Design of superior spider silk: from nanostructure to mechanical properties

Spider dragline silk is of practical interest because of its excellent mechanical properties. However, the structure of this material is still largely unknown. In this article, we report what we believe is a new model of the hierarchical structure of silk based on scanning electron microscope and atomic force microscope images. This hierarchical structure includes beta-sheet, polypeptide chain network, and silk fibril. It turns out that an exceptionally high strength of the spider dragline silk can be obtained by decreasing the size of the crystalline nodes in the polypeptide chain network while increasing the degree of orientation of the crystalline nodes. Based on this understanding, how the reeling speed affects mechanical properties of spider dragline silk can be understood properly. Hopefully, the understanding obtained in this study will shed light on the formation of spider silk, and consequently, on the principles for the design of ultrastrong silk.     

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.     

The effect of spinning conditions on the mechanics of a spider's dragline silk.

We studied the mechanical properties of dragline threads of the edible golden silk spider Nephila edulis that are produced under spinning speeds ranging from 0.1 to 400 mm s(-1) and temperatures ranging from 5 to 40 degrees C. These conditions affected the silk in all of the mechanical traits we tested (strain at breaking, breaking energy, initial Young's modulus and point of yielding). We argue that both trade-offs (between mechanical properties) and constraints (in the manufacturing process) have a large role in defining spider silk fibres.     

Spider silk aging: initial improvement in a high performance material followed by slow degradation

Spider silk possesses a unique combination of high tensile strength and elasticity resulting in extraordinarily tough fibers, compared with the best synthetic materials. However, the potential application of spider silk and biomimetic fibers depends upon retention of their high performance under a variety of conditions. Here, we report on changes in the mechanical properties of dragline and capture silk fibers from several spider species over periods up to 4 years of benign aging. We find an improvement in mechanical performance of silk fibers during the first year of aging. Fibers rapidly decrease in diameter, suggesting an increase in structural alignment and organization of molecules. One-year old silk also is stiffer and has higher stress at yield than fresh silk, whereas breaking force, elasticity, and toughness either improve or are unaffected by early aging. However, 4-year old silk shows signs of degradation as the breaking load, elasticity, and toughness are all lower than in fresh silk. Aging, however, does not reduce the tensile strength of silk. These data suggest initially rapid reorganization and tighter packaging of molecules within the fiber, followed by longer-term decomposition. We hypothesize that possibly the breakdown of amino acids via emission of ammonia gas, as is seen in long-term aging of museum silkworm fabrics, may contribute. Degradation of spider silk under benign conditions may be a concern for efforts to construct and utilize biomimetic silk analogs. However, our findings suggest an initial improvement in mechanical performance and that even old spider silk still retains impressive mechanical performance. J. Exp. Zool. 309A:494-504, 2008. (c) 2008 Wiley-Liss, Inc.     

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.     

Interactions between Spider Silk and Cells – NIH/3T3 Fibroblasts Seeded on Miniature Weaving Frames

Background

Several materials have been used for tissue engineering purposes, since the ideal matrix depends on the desired tissue. Silk biomaterials have come to focus due to their great mechanical properties. As untreated silkworm silk has been found to be quite immunogenic, an alternative could be spider silk. Not only does it own unique mechanical properties, its biocompatibility has been shown already in vivo. In our study, we used native spider dragline silk which is known as the strongest fibre in nature.

Methodology/Principal Findings

Steel frames were originally designed and manufactured and woven with spider silk, harvesting dragline silk directly out of the animal. After sterilization, scaffolds were seeded with fibroblasts to analyse cell proliferation and adhesion. Analysis of cell morphology and actin filament alignment clearly revealed adherence. Proliferation was measured by cell count as well as determination of relative fluorescence each after 1, 2, 3, and 5 days. Cell counts for native spider silk were also compared with those for trypsin-digested spider silk. Spider silk specimens displayed less proliferation than collagen- and fibronectin-coated cover slips, enzymatic treatment reduced adhesion and proliferation rates tendentially though not significantly. Nevertheless, proliferation could be proven with high significance (p<0.01).

