumen as a semi?liquid gel. Polymerization is based on weak molecular interactions between repetitive amino acid motifs present in one or more silk proteins; cross?linking by disulfide bonds may be important in the silks spun under water. The mechanism of long?term storage of the silk dope inside the glands and its conversion into the silk fiber during spinning is not fully understood. The conversion occurs within seconds at ambient temperature and pressure, under minimal drawing force and in some cases under water. The silk filament is largely built of proteins called fibroins and in Lepidoptera and Trichoptera coated by glue?type proteins known as sericins. Silks often contain small amounts of additional proteins of poorly known function. The silk components controlling dope storage and filament formation seem to be conserved at the level of orders, while the nature of polymerizing motifs in the fibroins, which determine the physical properties of silk, differ at the level of family and even genus. Most silks are based on fibroin β?sheets interrupted with other structures such as α?helices but the silk proteins of certain sawflies have predominantly a collagen?like or polyglycine II arrangement and the silks of social Hymenoptera are formed from proteins in a coiled coil arrangement.     

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.     

New process to form a silk fibroin porous 3-D structure

A new process to form fibroin spongy porous 3-D structure is reported herein. The process involves freezing and thawing fibroin aqueous solution in the presence of a small amount of an organic solvent. The process requires no freeze-drying, chemical cross-linking, or the aid of other polymeric materials. The solvent concentration, fibroin concentration, freezing temperature, and freezing duration affect the sponge formation, its porous structure, and its mechanical properties. Measurements by XRD and FTIR indicate that silk I and silk II crystalline structures exist in the fibroin sponge and that the secondary structure of fibroin is transformed to a beta-sheet from a random coil during this process. The tensile strength decreased slightly, but the fibroin sponge showed no deformation after autoclaving. Therefore, the fibroin sponge was sterilized using an autoclave. For 3 weeks, MC3T3 cells proliferated in the sterilized fibroin sponge. The fibroin sponge formed by this new process is applicable as a tissue-engineering scaffold because it is formed from biocompatible pure silk fibroin and offers both porous structure and mechanical properties that are suitable for cell growth and handling.     

Some observations on the structure and function of the spinning apparatus in the silkworm Bombyx mori

Silkworm silk has outstanding mechanical properties despite being spun at room temperature and from aqueous solution. Although it has been proposed that fiber formation is mainly induced by shearing and extensional flow in the spinneret, the detailed structure and function of the spinning apparatus of Bombyx mori silkworms are still not fully elucidated. In this paper we describe three aspects of the functional microanatomy of the spinning apparatus: changes in the diameter of the silk gland duct with distance along the duct, how the birefringence of the fibroin changes as it flows down the duct, and the detailed three-dimensional structure of the silk press and related structures. The existence of a double escaped nematic liquid crystal texture in the fibroin in a region of the duct is described. After this region the birefringence suddenly disappeared until the start of an internal draw down taper which commenced just before the silk press. In the internal draw down taper the birefringence increased dramatically to an asymptotic value as a thread was drawn from the fibroin gel. The structure of the silk press suggests that it acts as a restriction die whose diameter can be regulated.     

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.     

Flexibility regeneration of silk fibroin in vitro

Although natural silk fibers have excellent strength and flexibility, the regenerated silk materials generally become brittle in the dry state. How to reconstruct the flexibility for silk fibroin has bewildered scientists for many years. In the present study, the flexible regenerated silk fibroin films were achieved by simulating the natural forming and spinning process. Silk fibroin films composed of silk I structure were first prepared by slow drying process. Then, the silk fibroin films were stretched in the wet state, following the structural transition from silk I to silk II. The difference between the flexible film and different brittle regenerated films was investigated to reveal the critical factors in regulating the flexibility of regenerated silk materials. Compared with the methanol-treated silk films, although having similar silk II structure and water content, the flexible silk films contained more bound water rather than free water, implying the great influence of bound water on the flexibility. Then, further studies revealed that the distribution of bound water was also a critical factor in improving silk flexibility in the dry state, which could be regulated by the nanoassembly of silk fibroin. Importantly, the results further elucidate the relation between mechanical properties and silk fibroin structures, pointing to a new mode of generating new types of silk materials with enhanced mechanical properties in the dry state, which would facilitate the fabrication and application of regenerated silk fibroin materials in different fields.     

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.     

Structural studies of Bombyx mori silk fibroin during regeneration from solutions and wet fiber spinning

Regenerated silk fibroin materials show properties dependent on the methods used to process them. The molecular structures of B. mori silk fibroin both in solution and in solid states were studied and compared using X-ray diffraction, FTIR, and (13)C NMR spectroscopy. Some portion of fibroin protein molecules dissolved in formic acid already have a beta-sheet structure, whereas those dissolved in TFA have some helical conformation. Moreover, fibroin molecules were spontaneously assembled into an ordered structure as the acidic solvents were removed from the fibroin-acidic solvent systems. This may be responsible for the improved physical properties of regenerated fibroin materials from acidic solvents. Regenerated fibroin materials have shown poor mechanical properties and brittleness compared to their original form. These problems were technically solved by improving the fiber forming process according to a method reported here. The regenerated fibroin fibers showed much better mechanical properties compared to the native silk fiber and their physical and chemical properties were characterized by X-ray diffraction, solid state (13)C NMR spectroscopy, SinTech tensile testing, and SEM.