Nanostructural features of a spider dragline silk as revealed by electron and X-ray diffraction studies

Transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used to examine the nanostructure of a natural polymer—a spider dragline silk—that has potential applications as an engineering material. The silk studied was collected from the cob-web weaving spider Latrodectus hesperus. Single crystal and polycrystalline electron diffraction patterns indicate the presence of crystals with a bimodal size distribution, in the range of 2 nm and 40-120 nm. The chain axis of the smaller crystals is more strictly aligned with the fiber axis than that of the larger crystals. Lattice parameters for the orthogonal unit cell are: a=9.4 Å (interchain), b=7.0 Å (dipeptide, fiber axis) and c=10.8 Å (intersheet). A fine structure in single crystal electron diffraction patterns indicates possible composition-dependent lattice strains. Results of tensile tests of the spider dragline silk are reported, and a simple model is presented linking the observed nanostructural features to the force-elongation response of this material.     

Evidence of Decoupling Protein Structure from Spidroin Expression in Spider Dragline Silks

The exceptional strength and extensibility of spider dragline silk have been thought to be facilitated by two spidroins, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2), under the assumption that protein secondary structures are coupled with the expressed spidroins. We tested this assumption for the dragline silk of three co-existing Australian spiders, Argiope keyserlingi, Latrodectus hasselti and Nephila plumipes. We found that silk amino acid compositions did not differ among spiders collected in May. We extended these analyses temporally and found the amino acid compositions of A. keyserlingi silks to differ when collected in May compared to November, while those of L. hasselti did not. To ascertain whether their secondary structures were decoupled from spidroin expression, we performed solid-state nuclear magnetic resonance spectroscopy (NMR) analysis on the silks of all spiders collected in May. We found the distribution of alanine toward β-sheet and _3,10helix/random coil conformations differed between species, as did their relative crystallinities, with A. keyserlingi having the greatest _3,10helix/random coil composition and N. plumipes the greatest crystallinity. The protein secondary structures correlated with the mechanical properties for each of the silks better than the amino acid compositions. Our findings suggested that a differential distribution of alanine during spinning could decouple secondary structures from spidroin expression ensuring that silks of desirable mechanical properties are consistently produced. Alternative explanations include the possibility that other spidroins were incorporated into some silks.     

Comprehensive Proteomic Analysis of Spider Dragline Silk from Black Widows: A Recipe to Build Synthetic Silk Fibers

The outstanding material properties of spider dragline silk fibers have been attributed to two spidroins, major ampullate spidroins 1 and 2 (MaSp1 and MaSp2). Although dragline silk fibers have been treated with different chemical solvents to elucidate the relationship between protein structure and fiber mechanics, there has not been a comprehensive proteomic analysis of the major ampullate (MA) gland, its spinning dope, and dragline silk using a wide range of chaotropic agents, inorganic salts, and fluorinated alcohols to elucidate their complete molecular constituents. In these studies, we perform in-solution tryptic digestions of solubilized MA glands, spinning dope and dragline silk fibers using five different solvents, followed by nano liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis with an Orbitrap Fusion™ Tribrid™. To improve protein identification, we employed three different tryptic peptide fragmentation modes, which included collision-induced dissociation (CID), electron transfer dissociation (ETD), and high energy collision dissociation (HCD) to discover proteins involved in the silk assembly pathway and silk fiber. In addition to MaSp1 and MaSp2, we confirmed the presence of a third spidroin, aciniform spidroin 1 (AcSp1), widely recognized as the major constituent of wrapping silk, as a product of dragline silk. Our findings also reveal that MA glands, spinning dope, and dragline silk contain at least seven common proteins: three members of the Cysteine-Rich Protein Family (CRP1, CRP2 and CRP4), cysteine-rich secretory protein 3 (CRISP3), fasciclin and two uncharacterized proteins. In summary, this study provides a proteomic blueprint to construct synthetic silk fibers that most closely mimic natural fibers.     

