Mechanical Response of Silk Crystalline Units from Force-Distribution Analysis

The outstanding mechanical toughness of silk fibers is thought to be caused by embedded crystalline units acting as cross links of silk proteins in the fiber. Here, we examine the robustness of these highly ordered β-sheet structures by molecular dynamics simulations and finite element analysis. Structural parameters and stress-strain relationships of four different models, from spider and Bombyx mori silk peptides, in antiparallel and parallel arrangement, were determined and found to be in good agreement with x-ray diffraction data. Rupture forces exceed those of any previously examined globular protein many times over, with spider silk (poly-alanine) slightly outperforming Bombyx mori silk ((Gly-Ala)_n). All-atom force distribution analysis reveals both intrasheet hydrogen-bonding and intersheet side-chain interactions to contribute to stability to similar extent. In combination with finite element analysis of simplified β-sheet skeletons, we could ascribe the distinct force distribution pattern of the antiparallel and parallel silk crystalline units to the difference in hydrogen-bond geometry, featuring an in-line or zigzag arrangement, respectively. Hydrogen-bond strength was higher in antiparallel models, and ultimately resulted in higher stiffness of the crystal, compensating the effect of the mechanically disadvantageous in-line hydrogen-bond geometry. Atomistic and coarse-grained force distribution patterns can thus explain differences in mechanical response of silk crystals, opening up the road to predict full fiber mechanics.     

Molecular chain orientation in supercontracted and re-extended spider silk.

The dragline silk from Nephila clavipes was studied by wide angle X-ray diffraction in its original state, after supercontraction to L/Lo = 0.46, and during re-extension to its original length Lo. The fibers were carefully dried before each exposure. The molecular orientation in the crystalline regions is found to follow the simple predictions of affine deformation, indicating that the crystals act as inert rigid filler particles. The crystals retain considerable orientation after supercontraction, when non-crystalline orientation is weak. This shows that crystallization occurs after orientation as the fiber forms. The oriented amorphous material, treated as a phase of constant volume fraction, also follows affine deformation. These results do not contain any indication of a special structure in the protein fiber.     

Rate-Dependent Behavior of the Amorphous Phase of Spider Dragline Silk

The time-dependent stress-strain behavior of spider dragline silk was already observed decades ago, and has been attributed to the disordered sequences in silk proteins, which compose the soft amorphous matrix. However, the actual molecular origin and magnitude of internal friction within the amorphous matrix has remained inaccessible, because experimentally decomposing the mechanical response of the amorphous matrix from the embedded crystalline units is challenging. Here, we used atomistic molecular dynamics simulations to obtain friction forces for the relative sliding of peptide chains of Araneus diadematus spider silk within bundles of these chains as a representative unit of the amorphous matrix in silk fibers. We computed the friction coefficient and coefficient of viscosity of the amorphous phase to be in the order of 10⁻⁶ Ns/m and 10⁴ Ns/m², respectively, by extrapolating our simulation data to the viscous limit. Finally, we used a finite element method for the amorphous phase, solely based on parameters derived from molecular dynamics simulations including the newly determined coefficient of viscosity. With this model the time scales of stress relaxation, creep, and hysteresis were assessed, and found to be in line with the macroscopic time-dependent response of silk fibers. Our results suggest the amorphous phase to be the primary source of viscosity in silk and open up the avenue for finite element method studies of silk fiber mechanics including viscous effects.     

Rate-Dependent Behavior of the Amorphous Phase of Spider Dragline Silk

The time-dependent stress-strain behavior of spider dragline silk was already observed decades ago, and has been attributed to the disordered sequences in silk proteins, which compose the soft amorphous matrix. However, the actual molecular origin and magnitude of internal friction within the amorphous matrix has remained inaccessible, because experimentally decomposing the mechanical response of the amorphous matrix from the embedded crystalline units is challenging. Here, we used atomistic molecular dynamics simulations to obtain friction forces for the relative sliding of peptide chains of Araneus diadematus spider silk within bundles of these chains as a representative unit of the amorphous matrix in silk fibers. We computed the friction coefficient and coefficient of viscosity of the amorphous phase to be in the order of 10⁻⁶ Ns/m and 10⁴ Ns/m², respectively, by extrapolating our simulation data to the viscous limit. Finally, we used a finite element method for the amorphous phase, solely based on parameters derived from molecular dynamics simulations including the newly determined coefficient of viscosity. With this model the time scales of stress relaxation, creep, and hysteresis were assessed, and found to be in line with the macroscopic time-dependent response of silk fibers. Our results suggest the amorphous phase to be the primary source of viscosity in silk and open up the avenue for finite element method studies of silk fiber mechanics including viscous effects.     

