Major Ampullate Silk Fibers Research Articles

Helium Ion Microscopy and Sectioning of Spider Silk.

Focused ion beams have recently emerged as a powerful tool for ultrastructural imaging of biological samples. In this article, we show that helium ion microscopy (HIM), in combination with ion milling, can be used to visualize the inner structure of both major and minor ampullate silk fibers of the orb-web weaving spider Nephila madagascariensis. The internal nanofibrils were imaged in pristine silk fibers, with little or no damage to the sample structure observed. Furthermore, a method to cut/rupture the fibers using He+ ions combined with internal sample tension is presented. This showed that the stretching and rupturing of spider silk is a highly dynamic process with considerable material reorganization.

The Spider Silk Standardization Initiative (S3I): A powerful tool to harness biological variability and to systematize the characterization of major ampullate silk fibers spun by spiders from suburban Sydney, Australia

The true stress-true strain curves of 11 Australian spider species from the Entelegynae lineage were tensile tested and classified based on the values of the alignment parameter, α*, in the framework of the Spider Silk Standardization Initiative (S3I). The application of the S3I methodology allowed the determination of the alignment parameter in all cases, and were found to range between α* = 0.03 and α* = 0.65. These data, in combination with previous results on other species included in the Initiative, were exploited to illustrate the potential of this approach by testing two simple hypotheses on the distribution of the alignment parameter throughout the lineage: (1) whether a uniform distribution may be compatible with the values obtained from the studied species, and (2) whether any trend may be established between the distribution of the α* parameter and phylogeny. In this regard, the lowest values of the α* parameter are found in some representatives of the Araneidae group, and larger values seem to be found as the evolutionary distance from this group increases. However, a significant number of outliers to this apparent general trend in terms of the values of the α* parameter are described.

Differences in the Elastomeric Behavior of Polyglycine-Rich Regions of Spidroin 1 and 2 Proteins

Two different polyglycine-rich fragments were selected as representatives of major ampullate gland spidroins (MaSp) 1 and 2 types, and their behavior in a water-saturated environment was simulated within the framework of molecular dynamics (MD). The selected fragments are found in the sequences of the proteins MaSp1a and MaSp2.2a of Argiope aurantia with respective lengths of 36 amino acids (MaSp1a) and 50 amino acids (MaSp2.2s). The simulation took the fully extended β-pleated conformation as reference, and MD was used to determine the equilibrium configuration in the absence of external forces. Subsequently, MD were employed to calculate the variation in the distance between the ends of the fragments when subjected to an increasing force. Both fragments show an elastomeric behavior that can be modeled as a freely jointed chain with links of comparable length, and a larger number of links in the spidroin 2 fragment. It is found, however, that the maximum recovery force recorded from the spidroin 2 peptide (Fmax ≈ 400 pN) is found to be significantly larger than that of the spidroin 1 (Fmax ≈ 250 pN). The increase in the recovery force of the spidroin 2 polyglycine-rich fragment may be correlated with the larger values observed in the strain at breaking of major ampullate silk fibers spun by Araneoidea species, which contain spidroin 2 proteins, compared to the material produced by spider species that lack these spidroins (RTA-clade).

The Spider Silk Standardization Initiative (S3I): A Powerful Tool to Harness Biological Variability and to Systematize the Characterization of Major Ampullate Silk Fibers Spun by Spiders from Suburban Sydney, Australia

The Spider Silk Standardization Initiative (S3I): A Powerful Tool to Harness Biological Variability and to Systematize the Characterization of Major Ampullate Silk Fibers Spun by Spiders from Suburban Sydney, Australia

Mesoscale structures in amorphous silks from a spider\u2019s orb-web

Of the 7–8 silk fibers making up an orb-web only the hierarchical structural organization of semicrystalline radial fibers -composed of major ampullate silk- has been studied in detail, given its fascinating mechanical features. While major ampullate silk’s nanofibrillar morphology is well established, knowhow on mesoscale (> 50–100 nm) assembly and its contribution to mechanical performance is limited. Much less is known on the hierarchical structural organization of other, generally less crystalline fibers contributing to an orb-webs’ function. Here we show by scanning X-ray micro&nanodiffraction that two fully amorphous, fine silk fibers from the center of an orb-web have different mesoscale features. One of the fibers has a fibrillar composite structure resembling stiff egg case silk. The other fiber has a skin–core structure based on a nanofibrillar ribbon wound around a disordered core. A fraction of nanofibrils appears to have assembled into mesoscale fibrils. This fiber becomes readily attached to the coat of major ampullate silk fibers. We observe that a detached fiber has ripped out the glycoprotein skin-layer containing polyglycine II nanocrystallites. The anchoring of the fiber in the coat suggests that it could serve for strengthening the tension and cohesion of major ampullate silk fibers.

