Silk Formation Research Articles

Pressure- and humidity-induced structural transition of silk fibroin

Spiders and silkworms convert silk fibroin solution into fibers by adjusting physiological conditions such as water content and internal pressure. Water molecules are known to plasticize molecular chains of silk fibroin, affording β-sheet structures. However, the effect of pressure on the crystallization of silk fibroin and the differences between pressure- and water-induced crystallization in spider and silkworm silk remain unexplained. Herein, the effect of pressure and humidity on the crystallization of spider and silkworm silk amorphous films is investigated. Despite not inducing irreversible crystallization of spider silk, pressure promotes β-sheet formation of silkworm silk. In contrast, water induces crystallization of both spider and silkworm silk. Pressure-induced crystallization results in larger crystallinity but smaller crystallite size in comparison to water-induced crystallization. The formation of β-sheet structures enhances breaking strength and modulus of the silk films, which paves the way toward silk-based structural materials.

Formation of natural silk and progress in artificial spinning

Natural silks, produced by spiders and silkworms, are excellent materials with marvelous mechanical properties, biocompatibility and biodegradability, and widely used in the fields of textile, optics, electronics, biomedicine and environmental engineering. So far, there are many spinning methods to improve the mechanical properties of artificial fibers, such as wet spinning, dry spinning, and electrospinning. However, the performance of most artificial fibers is still inferior to natural silks. It is important to understand the correlations between hierarchical structures and performance in the field of artificial spinning. This review introduces the formation of natural silks, the relationship between the mechanical properties of silks and the hierarchical structure, the research progress in artificial spinning, and the application of silks.

Tailorable hydrogel of gelatin with silk fibroin and its activation/crosslinking for enhanced proliferation of fibroblast cells

Gelatin based hydrogel (Gel) possess remarkable cytocompatibility profile rendering it appropriate for tissue engineering applications. Herein, the questionable mechanical property of Gel was tuned by tailoring with different loading concentrations of silk fibroin (SF). The as tailored matrix was reconnoitred for its physico-mechanical, chemical and biological properties in order to investigate the effect of SF loading. Ethanol treatment lead to enhance β-sheet formation of silk and subsequently, carbodiimide coupling was deployed to covalently crosslink the matrix. Substantial increase in cohesive energy with amplifying concentration of SF in the Gel matrix. As evidenced by Raman spectroscopy, right shift of the Amide I peak and stretching of COO− confirms activation of fibroin moiety along with crosslinking of gelatin, respectively. Moreover, with addition of SF, surface properties were tuned to attain maximum cell adhesion and proliferation. Further, MTT assay corroborated the same with definite increase in mitochondrial activities of L929 fibroblast cells for SF containing matrix as compared to its bare counterfeit while enhanced proliferation was confirmed by Rhodamine-DAPI staining. The alteration in mechanical and textural tribology of SF tailored Gel matrix certainly portrayed improved stability along with excellent cytocompatibility thus making it a plausible alternative in regenerative medicine.

Silk as templates for hydroxyapatite biomineralization: A comparative study of Bombyx mori and Antheraea pernyi silkworm silks

Silk is extensively investigated in bone tissue engineering due to its extraordinary mechanical properties and ability to regulate biomineralization. Protein templates regulate biomineralization process through chemical interaction with ions. However, the effect of structural differences in silk fibroin on biomineralization has not been studied in detail. In this study, Antheraea pernyi silk fibroin (ASF) and Bombyx mori silk fibroin (BSF) fibers were used as templates to study the effect of silk species on biomineralization. The results showed that silk fibroin could induce the formation of calcium-deficient hydroxyapatite in simulated body fluid (SBF), and the SBF treatment resulted in the formation of silk I crystals. Compared with BSF, ASF exhibited a higher ability to induce mineralization, which may depend on the differences in hydrophilic amorphous fractions between ASF and BSF. The amorphous fractions of ASF contain more acidic amino acids, which can provide more nucleation sites in the initial stage of mineralization, resulting in faster mineralization process and more mineral deposits. This study decodes the key role of silk fibroin fractions on biomineralization, and provides deeper insights for the study of silk fibroin as biomineralization template and bone repair materials.