Viscous Friction between Crystalline and Amorphous Phase of Dragline Silk

The hierarchical structure of spider dragline silk is composed of two major constituents, the amorphous phase and crystalline units, and its mechanical response has been attributed to these prime constituents. Silk mechanics, however, might also be influenced by the resistance against sliding of these two phases relative to each other under load. We here used atomistic molecular dynamics (MD) simulations to obtain friction forces for the relative sliding of the amorphous phase and crystalline units of Araneus diadematus spider silk. We computed the coefficient of viscosity of this interface to be in the order of 10² Ns/m² by extrapolating our simulation data to the viscous limit. Interestingly, this value is two orders of magnitude smaller than the coefficient of viscosity within the amorphous phase. This suggests that sliding along a planar and homogeneous surface of straight polyalanine chains is much less hindered than within entangled disordered chains. Finally, in a simple finite element model, which is based on parameters determined from MD simulations including the newly deduced coefficient of viscosity, we assessed the frictional behavior between these two components for the experimental range of relative pulling velocities. We found that a perfectly relative horizontal motion has no significant resistance against sliding, however, slightly inclined loading causes measurable resistance. Our analysis paves the way towards a finite element model of silk fibers in which crystalline units can slide, move and rearrange themselves in the fiber during loading.     

Modeling of mechanical properties and structural design of spider web

With a unique combination of strength and toughness among materials, spider silk is the model for engineering materials. This paper presents the stress-strain behavior of Nephila clavipes spider silk under tension, transverse compression, and torsional deformation obtained by a battery of micro testing equipment. The experimental results showed significantly higher toughness than the state-of-the-art fibers in tension and in transverse compression. Higher shear modulus was also observed for the spider silk comparing to other liquid crystalline fibers such as aramid fibers. On the basis of the experimental results finite element analysis is used to simulate static and dynamic properties of spider web and to explore the role of both material properties and architectural design in its structural integrity and mechanical performance.     

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.     

Tunable silk: using microfluidics to fabricate silk fibers with controllable properties

Despite widespread use of silk, it remains a significant challenge to fabricate fibers with properties similar to native silk. It has recently been recognized that the key to tuning silk fiber properties lies in controlling internal structure of assembled β-sheets. We report an advance in the precise control of silk fiber formation with control of properties via microfluidic solution spinning. We use an experimental approach combined with modeling to accurately predict and independently tune fiber properties including Young's modulus and diameter to customize fibers. This is the first reported microfluidic approach capable of fabricating functional fibers with predictable properties and provides new insight into the structural transformations responsible for the unique properties of silk. Unlike bulk processes, our method facilitates the rapid and inexpensive fabrication of fibers from small volumes (50 μL) that can be characterized to investigate sequence-structure-property relationships to optimize recombinant silk technology to match and exceed natural silk properties.     

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.     

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.     

A model for the structure of the C-terminal domain of dragline spider silk and the role of its conserved cysteine

Dragline spider silk fibers have extraordinary attributes as biomaterials of superior strength and toughness. Previously we have shown that the conserved C-terminal domain of a dragline spider silk protein is necessary for directing oriented microfiber formation. Here we present for the first time a state-of-the-art model of the three-dimensional structure of this domain, and, by comparing several dragline proteins, identify its key evolutionarily conserved features. Further, using the baculovirus expression system, we produced recombinant proteins that are mutated in the unique cysteine residue present in the domain. While a conservative mutation to serine allows fiber formation, thus demonstrating that there is no need for disulfide bond formation in this system, a mutation to arginine significantly alters the local surface properties, preventing fiber formation. These experimental results are in agreement with our model, wherein the cysteine is localized in a highly conserved hydrophobic loop that we predict to be important for the protein-protein interactions of this domain and hence also for fiber formation.     

Synthetic spider silk fibers spun from Pyriform Spidroin 2, a glue silk protein discovered in orb-weaving spider attachment discs

Spider attachment disc silk fibers are spun into a viscous liquid that rapidly solidifies, gluing dragline silk fibers to substrates for locomotion or web construction. Here we report the identification and artificial spinning of a novel attachment disc glue silk fibroin, Pyriform Spidroin 2 (PySp2), from the golden orb weaver Nephila clavipes . MS studies support PySp2 is a constituent of the pyriform gland that is spun into attachment discs. Analysis of the PySp2 protein architecture reveals sequence divergence relative to the other silk family members, including the cob weaver glue silk fibroin PySp1. PySp2 contains internal block repeats that consist of two subrepeat units: one dominated by Ser, Gln, and Ala and the other Pro-rich. Artificial spinning of recombinant PySp2 truncations shows that the Ser-Gln-Ala-rich subrepeat is sufficient for the assembly of polymeric subunits and subsequent fiber formation. These studies support that both orb- and cob-weaving spiders have evolved highly polar block-repeat sequences with the ability to self-assemble into fibers, suggesting a strategy to allow fiber fabrication in the liquid environment of the attachment discs.     