Spider Silk-CBD-Cellulose Nanocrystal Composites: Mechanism of Assembly

The fabrication of cellulose-spider silk bio-nanocomposites comprised of cellulose nanocrystals (CNCs) and recombinant spider silk protein fused to a cellulose binding domain (CBD) is described. Silk-CBD successfully binds cellulose, and unlike recombinant silk alone, silk-CBD self-assembles into microfibrils even in the absence of CNCs. Silk-CBD-CNC composite sponges and films show changes in internal structure and CNC alignment related to the addition of silk-CBD. The silk-CBD sponges exhibit improved thermal and structural characteristics in comparison to control recombinant spider silk sponges. The glass transition temperature (Tg) of the silk-CBD sponge was higher than the control silk sponge and similar to native dragline spider silk fibers. Gel filtration analysis, dynamic light scattering (DLS), small angle X-ray scattering (SAXS) and cryo-transmission electron microscopy (TEM) indicated that silk-CBD, but not the recombinant silk control, formed a nematic liquid crystalline phase similar to that observed in native spider silk during the silk spinning process. Silk-CBD microfibrils spontaneously formed in solution upon ultrasonication. We suggest a model for silk-CBD assembly that implicates CBD in the central role of driving the dimerization of spider silk monomers, a process essential to the molecular assembly of spider-silk nanofibers and silk-CNC composites.     

Exploring the backbone dynamics of native spider silk proteins in Black Widow silk glands with solution-state NMR spectroscopy

Spider dragline silk is an outstanding biopolymer with a strength that exceeds steel by weight and a toughness greater than high-performance fibers like Kevlar. For this reason, understanding how a spider converts the gel-like, aqueous protein spinning dope within the major ampullate (MA) gland into a super fiber is of great importance for developing future biomaterials based on spider silk. In this work, the initial state of the silk proteins within Black Widow MA glands was probed with solution-state NMR spectroscopy. ¹⁵N relaxation parameters, T₁, T₂ and ¹⁵N-{¹H} steady-state NOE were measured for twelve backbone environments at two spectrometer frequencies, 500 and 800 MHz. The NMR relaxation parameters extracted for all twelve environments are consistent with MA silk protein backbone dynamics on the fast sub-nanosecond timescale. Therefore, it is concluded that the repetitive core of spider MA proteins are in an unfolded, highly flexible state in the MA gland.     

Self-tightening of spider silk fibers induced by moisture

Spider dragline silk has a unique combination of desirable mechanical properties—low density, high tensile strength and large elongation until breaking—that makes it attractive from an engineering perspective [Nature 410 (2001) 541]. Nevertheless, this outstanding performance is threatened by the way mechanical properties are affected by a wet environment, particularly if the stress of these fibers can relax when exposed to moisture. Tests on spider dragline silk (Argiope trifasciata) performed by the authors have shown that when the fiber is clamped and exposed to a wet enough environment non-vanishing supercontraction forces develop. When the moisture is removed the residual stresses increase, and this effect has proven long lasting, as the fiber remains stressed for hours. In addition, the tensile properties of the fiber remain unaffected by the residual stresses build up after removing the moisture or after a wetting and drying cycle. These tests give support to the thesis that supercontraction helps to keep the spider webs tight and opens new applications for synthetic analogs.     

A new silk: Mechanical, compositional, and morphological characterization of leafhopper (Kahaono montana) silk

The mechanical properties, amino acid composition, internal morphology, and solvent-induced interaction of silk produced by the endemic Australian leafhopper, Kahaono montana Evans (Hemiptera: Cicadellidae) were studied. Ion plasma etching/scanning electron microscopy examination of the internal morphology revealed a skin-core structure, with bands in the core region aligned regularly in a transverse direction to the fibre axis, separated by a nominal spacing of 100 nm. The internal structure of the silk was compared with those from spider Eriophora transmarina (Keyserling) (Araneida: Araneidae) radial thread and silkworm (Bombyx mori). The amino acid composition of K. montana silk was determined using HPLC, and was found to be dominated by small amino acids: Serine, alanine and glycine. The silk-solvent interaction was tested using selected aqueous, organic and surfactant solutions, and the solubility of the silk was found depend primarily on the pH and ionic strength of the solvent. Tensile tests showed that the silk has considerably weaker mechanical properties than spider silk and silkworm silk. The differences in mechanical properties of K. montana silk compared with spider and silkworm silk are attributed to the distinction in amino acid composition ratio and internal morphology, and are likely to reflect the functions of the silks in these species.     