Engineered a novel pH-sensitive short major ampullate spidroin

The pH diversification has been proved as an important factor affecting the self-assembly of spidroin. Herein, we constructed a novel spider silk protein (NT-MaSp1s-CT) with the pH-dependent secondary structures, containing pH-sensitive N-terminal, C-terminal domains and a repeated core region with merely 191 amino acids. Then pH sensitivity of NT-MaSp1s-CT was detected at different pH conditions and NT-MaSp1s-CT displayed pH-dependent conformational transitions consistent with rational designed objective. Besides, the micelles theory was employed to inquiry the assembly mechanism of NT-MaSp1s-CT in high concentration spinning dope. As expected, NT-MaSp1s-CT protein can be spun into continuous and uniform fibers with the pH ranging from 2 to 11, which is the largest pH boundary for artificial spider silk formation, simplifying the assembly conditions and paving a broad path for spinning process. Moreover, the hemolysis and cytotoxicity of NT-MaSp1s-CT fibers were also determined and the novel fibers exhibit excellent biocompatibility, providing wider potential applications in the biomedical and pharmaceutical fields.

Structure of the N-terminal domain of Euprosthenops australis dragline silk suggests that conversion of spidroin dope to spider silk involves a conserved asymmetric dimer intermediate.

Spider silk is a biomaterial with exceptional mechanical toughness, and there is great interest in developing biomimetic methods to produce engineered spider silk-based materials. However, the mechanisms that regulate the conversion of spider silk proteins (spidroins) from highly soluble dope into silk are not completely understood. The N-terminal domain (NT) of Euprosthenops australis dragline silk protein undergoes conformational and quaternary-structure changes from a monomer at a pH above 7 to a homodimer at lower pH values. Conversion from the monomer to the dimer requires the protonation of three conserved glutamic acid residues, resulting in a low-pH `locked' dimer stabilized by symmetric electrostatic interactions at the poles of the dimer. The detailed molecular events during this transition are still unresolved. Here, a 2.1 Å resolution crystal structure of an NT T61A mutant in an alternative, asymmetric, dimer form in which the electrostatic interactions at one of the poles are dramatically different from those in symmetrical dimers is presented. A similar asymmetric dimer structure from dragline silk of Nephila clavipes has previously been described. It is suggested that asymmetric dimers represent a conserved intermediate state in spider silk formation, and a revised `lock-and-trigger' mechanism for spider silk formation is presented.

Disruption of the Metal Ion Environment by EDTA for Silk Formation Affects the Mechanical Properties of Silkworm Silk.

Silk fiber has become a research focus because of its comprehensive mechanical properties. Metal ions can influence the conformational transition of silk fibroin. Current research is mainly focused on the role of a single ion, rather than the whole metal ion environment. Here, we report the effects of the overall metal ion environment on the secondary structure and mechanical properties of silk fibers after direct injection and feeding of silkworms with EDTA. The metal composition of the hemolymph, silk gland, and silk fiber changed significantly post EDTA treatment. Synchrotron FTIR analysis indicated that the secondary structure of silk fiber after EDTA treatment changed dramatically; particularly, the β-sheets decreased and the β-turns increased. Post EDTA treatment, the silk fiber had significantly decreased strength, Young’s modulus, and toughness as compared with the control groups, while the strain exhibited no obvious change. These changes can be attributed to the change in the metal ion environment in the silk fibroin and sericin in the silk gland. Our investigation provides a new theoretical basis for the natural silk spinning process, and our findings could help develop a method to modify the mechanical properties of silk fiber using metal ions.

Inducible Fibril Formation of Silk-Elastin Diblocks.