Conclusion/Significance

Native spider silk does not require any modification to its application as a biomaterial that can rival any artificial material in terms of cell growth promoting properties. We could show adhesion mechanics on intracellular level. Additionally, proliferation kinetics were higher than in enzymatically digested controls, indicating that spider silk does not require modification. Recent findings concerning reduction of cell proliferation after exposure could not be met. As biotechnological production of the hierarchical composition of native spider silk fibres is still a challenge, our study has a pioneer role in researching cellular mechanics on native spider silk fibres.     

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.     

Synthetic Spider Silk Production on a Laboratory Scale

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.     

蜘蛛丝蛋白研究进展

由于蜘蛛丝蛋白分子高度重复的一级结构、特殊的溶解特性和分子折叠行为以及具有形成非凡力学特性丝纤维的能力而引人注目。本文从蛛丝蛋白基因、天然蛛丝形成过程、蛛丝蛋白的基因工程生产及蛛丝蛋白的应用前景等几个方面着重介绍了近20年来对蛛丝蛋白的研究进展。围绕蛛丝蛋白展开的研究将有助于揭示蛋白质一级结构、蛋白质分子折叠与蛋白质大分子特性之间的内在联系。     

Bundles of Spider Silk,Braided into Sutures,Resist Basic Cyclic Tests: Potential Use for Flexor Tendon Repair

Repair success for injuries to the flexor tendon in the hand is often limited by the in vivo behaviour of the suture used for repair. Common problems associated with the choice of suture material include increased risk of infection, foreign body reactions, and inappropriate mechanical responses, particularly decreases in mechanical properties over time. Improved suture materials are therefore needed. As high-performance materials with excellent tensile strength, spider silk fibres are an extremely promising candidate for use in surgical sutures. However, the mechanical behaviour of sutures comprised of individual silk fibres braided together has not been thoroughly investigated. In the present study, we characterise the maximum tensile strength, stress, strain, elastic modulus, and fatigue response of silk sutures produced using different braiding methods to investigate the influence of braiding on the tensile properties of the sutures. The mechanical properties of conventional surgical sutures are also characterised to assess whether silk offers any advantages over conventional suture materials. The results demonstrate that braiding single spider silk fibres together produces strong sutures with excellent fatigue behaviour; the braided silk sutures exhibited tensile strengths comparable to those of conventional sutures and no loss of strength over 1000 fatigue cycles. In addition, the braiding technique had a significant influence on the tensile properties of the braided silk sutures. These results suggest that braided spider silk could be suitable for use as sutures in flexor tendon repair, providing similar tensile behaviour and improved fatigue properties compared with conventional suture materials.     

Microbial production of amino acid-modified spider dragline silk protein with intensively improved mechanical properties

Spider dragline silk is a remarkably strong fiber with impressive mechanical properties, which were thought to result from the specific structures of the underlying proteins and their molecular size. In this study, silk protein 11R26 from the dragline silk protein of Nephila clavipes was used to analyze the potential effects of the special amino acids on the function of 11R26. Three protein derivatives, ZF4, ZF5, and ZF6, were obtained by site-directed mutagenesis, based on the sequence of 11R26, and among these derivatives, serine was replaced with cysteine, isoleucine, and arginine, respectively. After these were expressed and purified, the mechanical performance of the fibers derived from the four proteins was tested. Both hardness and average elastic modulus of ZF4 fiber increased 2.2 times compared with those of 11R26. The number of disulfide bonds in ZF4 protein was 4.67 times that of 11R26, which implied that disulfide bonds outside the poly-Ala region affect the mechanical properties of spider silk more efficiently. The results indicated that the mechanical performances of spider silk proteins with small molecular size can be enhanced by modification of the amino acids residues. Our research not only has shown the feasibility of large-scale production of spider silk proteins but also provides valuable information for protein rational design.     