Strain-Dependent Structural Changes in Major and Minor Ampullate Spider Silk Revealed by Two-Photon Excitation Polarization.

Spider silk's mechanical properties make it an interesting material for many industrial applications. The structure and nanoscopic organization of its proteins are the basis of these qualities. In this study, the emission maxima of the autofluorescence from the protein core of major and minor ampullate silk fibers from the orb-web-weaving spider Nephila madagascariensis are determined and found to be 534 ± 11 and 547 ± 19 nm, respectively. Molecular conformational changes during applied strain are observed in both fiber types using two-photon excitation polarization measurements. Our findings showed that within the fibers the autofluorescent dipoles are separated into two distinct populations, one randomly orientated (amorphous regions) and one with aligned dipoles as found in crystalline structures. The crystalline-amorphous ratio was determined, and it was found that the crystalline dipoles made up around 30 and 20% of the autofluorescent dipoles in major and minor ampullate silk fibers, respectively. Using two-photon polarization measurements, it is possible to directly observe that the major and minor ampullate silk fibers structurally adapt to the applied stress, as well as discern different molecular conformational changes between major and minor ampullates. It was seen that the crystalline-amorphous ratio increased, with up to 9% for major fibers and 6% for minor fibers, as strain was applied, suggesting a conformational adaptation of the fiber, interpreted as noncrystalline 310-helices transforming into crystalline β-sheets.

A Leishmania secretion system for the expression of major ampullate spidroin mimics.

Spider major ampullate silk fibers have been shown to display a unique combination of relatively high fracture strength and toughness compared to other fibers and show potential for tissue engineering scaffolds. While it is not possible to mass produce native spider silks, the potential ability to produce fibers from recombinant spider silk fibers could allow for an increased innovation rate within tissue engineering and regenerative medicine. In this pilot study, we improved upon a prior fabrication route by both changing the expression host and additives to the fiber pulling precursor solution to improve the performance of fibers. The new expression host for producing spidroin protein mimics, protozoan parasite Leishmania tarentolae, has numerous advantages including a relatively low cost of culture, rapid growth rate and a tractable secretion pathway. Tensile testing of hand pulled fibers produced from these spidroin-like proteins demonstrated that additives could significantly modify the fiber’s mechanical and/or antimicrobial properties. Cross-linking the proteins with glutaraldehyde before fiber pulling resulted in a relative increase in tensile strength and decrease in ductility. The addition of ampicillin into the spinning solution resulted in the fibers being able to inhibit bacterial growth.

Material properties of evolutionary diverse spider silks described by variation in a single structural parameter.

Spider major ampullate gland silks (MAS) vary greatly in material properties among species but, this variation is shown here to be confined to evolutionary shifts along a single universal performance trajectory. This reveals an underlying design principle that is maintained across large changes in both spider ecology and silk chemistry. Persistence of this design principle becomes apparent after the material properties are defined relative to the true alignment parameter, which describes the orientation and stretching of the protein chains in the silk fiber. Our results show that the mechanical behavior of all Entelegynae major ampullate silk fibers, under any conditions, are described by this single parameter that connects the sequential action of three deformation micromechanisms during stretching: stressing of protein-protein hydrogen bonds, rotation of the β-nanocrystals and growth of the ordered fraction. Conservation of these traits for over 230 million years is an indication of the optimal design of the material and gives valuable clues for the production of biomimetic counterparts based on major ampullate spider silk.

Proteomic Evidence for Components of Spider Silk Synthesis from Black Widow Silk Glands and Fibers.

Spider silk research has largely focused on spidroins, proteins that are the primary components of spider silk fibers. Although a number of spidroins have been characterized, other types of proteins associated with silk synthesis are virtually unknown. Previous analyses of tissue-specific RNA-seq libraries identified 647 predicted genes that were differentially expressed in silk glands of the Western black widow, Latrodectus hesperus. Only ∼5% of these silk-gland specific transcripts (SSTs) encode spidroins; although the remaining predicted genes presumably encode other proteins associated with silk production, this is mostly unverified. Here, we used proteomic analysis of multiple silk glands and dragline silk fiber to investigate the translation of the differentially expressed genes. We find 48 proteins encoded by the differentially expressed transcripts in L. hesperus major ampullate, minor ampullate, and tubuliform silk glands and detect 17 SST encoded proteins in major ampullate silk fibers. The observed proteins include known silk-related proteins, but most are uncharacterized, with no annotation. These unannotated proteins likely include novel silk-associated proteins. Major and minor ampullate glands have the highest overlap of identified proteins, consistent with their shared, distinctive ampullate shape and the overlapping functions of major and minor ampullate silks. Our study substantiates and prioritizes predictions from differential expression analysis of spider silk gland transcriptomes.