Silk–elastin block copolymers have such physical and biological properties that make them attractive biomaterials for applications ranging from tissue regeneration to drug delivery. Silk–elastin block copolymers that only assemble into fibrils at high concentrations can be used for a template-induced fibril assembly. This can be achieved by additionally including template-binding blocks that promote high local concentrations of polymers on the template, leading to a template-induced fibril assembly. We hypothesize that template-inducible silk-fibril formation, and hence high critical concentrations for fibril formation, requires careful tuning of the block lengths, to be close to a critical set of block lengths that separates fibril forming from nonfibril forming polymer architectures. Therefore, we explore herein the impact of tuning block lengths for silk–elastin diblock polypeptides on fibril formation. For silk–elastin diblocks ESm–SQn, in which the elastin pentamer repeat is ES = GSGVP and the crystallizable silk octamer repeat is SQ = GAGAGAGQ, we find that no fibril formation occurs for n = 6 but that the n = 10 and 14 diblocks do show concentration-dependent fibril formation. For n = 14 diblocks, no effect is observed of the length m (with m = 40, 60, 80) of the amorphous block on the lengths of the fibrils. In contrast, for the n = 10 diblocks that are closest to the critical boundary for fibril formation, we find that long amorphous blocks (m = 80) oppose the growth of fibrils at low concentrations, making them suitable for engineering template-inducible fibril formation.

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure.

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group's method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Conformation and dynamics of soluble repetitive domain elucidates the initial \u03b2-sheet formation of spider silk

The β-sheet is the key structure underlying the excellent mechanical properties of spider silk. However, the comprehensive mechanism underlying β-sheet formation from soluble silk proteins during the transition into insoluble stable fibers has not been elucidated. Notably, the assembly of repetitive domains that dominate the length of the protein chains and structural features within the spun fibers has not been clarified. Here we determine the conformation and dynamics of the soluble precursor of the repetitive domain of spider silk using solution-state NMR, far-UV circular dichroism and vibrational circular dichroism. The soluble repetitive domain contains two major populations: ~65% random coil and ~24% polyproline type II helix (PPII helix). The PPII helix conformation in the glycine-rich region is proposed as a soluble prefibrillar region that subsequently undergoes intramolecular interactions. These findings unravel the mechanism underlying the initial step of β-sheet formation, which is an extremely rapid process during spider silk assembly.

Bmo-miR-275 down-regulates expression of Bombyx mori sericin gene 2 in vitro.

We hypothesized that bmo-miR-275 has a potential regulatory function regarding the expression of sericin gene 2 (BmSer-2). First, we examined the expression of bmo-miR-275 and its target gene BmSer-2 in seven different tissues from 5th instar day-3 silkworm larvae. The results showed that they were both specifically expressed in the middle silk gland, implying that spatio-temporal conditions are required for bmo-miR-275 to regulate the expression of BmSer-2. To test this hypothesis, we constructed a pri-bmo-miR-275 expressing plasmid pcDNA3.0 [ie1-egfp-pri-bmo-miR-275-SV40] and BmSer-2-3´UTR recombinant reporter plasmids pGL3.0 [A3-luc-Ser-2-3′ UTR-SV40]. Finally, BmN cells were harvested and luciferase activity was detected. Results showed that luciferase activity was reduced significantly (P<0.05) in BmN cells co-transfected with pcDNA3.0 [ie1-egfp-pri-bmo-miR-275-SV40] and pGL3.0 [A3-luc-Ser-2-3’UTR-SV40], suggesting that bmo-miR-275 can down-regulate the expression of BmSer-2 in vitro. Our results improve the understanding of the regulatory function of Bombyx mori miRNA on the expression of genes regulating silk formation.

From EST to novel spider silk gene identification for production of spidroin-based biomaterials

A cDNA library from a pool of all the seven silk glands from a tropical spider species was constructed. More than 1000 expressed sequence tag (EST) clones were created. Almost 65% of the EST clones were identified and around 50% were annotated. The cellular and functional distribution of the EST clones indicated high protein synthesis activity in spider silk glands. Novel clones with repetitive amino acid sequences, which is one of the most important characteristics of spider silk genes, were isolated. One of these clones, namely TuSp2 in current research, contains two almost identical fragments with one short C-terminal domain. Reverse transcription (RT) PCR and expression analysis showed that it is expressed in the tubuliform gland and involved in eggcase silk formation. Furthermore, its single repetitive domain can be induced to form various types of materials, including macroscopic fibers, transparent film and translucent hydrogel. This study implies promising potentials for future identification of novel spidroins and development of new spidroin-based biomaterials.

