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.     

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.     

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。     

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.     

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.     

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.     

蜘蛛拖丝蛋白基因的构建及在大肠杆菌中的表达

蜘蛛大壶腹线产生的拖丝是非常优良的纤维蛋白, 具有独特的强度和弹性。基于拖丝蛋白高度重复序列和部分cDNA序列, 合成蜘蛛拖丝蛋白基因单体, 通过头尾相连的构建策略, 得到拖丝蛋白多聚体, 与原核高效表达载体pET30a(+)连接, 转化大肠杆菌BLR(DE3), 用IPTG诱导表达。 表达产物经His.Bind树脂金属螯合亲和层析一步纯化, 纯度达90%以上, 表达量为20mg/L。SDS-PAGE和蛋白质印迹图谱显示表达产物分子量为37kD, 其值与氨基酸组分分析结果与理论推算值基本符合。      

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.     

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.     

Spider Glue Proteins Have Distinct Architectures Compared with Traditional Spidroin Family Members

Adhesive spider glues are required to perform a variety of tasks, including web construction, prey capture, and locomotion. To date, little is known regarding the molecular and structural features of spider glue proteins, in particular bioadhesives that interconnect dragline or scaffolding silks during three-dimensional web construction. Here we use biochemical and structural approaches to identify and characterize two aggregate gland specific gene products, AgSF1 and AgSF2, and demonstrate that these proteins co-localize to the connection joints of both webs and wrapping silks spun from the black widow spider, Latrodectus hesperus. Protein architectures are markedly divergent between AgSF1 and AgSF2, as well as traditional spider silk fibroin family members, suggesting connection joints consist of a complex proteinaceous network. AgSF2 represents a nonglycosylated 40-kDa protein that has novel internal amino acid block repeats with the consensus sequence NVNVN embedded in a glycine-rich matrix. Analysis of the amino acid sequence of AgSF1 reveals pentameric QPGSG iterations that are similar to conserved modular elements within mammalian elastin, a rubber-like elastomeric protein that interfaces with collagen. Wet-spinning methodology using purified recombinant proteins show AgSF1 has the potential to self-assemble into fibers. X-ray fiber diffraction studies performed on these synthetic fibers reveal the presence of noncrystalline domains that resemble classical rubber networks. Collectively, these data support that the aggregate gland serves to extrude a protein mixture that contains substances that allow for the self-assembly of fiber-like structures that interface with dragline silks to mediate prey capture.     

Spider silks from plants – a challenge to create native-sized spidroins

Silk threads from spiders exhibit extraordinary mechanical properties, such as superior toughness and elasticity. Spider silks consist of several different large repetitive proteins that act as the basic materials responsible for these outstanding features. The production of spider silk protein variants in plants opens up new horizons in the production and functional investigation that enable the use of spider silks in innovative material development, nanotechnology and biomedicine in the future. This review summarizes and discusses production of spider silk protein variants in plants, especially with regards to plant expression systems, purification strategies, and characteristics of spider silk variants. Furthermore, the challenge of producing native-sized recombinant spidroins in planta is outlined, presenting three different strategies for achieving these high repetitive proteins with the help of non-repetitive C-terminal domains, crosslinking transglutaminase, and self-linking inteins. The potential of these fascinating proteins in medicine is also highlighted.     

Nanoparticle self-assembly by a highly stable recombinant spider wrapping silk protein subunit

Artificial spider silk proteins may form fibers with exceptional strength and elasticity. Wrapping silk, or aciniform silk, is the toughest of the spider silks, and has a very different protein composition than other spider silks. Here, we present the characterization of an aciniform protein (AcSp1) subunit named W₁, consisting of one AcSp1 199 residue repeat unit from Argiope trifasciata. The structural integrity of recombinant W₁ is demonstrated in a variety of buffer conditions and time points. Furthermore, we show that W₁ has a high thermal stability with reversible denaturation at ∼71 °C and forms self-assembled nanoparticle in near-physiological conditions. W₁ therefore represents a highly stable and structurally robust module for protein-based nanoparticle formation.     

The evolution of cryptic spider silk: a behavioral test

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

How sequence determines elasticity of disordered proteins

How nature tunes sequences of disordered protein to yield the desired coiling properties is not yet well understood. To shed light on the relationship between protein sequence and elasticity, we here investigate four different natural disordered proteins with elastomeric function, namely: FG repeats in the nucleoporins; resilin in the wing tendon of dragonfly; PPAK in the muscle protein titin; and spider silk. We obtain force-extension curves for these proteins from extensive explicit solvent molecular dynamics simulations, which we compare to purely entropic coiling by modeling the four proteins as entropic chains. Although proline and glycine content are in general indicators for the entropic elasticity as expected, divergence from simple additivity is observed. Namely, coiling propensities correlate with polyproline II content more strongly than with proline content, and given a preponderance of glycines for sufficient backbone flexibility, nonlocal interactions such as electrostatic forces can result in strongly enhanced coiling, which results for the case of resilin in a distinct hump in the force-extension curve. Our results, which are directly testable by force spectroscopy experiments, shed light on how evolution has designed unfolded elastomeric proteins for different functions.     