Characterizing the secondary protein structure of black widow dragline silk using solid-state NMR and X-ray diffraction.

This study provides a detailed secondary structural characterization of major ampullate dragline silk from Latrodectus hesperus (black widow) spiders. X-ray diffraction results show that the structure of black widow major ampullate silk fibers is comprised of stacked β-sheet nanocrystallites oriented parallel to the fiber axis and an amorphous region with oriented (anisotropic) and isotropic components. The combination of two-dimensional (2D) (13)C-(13)C through-space and through-bond solid-state NMR experiments provide chemical shifts that are used to determine detailed information about the amino acid motif secondary structure in black widow spider dragline silk. Individual amino acids are incorporated into different repetitive motifs that make up the majority of this protein-based biopolymer. From the solid-state NMR measurements, we assign distinct secondary conformations to each repetitive amino acid motif and, hence, to the amino acids that make up the motifs. Specifically, alanine is incorporated in β-sheet (poly(Alan) and poly(Gly-Ala)), 3(1)-helix (poly(Gly-Gly-Xaa), and α-helix (poly(Gln-Gln-Ala-Tyr)) components. Glycine is determined to be in β-sheet (poly(Gly-Ala)) and 3(1)-helical (poly(Gly-Gly-X(aa))) regions, while serine is present in β-sheet (poly(Gly-Ala-Ser)), 3(1)-helix (poly(Gly-Gly-Ser)), and β-turn (poly(Gly-Pro-Ser)) structures. These various motif-specific secondary structural elements are quantitatively correlated to the primary amino acid sequence of major ampullate spidroin 1 and 2 (MaSp1 and MaSp2) and are shown to form a self-consistent model for black widow dragline silk.

Structure and Mechanical Properties of Spider Silk Films at the Air–Water Interface

The kinetics of adsorption of solubilized spider major ampullate (MA) silk fibers at the air-water interface and the molecular structure and mechanical properties of the interfacial films formed have been studied using various physical techniques. The data show that Nephila clavipes MA proteins progressively adsorb at the interface and ultimately form a highly cohesive thin film. In situ infrared spectroscopy shows that as soon as they reach the interface the proteins predominantly form β sheets. The protein secondary structure does not change significantly as the film grows, and the amount of β sheet is the same as that of the natural fiber. This suggests that the final β-sheet content is mainly dictated by the primary structure and not by the underlying formation process. The measure of the shear elastic constant at low strain reveals a very strong, viscous, cohesive assembly. The β sheets seem to form cross-links dispersed within an intermolecular network, thus probably playing a major role in the film strength. More importantly, the molecular weight seems to be a crucial factor because interfacial films made from the natural proteins are ~7 times stronger and ~3 times more viscous than those obtained previously with shorter recombinant proteins. Brewster angle microscopy at the air-water interface and transmission electron microscopy of transferred films have revealed a homogeneous organization on the micrometer scale. The images suggest that the structural assembly at the air-water interface leads to the formation of macroscopically solid and highly cohesive networks. Overall, the results suggest that natural spider silk proteins, although sharing similarities with recombinant proteins, have the particular ability to self-assemble into ordered materials with exceptional mechanical properties.

The role of salt and shear on the storage and assembly of spider silk proteins

Major ampullate silk fibers of orb web-weaving spiders have impressive mechanical properties due to the fact that the underlying proteins partially fold into helical/amorphous structures, yielding relatively elastic matrices that are toughened by anisotropic nanoparticulate inclusions (formed from stacks of beta-sheets of the same proteins). In vivo the transition from soluble protein to solid fibers involves a combination of chemical and mechanical stimuli (such as ion exchange, extraction of water and shear forces). Here we elucidate the effects of such stimuli on the in vitro aggregation of engineered and recombinantly produced major ampullate silk-like proteins (focusing on structure-function relationships with respect to their primary structures), and discuss their relevance to the storage and assembly of spider silk proteins in vivo.