Plasticity in major ampullate silk production in relation to spider phylogeny and ecology

Spider major ampullate silk is a high-performance biomaterial that has received much attention. However, most studies ignore plasticity in silk properties. A better understanding of silk plasticity could clarify the relative importance of chemical composition versus processing of silk dope for silk properties. It could also provide insight into how control of silk properties relates to spider ecology and silk uses. We compared silk plasticity (defined as variation in the properties of silk spun by a spider under different conditions) between three spider clades in relation to their anatomy and silk biochemistry. We found that silk plasticity exists in RTA clade and orbicularian spiders, two clades that differ in their silk biochemistry. Orbiculariae seem less dependent on external spinning conditions. They probably use a valve in their spinning duct to control friction forces and speed during spinning. Our results suggest that plasticity results from different processing of the silk dope in the spinning duct. Orbicularian spiders seem to display better control of silk properties, perhaps in relation to their more complex spinning duct valve.     

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.     

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.     

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。     

Hexagonal columnar liquid crystal in the cells secreting spider silk

The liquid crystallinity of spider dragline silk dope is thought to be important for both the spinning process and the extreme mechanical properties of the final thread. Although the formation of the liquid crystalline units is poorly understood, it has been suggested that spider silk proteins are secreted in a random coil and then aggregate end-to-end into rod-shaped units to form supramolecular liquid crystals. However, evidence presented here from transmission electron microscopy indicates that coat protein of the dragline silk of a Nephila spider is stored as hexagonal columnar liquid crystals within the intracellular secretory vesicles. This implies that this component is already folded into short rods within the gland cells and forms molecular rather than supramolecular liquid crystals.     

Spider capture silk: performance implications of variation in an exceptional biomaterial

Spiders and their silk are an excellent system for connecting the properties of biological materials to organismal ecology. Orb-weaving spiders spin sticky capture threads that are moderately strong but exceptionally extensible, resulting in fibers that can absorb remarkable amounts of energy. These tough fibers are thought to be adapted for arresting flying insects. Using tensile testing, we ask whether patterns can be discerned in the evolution of silk material properties and the ecological uses of spider capture fibers. Here, we present a large comparative data set that allows examination of capture silk properties across orb-weaving spider species. We find that material properties vary greatly across species. Notably, extensibility, strength, and toughness all vary approximately sixfold across species. These material differences, along with variation in fiber size, dictate that the mechanical performance of capture threads, the energy and force required to break fibers, varies by more than an order of magnitude across species. Furthermore, some material and mechanical properties are evolutionarily correlated. For example, species that spin small diameter fibers tend to have tougher silk, suggesting compensation to maintain breaking energy. There is also a negative correlation between strength and extensibility across species, indicating a potential evolutionary trade-off. The different properties of these capture silks should lead to differences in the performance of orb webs during prey capture and help to define feeding niches in spiders.     

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.     

In Vitro Evaluation of Spider Silk Meshes as a Potential Biomaterial for Bladder Reconstruction

Reconstruction of the bladder by means of both natural and synthetic materials remains a challenge due to severe adverse effects such as mechanical failure. Here we investigate the application of spider major ampullate gland-derived dragline silk from the Nephila edulis spider, a natural biomaterial with outstanding mechanical properties and a slow degradation rate, as a potential scaffold for bladder reconstruction by studying the cellular response of primary bladder cells to this biomaterial. We demonstrate that spider silk without any additional biological coating supports adhesion and growth of primary human urothelial cells (HUCs), which are multipotent bladder cells able to differentiate into the various epithelial layers of the bladder. HUCs cultured on spider silk did not show significant changes in the expression of various epithelial-to-mesenchymal transition and fibrosis associated genes, and demonstrated only slight reduction in the expression of adhesion and cellular differentiation genes. Furthermore, flow cytometric analysis showed that most of the silk-exposed HUCs maintain an undifferentiated immunophenotype. These results demonstrate that spider silk from the Nephila edulis spider supports adhesion, survival and growth of HUCs without significantly altering their cellular properties making this type of material a suitable candidate for being tested in pre-clinical models for bladder reconstruction.