Solid-state NMR relaxation studies of Australian spider silks

Solid-state NMR techniques were used to study two different types of spider silk from two Australian orb-web spider species, Nephila edulis and Argiope keyserlingi. A comparison of (13)C-T(1) and (1)H-T(1rho) solid-state NMR relaxation data of the Ala Calpha, Ala Cbeta, Gly Calpha, and carbonyl resonances revealed subtle differences between dragline and cocoon silk. (13)C-T(1rho) and (1)H-T(1) relaxation experiments showed significant differences between silks of the two species with possible structural variations. Comparison of our data to previous (13)C-T(1) relaxation studies of silk from Nephila clavipes (A. Simmons et al., Macromolecules, 1994, Vol. 27, pp. 5235-5237) also supports the finding that differences in molecular mobility of dragline silk exist between species. Interspecies differences in silk structure may be due to different functional properties. Relaxation studies performed on wet (supercontracted) and dry silks showed that the degree of hydration affects relaxation properties, and hence changes in molecular mobility are correlated with functional properties of silk.     

Contribution of cell surface hydrophobicity protein 1 (Csh1p) to virulence of hydrophobic Candida albicans serotype A cells

The CSH1 gene product is the first protein implicated to affect the phenotype of cell surface hydrophobicity in Candida albicans. Ablation of expression of CSH1 resulted in a 75% loss of the cell surface hydrophobicity (CSH) phenotype. When the C. albicans csh1 knockout derivative was cultured from frozen stocks, it had reacquired CSH levels similar to the parent strain and isogenic reintegrant in the absence of Csh1p re-expression through an unknown mechanism. Prior to reacquisition of CSH, the knockout was less adherent to fibronectin than the parent. Comparison of the csh1 knockout and CSH1 reintegrant in a hematogenous dissemination model allows analysis of Csh1p contribution to virulence using matched strains with similar levels of CSH. No statistical significance between the knockout and reintegrant was found in virulence based on median day of survival, although a reproducible delay in onset of lethal infection for the knockout was observed. A modest difference in mucosal colonization in a vaginal infection model was also observed between the knockout and reintegrant. The present study demonstrates that Csh1p contributes to virulence of C. albicans in mice, but other gene products also contribute to the CSH phenotype and virulence.     

Molecular characterization and evolutionary study of spider tubuliform (eggcase) silk protein

As a result of hundreds of millions of years of evolution, orb-web-weaving spiders have developed the use of seven different silks produced by different abdominal glands for various functions. Tubuliform silk (eggcase silk) is unique among these spider silks due to its high serine and very low glycine content. In addition, tubuliform silk is the only silk produced just during a short period of time, the reproductive season, in the spider's life. To understand the molecular characteristics of the proteins composing this silk, we constructed tubuliform-gland-specific cDNA libraries from three different spider families, Nephila clavipes, Argiope aurantia, and Araneus gemmoides. Sequencing of tubuliform silk cDNAs reveals the repetitive architecture of its coding sequence and novel amino acid motifs. The inferred protein, tubuliform spidroin 1 (TuSp1), contains highly homogenized repeats in all three spiders. Amino acid composition comparison of the predicted tubuliform silk protein sequence to tubuliform silk indicates that TuSp1 is the major component of tubuliform silk. Repeat unit alignment of TuSp1 among three spider species shows high sequence conservation among tubuliform silk protein orthologue groups. Sequence comparison among TuSp1 repetitive units within species suggests intragenic concerted evolution, presumably through gene conversion and unequal crossover events. Comparative analysis demonstrates that TuSp1 represents a new orthologue in the spider silk gene family.     

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.     

Evidence for diet effects on the composition of silk proteins produced by spiders

Silks are highly expressed, secreted proteins that represent a substantial metabolic cost to the insects and spiders that produce them. Female spiders in the superfamily Araneoidea (the orb-spinning spiders and their close relatives) spin six different kinds of silk (three fibroins and three fibrous protein glues) that differ in amino acid content and protein structure. In addition to this diversity in silks produced by different glands, we found that individual spiders of the same species can spin dragline silks (drawn from the spider's ampullate gland) that vary in content as well. Freely foraging ARGIOPE: argentata (Araneae: Araneoidea), collected from 13 Caribbean islands, produced dragline silk that showed an inverse relationship between the amount of serine and glycine they contained. X-ray microdiffraction of the silks localized these differences to the amorphous regions of the protein that are thought to lend silks their elasticity. The crystalline regions of the proteins, which lend silks their strength, were unaffected. Laboratory experiments with ARGIOPE: keyserlingi suggested that variation in silk composition reflects the type of prey the spiders were fed but not the total amount of prey they received. Hence, it may be that the amino acid content (and perhaps the mechanical properties) of dragline silk spun by ARGIOPE: directly reflect the spiders' diet. The ability to vary silk composition and, possibly, function is particularly important for organisms that disperse broadly, such as Argiope, and that occupy diverse habitats with diverse populations of prey.