Development of silk-like materials based on Bombyx mori and Nephila clavipes dragline silk fibroins

In order to develop new silk-like materials in the form of fiber and non-woven nano-fiber, this study synthesized a new silk-like protein by selecting the sequence from the crystalline region of Bombyx mori silk fibroin, (GAGSGA)₆, to imitate the processing condition of the silk fibroin with the combination of the sequence YGGLGSQGAGRG, the hydrophilic motif of spider dragline silk which was considered as the origin of supercontraction of spider dragline silk (Yang Z, et al. J Am Chem Soc 2000;122: 9019-25). The CD pattern of the silk-like protein in hexafluoroacetone (HFA) indicates that it takes helical structure. The solid-state structures of the silk-like protein before and after methanol treatment were determined to be random coil and Silk II structure (mainly β-sheet structure), respectively, using ¹³C CP/MAS NMR. The ¹³C CP/MAS NMR spectra of the protein after methanol treatment in hydrated state showed that the random coil peaks from Ala Cβ and Ser Cβ carbons become sharper compared with the corresponding peaks in the dry state. The 2D-WISE spectra in the hydrated state also showed increase of the narrow components for these carbons. Thus, water molecules are relatively easy to access at the random coil regions of the protein. The fiber formation of the silk-like protein was possible with wet-spinning or electrospinning methods using HFA as dope solvent and methanol as coagulation solvent.     

Major Ampullate Spider Silk with Indistinguishable Spidroin Dope Conformations Leads to Different Fiber Molecular Structures

To plentifully benefit from its properties (mechanical, optical, biological) and its potential to manufacture green materials, the structure of spider silk has to be known accurately. To this aim, the major ampullate (MA) silk of Araneus diadematus (AD) and Nephila clavipes (NC) has been compared quantitatively in the liquid and fiber states using Raman spectromicroscopy. The data show that the spidroin conformations of the two dopes are indistinguishable despite their specific amino acid composition. This result suggests that GlyGlyX and GlyProGlyXX amino acid motifs (X = Leu, Glu, Tyr, Ser, etc.) are conformationally equivalent due to the chain flexibility in the aqueous environment. Species-related sequence specificity is expressed more extensively in the fiber: the β-sheet content is lower and width of the orientation distribution of the carbonyl groups is broader for AD (29% and 58°, respectively) as compared to NC (37% and 51°, respectively). β-Sheet content values are close to the proportion of polyalanine segments, suggesting that β-sheet formation is mainly dictated by the spidroin sequence. The extent of molecular alignment seems to be related to the presence of proline (Pro) that may decrease conformational flexibility and inhibit chain extension and alignment upon drawing. It appears that besides the presence of Pro, secondary structure and molecular orientation contribute to the different mechanical properties of MA threads.     

Active control on molecular conformations and tensile properties of spider silk

The mechanical properties of spider dragline silk vary with the spinning conditions, and molecular conformation is one of the important factors for the strength and strain of materials. Four kinds of Araneus ventricosus spider dragline silk fibers, measured by Raman microscopic spectrometry, were produced under different conditions: (1) reeled at the rate of 2 cm/s; (2) secreted by a dropping spider from a 100‐cm‐high table; (3) spun by spiders raised in two different containers. The Raman spectra of these fibers showed that the spinning method and growing environment of spiders had evident influences on the molecular conformations and tensile properties of dragline silk, and the dragline silk obtained from a dropping spider contained the greatest number of molecules with highly oriented β‐sheet structures and gave higher stress/strain values. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 901–905, 2004     

Recombinant Silk Proteins with Additional Polyalanine Have Excellent Mechanical Properties

This paper explores the structures of exogenous protein molecules that can effectively improve the mechanical properties of silkworm silk. Several transgenic vectors fused with the silkworm fibroin light chain and type 3 repeats in different multiples of the ampullate dragline silk protein 1 (MaSp1) from black widow spider with different lengths of the polyalanine motifs were constructed for this study. Transgenic silkworms were successfully obtained by piggyBac-mediated microinjection. Molecular detection showed that foreign proteins were successfully secreted and contained within the cocoon shells. According to the prediction of PONDR^® VSL2 and PONDR^® VL-XT, the type 3 repeats and the polyalanine motif of the MaSp1 protein were amorphous. The results of FTIR analysis showed that the content of β-sheets in the silk of transgenic silkworms engineered with transgenic vectors with additional polyalanine was significantly higher than that of wild-type silkworm silk. Additionally, silk with a higher β-sheet content had better fracture strength and Young’s modulus. The mechanical properties of silk with longer chains of exogenous proteins were improved. In general, our results provide theoretical guidance and technical support for the large-scale production of excellent bionic silk.     