Distinct contributions of model MaSp1 and MaSp2 like peptides to the mechanical properties of synthetic major ampullate silk fibers as revealed in silico

All characterized major ampullate silks from orb-web weaving spiders are composites of primarily two different proteins: MaSp1 and MaSp2. The conserved association of MaSp1 and MaSp2 in these spider species, the highly conserved amino acid motifs, and variable ratios of MaSp1 to MaSp2 demonstrate the importance of both MaSp1 and MaSp2 to the strength and elasticity of the fiber. Computer simulated mechanical tests predicted differing roles for MaSp1 and MaSp2 in the mechanical properties of the fibers. Recombinant MaSp1 and MaSp2 proteins were blended and spun into fibers mimicking the computer-simulated conditions. Mechanical testing verified the differing roles of MaSp1 and MaSp2.

An Investigation of the Divergence of Major Ampullate Silk Fibers from Nephila clavipes and Argiope aurantia

An Investigation of the Divergence of Major Ampullate Silk Fibers from <i>Nephila clavipes</i> and <i>Argiope aurantia</i>

An Investigation of the Divergence of Major Ampullate Silk Fibers from Nephila clavipes and Argiope aurantia

The major ampullate fiber of both Nephila clavipes and Argiope aurantia is composed of two different proteins, MaSp1 and MaSp2. Each of these proteins has a highly conserved pattern of silk-associated amino acid motifs. The GPGXX motif is the only source of proline and is unique to MaSp2. On the basis of the percent of proline, Nephila clavipes major ampullate silk was calculated to consist of 19% MaSp2 and 81% MaSp1, while Argiope aurantia was calculated to have a significantly higher MaSp2 content of 59% with MaSp1 comprising the remaining 41%. To investigate the functional implications of the difference in protein composition, major ampullate silk fibers from Nephila clavipes and Argiope aurantia were mechanically tested and compared. Stress-strain curves produced from polynomial regression show that the two significant differences between major ampullate silk fibers from Nephila clavipes and Argiope aurantia are the average peak load stress and Young's modulus, with Argiope higher for both.

A Physico-Chemical Study of the Supercontraction of Spider Major Ampullate Silk Fibers

Methanol, ethanol, and isopropanol do not radially swell or produce supercontrac tion (SC) of spider major ampullate silk fibers. Their aqueous solutions do so at rates associated with water concentrations, but produce no levels of SC ratios other than those expected of water Itself. Saturated aqueous solutions of NaSCN and LiBr produce swelling and lowered birefringences of axially restrained fibers. On unre strained fibers NaSCN produces further SC. After SC, the wetted fibers can be stretched to their original lengths, and on drying they are in a stable state from which further SC and re-stretching cycles can be repeated. On both unrestrained major and minor ampullate fibers, LiBr causes irreversible gellation. X-ray diffraction measure ments show that the crystallite unit cell rotates due to macro distortion during SC, but is otherwise unchanged. The suggestion is that water ruptures hydrogen bonds between amino and carboxy groups, in the anti-parallel rippled sheet model of silk, to produce radial swelling and allow SC. The internal axial retractive force present in the wetted fibers, which is the driving force for SC, provides axial tension in the restrained condition and in turn prevents access to LiBr, which in the unrestrained condition brings about gellation.

Web Components Associated with the Major Ampullate Silk Fibers of Orb-Web-Building Spiders

Web Components Associated with the Major Ampullate Silk Fibers of Orb-Web-Building Spiders

Dimensions, Birefringences, and Force-Elongation Behavior of Major and Minor Ampullate Silk Fibers from Orb-Web-Spinning Spiders—The Effects of Wetting on these Properties

The effects of wetting by water were determined on the dimensions, birefringences, and force-elongation behavior of major and minor ampullate silk fibers from Araneus diadematus Clerck, and major only from Araneus sericatus Clerck and Araneus gemma (McCook) (all family Araneidae). The values obtained at room conditions (RC), in the wetted, restrained (WR) and wetted, axially unrestrained (WU) states, the ratios of these, between states, are reported upon. The unexpected supercontraction of major ampullate fibers, WU, is contrasted with the stabile behavior of those from the minor ampullate gland systems. When dried from the WR state, both types appear to recover their RC properties. But when dried slack from the WU state, the major ampullate fibers undergo the “drawing” phenomenon typical of many man-made protofibers. The techniques required in order to secure data on these fragile (diameters, 0.6–6.0 μm) fibers, are described in some detail.