Proteome profile of spinneret from the silkworm, Bombyx mori.

The silkworm spinneret is an important tissue for silk fibrillogenesis and spinning. All biochemical processes during silk fibrillogenesis are correlated with silk properties. Understanding the role of spinneret in silk fibrillogenesis may help to reveal the mechanism of silk fibrillogenesis as well as improve silk quality for commercial purposes. Thus, we profiled the proteome of silkworm spinneret. A total of 1572 proteins and 232 differential abundance proteins were identified. Silk fibrillogenesis-related proteins, such as cuticle proteins, ion-transporting proteins, muscular proteins, and energy metabolic proteins, were abundant in spinneret. Metabolic pathway and GO enrichment analyses revealed that the identified proteins were involved in energy metabolism, chitin binding, and cuticle construction. Active energy metabolism may provide abundant energy for the muscle contraction as well as ion and water exchange. The chitin binding and cuticle construction process may provide sufficient shear forces for silk formation. Our data suggest that silkworm spinneret provides a suitable physiological and biochemical environment for silk fibrillogenesis. These proteins are potential targets for improving silk quality in the silk industry. Data are available via ProteomeXchange with identifier PXD004455.

Mass spectrometry captures structural intermediates in protein fiber self-assembly.

Self-assembling proteins, the basis for a broad range of biological scaffolds, are challenging to study using most structural biology approaches. Here we show that mass spectrometry (MS) in combination with MD simulations captures structural features of short-lived oligomeric intermediates in spider silk formation, providing direct insights into its complex assembly process.

Insights into Silk Formation Process: Correlation of Mechanical Properties and Structural Evolution during Artificial Spinning of Silk Fibers.

The extraordinary comprehensive mechanical properties of animal silk (especially spider and silkworm silk) have led to extensive research on the underlying mechanisms involved. Herein, we selected various regenerated silk fibroin (RSF) fibers by choosing different postdraw conditions in a wet-spinning process developed in this laboratory to study their structure-property relationship. We use synchrotron radiation infrared and X-ray diffraction techniques to monitor the structural differences in these RSF fibers and correlate them with their mechanical properties. The results show that with the increase of post draw-down ratio, the β-sheet content, crystallinity, and molecular orientation in these RSF fibers increase while the crystalline size decreases. The relationship between structural changes and the draw-down ratio reflects the corresponding variation in mechanical properties, namely, an increase in breaking stress with a decline in breaking strain in relation to increases in draw-down ratio. Therefore, these results provide solid and direct evidence on the evolution of structure during the artificial spinning process and on how structure determines the final mechanical performance of silk fibers. We believe this study provides a good background on the relationship between microscopic structure and macroscopic properties in polymer science and may prove useful in the production of high performance materials, not only for silk fibers but also for other natural and synthetic polymeric materials.

Secondary Structure Transition and Critical Stress for a Model of Spider Silk Assembly.

Spiders spin their silk from an aqueous solution to a solid fiber in ambient conditions. However, to date, the assembly mechanism in the spider silk gland has not been satisfactorily explained. In this paper, we use molecular dynamics simulations to model Nephila clavipes MaSp1 dragline silk formation under shear flow and determine the secondary structure transitions leading to the experimentally observed fiber structures. While no experiments are performed on the silk fiber itself, insights from this polypeptide model can be transferred to the fiber scale. The novelty of this study lies in the calculation of the shear stress (300-700 MPa) required for fiber formation and identification of the amino acid residues involved in the transition. This is the first time that the shear stress has been quantified in connection with a secondary structure transition. By study of molecules containing varying numbers of contiguous MaSp1 repeats, we determine that the smallest molecule size giving rise to a "silk-like" structure contains six polyalanine repeats. Through a probability analysis of the secondary structure, we identify specific amino acids that transition from α-helix to β-sheet. In addition to portions of the polyalanine section, these amino acids include glycine, leucine, and glutamine. The stability of β-sheet structures appears to arise from a close proximity in space of helices in the initial spidroin state. Our results are in agreement with the forces exerted by spiders in the silking process and the experimentally determined global secondary structure of spidroin and pulled MaSp1 silk. Our study emphasizes the role of shear in the assembly process of silk and can guide the design of microfluidic devices that attempt to mimic the natural spinning process and predict molecular requirements for the next generation of silk-based functional materials.