Diversified Structural Basis of a Conserved Molecular Mechanism for pH‐Dependent Dimerization in Spider Silk N‐Terminal Domains

Conversion of spider silk proteins from soluble dope to insoluble fibers involves pH‐dependent dimerization of the N‐terminal domain (NT). This conversion is tightly regulated to prevent premature precipitation and enable rapid silk formation at the end of the duct. Three glutamic acid residues that mediate this process in the NT from Euprosthenops australis major ampullate spidroin 1 are well conserved among spidroins. However, NTs of minor ampullate spidroins from several species, including Araneus ventricosus (^AvMiSp NT), lack one of the glutamic acids. Here we investigate the pH‐dependent structural changes of ^AvMiSp NT, revealing that it uses the same mechanism but involves a non‐conserved glutamic acid residue instead. Homology modeling of the structures of other MiSp NTs suggests that these harbor different compensatory residues. This indicates that, despite sequence variations, the molecular mechanism underlying pH‐dependent dimerization of NT is conserved among different silk types.     

Cross-plane thermal transport in micrometer-thick spider silk films

This work reports on the first study of thermal transport capacity in the thickness direction (∼μm scale) for spider silk films. Fresh (minimally processed) and hexafluoroisopropanol (HFIP) films of Nephila clavipes and Latrodectus hesperus major ampullate silk are studied. Detailed Raman spectroscopy reveals that the fresh films have more crystalline secondary protein structures such as antiparallel β-sheets than the HFIP films for N. clavipes. For N. clavipes, the randomly distributed antiparallel β-sheets in fresh films have nearly no effect in improving thermal conductivity in comparison with HFIP films. For L. hesperus, the films mainly consist of α-helices and random coils while the fresh film has a higher concentration of α-helices. The higher concentration of α-helices in fresh films gives rise to a higher heat capacity than HFIP films, while the thermal conductivity shows little effect from the α-helices concentration. Thickened HFIP films are heated at different temperatures to study the effect of heat treatment on structure and thermal transport capacity. These experiments demonstrate that α-helices are formed by thermal treatment and that thermal effusivity increases with the appearance of α-helices in films.     

The color of dragline silk produced in captivity by the spider Nephila clavipes

Fifty-six N. clavipes spiders from the same region of Florida were kept in captivity under the same conditions and fed a similar diet of crickets. Their major ampulate glands were forcibly silked. Dates, silking times, and the colors of the dragline silk produced were recorded. The colors ranged from all white through various combinations of white and yellow upon different silkings to all yellow. If a spider had been producing white silk for at least 4 h, the color being produced could suddenly change to yellow 38% of the time. These observations indicate that factors beyond diet and environment influence the color of silk produced in captivity by forcible silking. They also indicate that the spiders store both pigmented and unpigmented silks and that some aspect of forcible silking precludes the spiders' choosing the color. The yellow and white silks exhibit similar exterior surface morphologies as well as similar tensile properties.     

Multiwalled carbon nanotubes incorporated bombyx mori silk nanofibers by electrospinning

In this work, the regenerated silk protein with multiwalled carbon nanotubes (MWNT) was successfully electrospun in formic acid to generate the hybrid silk nanofibers. The morphology, structure and mechanical properties of the resulting silk/MWNT hybrid nanofibers were characterized using field emission scanning electron microscope (FE-SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FI-IR), Raman spectroscopy, wide angle X-ray diffraction (WAXD) and tensile testing. Thermo analysis was also carried out. TEM results confirmed that MWNT were well incorporated into the silk fibers. Addition of MWNT into silk nanofibers resulted in an enhanced mechanical property depending on MWNT content.     