Molecular mechanics of silk nanostructures under varied mechanical loading

Spider dragline silk is a self-assembling tunable protein composite fiber that rivals many engineering fibers in tensile strength, extensibility, and toughness, making it one of the most versatile biocompatible materials and most inviting for synthetic mimicry. While experimental studies have shown that the peptide sequence and molecular structure of silk have a direct influence on the stiffness, toughness, and failure strength of silk, few molecular-level analyses of the nanostructure of silk assemblies, in particular, under variations of genetic sequences have been reported. In this study, atomistic-level structures of wildtype as well as modified MaSp1 protein from the Nephila clavipes spider dragline silk sequences, obtained using an in silico approach based on replica exchange molecular dynamics and explicit water molecular dynamics, are subjected to simulated nanomechanical testing using different force-control loading conditions including stretch, pull-out, and peel. The authors have explored the effects of the poly-alanine length of the N. clavipes MaSp1 peptide sequence and identify differences in nanomechanical loading conditions on the behavior of a unit cell of 15 strands with 840-990 total residues used to represent a cross-linking β-sheet crystal node in the network within a fibril of the dragline silk thread. The specific loading condition used, representing concepts derived from the protein network connectivity at larger scales, have a significant effect on the mechanical behavior. Our analysis incorporates stretching, pull-out, and peel testing to connect biochemical features to mechanical behavior. The method used in this study could find broad applications in de novo design of silk-like tunable materials for an array of applications.     

Genetic engineering of structural protein polymers.

Genetic and protein engineering are components of a new polymer chemistry that provide the tools for producing macromolecular polyamide copolymers of diversity and precision far beyond the current capabilities of synthetic polymer chemistry. The genetic machinery allows molecular control of chemical and physical chain properties. Nature utilizes this control to formulate protein polymers into materials with extraordinary mechanical properties, such as the strength and toughness of silk and the elasticity and resilience of mammalian elastin. The properties of these materials have been attributed to the presence of short repeating oligopeptide sequences contained in the proteins, fibroin, and elastin. We have produced homoblock protein polymers consisting exclusively of silk-like crystalline blocks and elastin-like flexible blocks. We have demonstrated that each homoblock polymer as produced by microbial fermentation exhibits measurable properties of crystallinity and elasticity. Additionally, we have produced alternating block copolymers of various amounts of silk-like and elastin-like blocks, ranging from a ratio of 1:4 to 2:1, respectively. The crystallinity of each copolymer varies with the amount of crystalline block interruptions. The production of fiber materials with custom-engineered mechanical properties is a potential outcome of this technology.     

Silk tape nanostructure and silk gland anatomy of trichoptera

Caddisflys (order Trichoptera) construct elaborate protective shelters and food harvesting nets with underwater adhesive silk. The silk fiber resembles a nanostructured tape composed of thousands of nanofibrils (～ 120 nm) oriented with the major axis of the fiber, which in turn are composed of spherical subunits. Weaker lateral interactions between nanofibrils allow the fiber to conform to surface topography and increase contact area. Highly phosphorylated (pSX)(4) motifs in H-fibroin blocks of positively charged basic residues are conserved across all three suborders of Trichoptera. Electrostatic interactions between the oppositely charged motifs could drive liquid-liquid phase separation of silk fiber precursors into a complex coacervates mesophase. Accessibility of phosphoserine to an anti-phosphoserine antibody is lower in the lumen of the silk gland storage region compared to the nascent fiber formed in the anterior conducting channel. The phosphorylated motifs may serve as a marker for the structural reorganization of the silk precursor mesophase into strongly refringent fibers. The structural change occurring at the transition into the conducting channel makes this region of special interest. Fiber formation from polyampholytic silk proteins in Trichoptera may suggest a new approach to create synthetic silk analogs from water-soluble precursors.     

Structure, mechanism and mechanical properties of pupal attachment in Greta oto (Lepidoptera: Nymphalidae: Ithomiinae)

The structure and mechanism of pupal attachment are described for the nymphalid Greta oto using electron microscopy, and high‐speed and time‐lapse photography. The cremaster is composed of a 3‐D array of hooked setae that engage with silk fibers spun into layers in a pad on the lower leaf surface. Each seta comprises a shaft terminating in a strongly curved hook, tipped with two lateral barbs. These hook into the silk pad, which is densely laid and built‐up in the central portion, flattening out peripherally. Time‐lapse photography showed that silk pad construction by fifth instar larvae is completed in four distinct spinning movements, producing a random fiber arrangement. It is proposed that such a fiber arrangement provides isotropic strength, giving greater flexibility to the attachment. The cremaster is attached to the silk pad by a series of lateral movements of the pupa's posterior abdomen. This movement, together with the shape of the setal hooks, is thought to be integral to the attachment process. Tensile loading tests showed that attachment failure is due to the breakage of the silk pad, which undergoes gradual destruction before releasing the cremaster. The attachment was found to have high tensile strength and fracture toughness, both of which suggest that it has evolved for the dual purpose of preventing the pupa being pulled from the leaf by a predator and preventing the attachment being weakened by wind, which causes the pupa to swing.     