In situ formation of silk-gelatin hybrid hydrogels for affinity-based growth factor sequestration and release

Facile formation of silk fibroin/gelatin-heparin hybrid hydrogels for affinity-based growth factor sequestration and release.

Transcriptomic Analysis of the Anterior Silk Gland in the Domestic Silkworm (Bombyx mori) - Insight into the Mechanism of Silk Formation and Spinning.

Silk proteins are synthesized in the middle and posterior silk glands of silkworms, then transit into the anterior of the silk gland, where the silk fibers are produced, stored and processed. The mechanism of formation and spinning of the silk fibers has not been fully elucidated, and transcriptome analyses specific to the anterior silk gland have not been reported. In the present study, we explored gene expression profiles in five regions of silk gland samples using the RNA-Seq method. As a result, there were 959,979,570 raw reads obtained, of which 583,068,172 reads were mapped to the silkworm genome. A total of 7419 genes were found to be expressed in terms of reads per kilobase of exon model per million mapped reads ≥ 5 in at least one sample. The gene numbers and expression levels of the expressed genes differed between these regions. The differentially expressed genes were analyzed, and 282 genes were detected as up-regulated in the anterior silk gland, compared with the other parts. Functions of these genes were addressed using the gene ontology and Kyoto Encyclopedia of Genes and Genomes databases, and seven key pathways were enriched. It suggested that the ion transportation, energy metabolism, protease inhibitors and cuticle proteins played essential roles in the process of silk formation and spinning in the anterior silk gland. In addition, 210 genes were found differently expressed between males and females, which should help to elucidate the mechanism of the quality difference in silk fibers from male and female silkworms.

Carbonic anhydrase generates a pH gradient in Bombyx mori silk glands

Silk is a protein of interest to both biological and industrial sciences. The silkworm, Bombyx mori, forms this protein into strong threads starting from soluble silk proteins using a number of biochemical and physical cues to allow the transition from liquid to fibrous silk. A pH gradient has been measured along the gland, but the methodology employed was not able to precisely determine the pH at specific regions of interest in the silk gland. Furthermore, the physiological mechanisms responsible for the generation of this pH gradient are unknown.In this study, concentric ion selective microelectrodes were used to determine the luminal pH of B. mori silk glands. A gradient from pH 8.2 to 7.2 was measured in the posterior silk gland, with a pH 7 throughout the middle silk gland, and a gradient from pH 6.8 to 6.2 in the beginning of the anterior silk gland where silk processing into fibers occurs. The small diameter of the most anterior region of the anterior silk gland prevented microelectrode access in this region. Using a histochemical method, the presence of active carbonic anhydrase was identified in the funnel and anterior silk gland of fifth instar larvae. The observed pH gradient collapsed upon addition of the carbonic anhydrase inhibitor methazolamide, confirming an essential role for this enzyme in pH regulation in the B. mori silk gland. Plastic embedding of whole silk glands allowed clear visualization of the morphology, including the identification of four distinct epithelial cell types in the gland and allowed correlations between silk gland morphology and silk stages of assembly related to the pH gradient.B. mori silk glands have four different epithelial cell types, one of which produces carbonic anhydrase. Carbonic anhydrase is necessary for the mechanism that generates an intraluminal pH gradient, which likely regulates the assembly of silk proteins and then the formation of fibers from soluble silk proteins. These new insights into native silk formation may lead to a more efficient production of artificial or regenerated silkworm silk fibers.