Partial deuteration probing structural changes in supercontracted spider silk

Polarized IR-spectroscopic and mechanical measurements are combined to analyse the conformational changes in hydrogenated and partially deuterated major ampullate spider silk of Nephila edulis. Special attention is given to supercontraction and to the case where the latter is hindered by mechanical constraints. The determination of the molecular order parameters of the different moieties proves that the amide hydrogen exchange is a selective process, taking place at the surface of β-sheet nanocrystals, implying that these regions are accessible by water. The mechanical properties are changing dramatically when the fiber is wet (“supercontraction”) due to the fact that the pre-stress of the chains interconnecting the nanocrystals is irreversibly released. In course of this a novel network of H-bonds is formed, a process which can be suppressed if supercontraction is hindered.     

Investigation of Synthetic Spider Silk Crystallinity and Alignment via Electrothermal,Pyroelectric, Literature XRD,and Tensile Techniques

The processes used to create synthetic spider silk greatly affect the properties of the produced fibers. This paper investigates the effect of process variations during artificial spinning on the thermal and mechanical properties of the produced silk. Property values are also compared to the ones of the natural dragline silk of the Nephila clavipes spider, and to unprocessed (as‐spun) synthetic silk. Structural characterization by scanning pyroelectric microscopy is employed to provide insight into the axial orientation of the crystalline regions of the fiber and is supported by X‐ray diffraction data. The results show that stretching and passage through liquid baths induce crystal formation and axial alignment in synthetic fibers, but with different structural organization than natural silks. Furthermore, an increase in thermal diffusivity and elastic modulus is observed with decreasing fiber diameter, trending toward properties of natural fiber. This effect seems to be related to silk fibers being subjected to a radial gradient during production.

     

Molecular order in spider major ampullate silk (dragline): Effects of spinning rate and post‐spin drawing

Optical birefringence measurements are used to characterize how the molecular order of spider (Nephila clavipes) major ampullate silk is affected by linear spinning rate, by the extent of post‐spin drawing, and by post‐spin drawing rate. Results are interpreted qualitatively in terms of a simple microstructural model, in which birefringence depends on both the overall degree of molecular orientation and the extent to which crystalline regions are present. In contrast to the behavior of conventional, synthetic polymers, birefringence is found to be an unreliable predictor of tensile stiffness: microstructural changes that lead to increased birefringence may leave stiffness unchanged or, in some cases, lower than before. It is unlikely that economic processing of silk‐like polymers into fiber that exhibits biomimetic tensile properties can be achieved with spinning followed by drawing, or with a single spinning step. Instead, spinning followed by thermochemical treatment under load may be needed to obtain the critical combination of molecular orientation and crystallinity in commercially satisfactory time scales. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 72: 895–903, 1999     

Effect of plasma treatment on the structure and allied textile properties of mulberry silk

Studies on the effect of nitrogen plasma on morphology and textile properties of mulberry silk fibers and fabrics have been conducted. The changes in the morphological structure have been monitored by transmission electron microscopy, X-ray diffraction, and internal reflection infrared spectroscopy. The changes in some of textile properties such as wettability, drying rate, and crease recovery owing to plasma treatment have also been investigated. It has been found that the surface of mulberry silk gets etched away, even affecting the crystalline region. This behavior is opposite to our findings on tassar silk. Therefore, an explanation of this differential behavior of mulberry with tasar on the basis of amino acid linkages vis-à-vis bonding, wettability, drying rate, and water retention capacity has been attempted. These results have been used to arrive at an understanding of internal structure of mulberry.     

Structures,mechanical properties and applications of silk fibroin materials

For centuries, Bombyx mori silkworm silk fibroin has been used as a high-end textile fiber. Beyond textiles, silk fibroin has also been used as a surgical suture material for decades, and is being further developed for various emerging biomedical applications. The facile and versatile processability of silk fibroin in native and regenerated forms makes it appealing in a range of applications that require a mechanically superior, biocompatible, biodegradable, and functionalizable material. In this review, we describe the current understandings of the constituents, structures, and mechanical properties of silk fibroin. Following that, we summarize the strategies to bring its mechanical performance closer to that of spider dragline silk. Next, we discuss how functionalization endows silk fibroin with desired functionalities and also the effects of functionalization on its mechanical properties. Finally, from the mechanical point of view, we discuss various matrices/morphologies of silk fibroin, and their respective applications in term of functionalities, mechanical properties and performance.