Transgenic modifications of silkworms as a means to obtain therapeutic biomolecules and protein fibers with exceptional properties

Transgenic modification of Bombyx mori silkworms is a benign approach for the production of silk fibers with extraordinary properties and also to generate therapeutic proteins and other biomolecules for various applications. Silk fibers with fluorescence lasting more than a year, natural protein fibers with strength and toughness exceeding that of spider silk, proteins and therapeutic biomolecules with exceptional properties have been developed using transgenic technology. The transgenic modifications have been done primarily by modifying the silk sericin and fibroin genes and also the silk producing glands. Although the genetic modifications were typically performed using the sericin 1 and other genes, newer techniques such as CRISPR/Cas9 have enabled successful modifications of both the fibroin H-chain and L-chain. Such modifications have led to the production of therapeutic proteins and other biomolecules in reasonable quantities at affordable costs for tissue engineering and other medical applications. Transgenically modified silkworms also have distinct and long-lasting fluorescence useful for bioimaging applications. This review presents an overview of the transgenic techniques for modifications of B. mori silkworms and the properties obtained due to such modifications with particular focus on production of growth factors, fluorescent proteins, and high performance protein fibers.     

蛋白质内含子介导蛛丝功能化平台的建立

为建立高效快捷的蛛丝功能化修饰平台,蛋白质内含子的反式剪接技术被首次应用于重组蛛丝的功能化修饰。在体外通过Ssp Dna B的反式剪接作用,在蛋白质水平上将12 k Da泛素相关修饰蛋白(SUMO)与蛛丝蛋白(W2CT)连接形成功能化蛛丝蛋白SUMOW2CT。修饰后SUMOW2CT与W2CT均能形成纳米至微米级的丝纤维,但SUMOW2CT自动成丝速度明显下降且产量约为W2CT的一半。与W2CT丝纤维(W)相似,SUMOW2CT丝纤维(UW)不具有超收缩能力和对2%SDS不耐受,但机械性能低于W2CT丝纤维。功能化蛋白SUMOW2CT形成的丝纤维中SUMO蛋白仍保持着正确三维结构,可被SUMO蛋白酶酶切。外源功能化蛋白质虽在一定程度上降低了丝的形成速度和机械性能,但修饰上的功能化蛋白仍保持着生物活性,表明断裂蛋白质内含子介导的蛛丝修饰平台成功建立,也为蛛丝的功能化修饰和应用奠定了坚实的技术基础。     

Review structure of silk by raman spectromicroscopy: from the spinning glands to the fibers

Raman spectroscopy has long been proved to be a useful tool to study the conformation of protein-based materials such as silk. Thanks to recent developments, linearly polarized Raman spectromicroscopy has appeared very efficient to characterize the molecular structure of native single silk fibers and spinning dopes because it can provide information relative to the protein secondary structure, molecular orientation, and amino acid composition. This review will describe recent advances in the study of the structure of silk by Raman spectromicroscopy. A particular emphasis is put on the spider dragline and silkworm cocoon threads, other fibers spun by orb-weaving spiders, the spinning dope contained in their silk glands and the effect of mechanical deformation. Taken together, the results of the literature show that Raman spectromicroscopy is particularly efficient to investigate all aspects of silk structure and production. The data provided can lead to a better understanding of the structure of the silk dope, transformations occurring during the spinning process, and structure and mechanical properties of native fibers.     

Reproducibility of the tensile properties of spider (Argiope trifasciata) silk obtained by forced silking

A modified forced silking procedure was developed to allow an accurate study of the tensile properties of spider (Argiope trifasciata) silk, especially the characterization of the variability of the tensile properties of forcibly silked fibers. The procedure involves an immobilization technique that does not require anesthetization of the spider, a mode of collection that allows immediate access to any silk sample with a minimum manipulation, and a technique to measure the diameters of the spider silk fibers systematically. The forcibly silked fibers obtained by this procedure show reproducible tensile properties in terms of force-displacement curves as well as stress-strain curves. Furthermore, reproducibility also extends to forcibly silked fibers obtained from different spiders when stress-strain is considered.