David P. Knight

Liquid crystalline spinning of spider silk.

Spider silk has outstanding mechanical properties despite being spun at close to ambient temperatures and pressures using water as the solvent. The spider achieves this feat of benign fibre processing by judiciously controlling the folding and crystallization of the main protein constituents, and by adding auxiliary compounds, to create a composite material of defined hierarchical structure. Because the ‘spinning dope’ (the material from which silk is spun) is liquid crystalline, spiders can draw it during extrusion into a hardened fibre using minimal forces. This process involves an unusual internal drawdown within the spiders spinneret that is not seen in industrial fibre processing, followed by a conventional external drawdown after the dope has left the spinneret. Successful copying of the spiders internal processing and precise control over protein folding, combined with knowledge of the gene sequences of its spinning dopes, could permit industrial production of silk-based fibres with unique properties under benign conditions.

Changes in element composition along the spinning duct in a Nephila spider.

Abstract. The silk gland of the golden orb spider Nephila edulis connects to the exit spigot through a long S-shaped duct that assists in the formation of the thread. Previous evidence suggests that the epithelium of the distal (last) part of the duct is specialized for ion transport and that a proton pump is involved in this process. Here, we present evidence from SEM (scanning electron microscope)-EDAX (energy dispersive X-ray) microanalysis of rapidly frozen material maintained at approximately –150°C and from the use of pH indicators that the element composition and pH change progressively as the dragline silk dope (spinning solution) passes down the duct to form the thread. Na+ and Cl– composition decreased while K+ and P and S increased. Indicators suggested that the pH dropped from 6.9±0.1 to 6.3±0.1. These novel findings suggest that the absorption of Na+ and secretion of the more chaotropic K+ may help the silk protein molecules to refold while the secretion of H+ may assist in this process and reduce the repulsive charges on them. This in turn may allow the molecules to approach one another more closely to crystallize. Thus precise control of the ionic environment within the spiders spinning duct may be important in forming a tough insoluble thread and when devising mimetic processes to spin silk proteins industrially.

Beta transition and stress-induced phase separation in the spinning of spider dragline silk.

Spider dragline silk is formed as the result of a remarkable transformation in which an aqueous dope solution is rapidly converted into an insoluble protein filament with outstanding mechanical properties. Microscopy on the spinning duct in Nephila edulis spiders suggests that this transformation involves a stress-induced formation of anti-parallel beta-sheets induced by extensional flow. Measurements of draw stress at different draw rates during silking confirm that a stress-induced phase transition occurs.

Conformation transition kinetics of Bombyx mori silk protein

Time‐resolved FTIR analysis was used to monitor the conformation transition induced by treating regenerated Bombyx mori silk fibroin films and solutions with different concentrations of ethanol. The resulting curves showing the kinetics of the transition for both films and fibroin solutions were influenced by the ethanol concentration. In addition, for silk fibroin solutions the protein concentration also had an effect on the kinetics. At low ethanol concentrations (for example, less than 40% v/v in the case of film), films and fibroin solutions showed a phase in which β‐sheets slowly formed at a rate dependent on the ethanol concentration. Reducing the concentration of the fibroin in solutions also slowed the formation of β‐sheets. These observations suggest that this phase represents a nucleation step. Such a nucleation phase was not seen in the conformation transition at ethanol concentrations > 40% in films or > 50% in silk fibroin solutions. Our results indicate that the ethanol‐induced conformation transition of silk fibroin in films and solutions is a three‐phase process. The first phase is the initiation of β‐sheet structure (nucleation), the second is a fast phase of β‐sheet growth while the third phase represents a slow perfection of previously formed β‐sheet structure. The nucleation step can be very fast or relatively slow, depending on factors that influence protein chain mobility and intermolecular hydrogen bond formation. The findings give support to the previous evidence that natural silk spinning in silkworms is nucleation‐dependent, and that silkworms (like spiders) use concentrated silk protein solutions, and careful control of the pH value and metallic ion content of the processing environment to speed up the nucleation step to produce a rapid conformation transition to convert the water soluble spinning dope to a tough solid silk fiber. Proteins 2007.

Silk production in a spider involves acid bath treatment

We studied physiological conditions during the spinning of dragline silk by the garden cross spider, Araneus diadematus. Silk is converted from the liquid feedstock in the gland into a solid thread via a tapering tubular duct and exits at a spigot. The distal part of the tubule appears specialized for ion transport and the management of the pH inside the lumen. Thus, it appears that spider silk in vivo, like some industrial polymers in vitro, is spun through an acid bath.

Spider Silk Proteins – Mechanical Property and Gene Sequence

Abstract Spiders spin up to seven different types of silk and each type possesses different mechanical properties. The reports on base sequences of spider silk protein genes have gained importance as the mechanical properties of silk fibers have been revealed. This review aims to link recent molecular data, often translated into amino acid sequences and predicted three dimensional structural motifs, to known mechanical properties.

Synchrotron FTIR Microspectroscopy of Single Natural Silk Fibers

Synchrotron FTIR (S-FTIR) microspectroscopy was used to monitor the silk protein conformation in a range of single natural silk fibers (domestic and wild silkworm and spider dragline silk). With the selection of suitable aperture size, we obtained high-resolution S-FTIR spectra capable of semiquantitative analysis of protein secondary structures. For the first time, we have determined from S-FTIR the β-sheet content in a range of natural single silk fibers, 28 ± 4, 23 ± 2, and 17 ± 4% in Bombyx mori, Antheraea pernyi, and Nephila edulis silks, respectively. The trend of β-sheet content in different silk fibers from the current study accords quite well with published data determined by XRD, Raman, and (13)C NMR. Our results indicate that the S-FTIR microspectroscopy method has considerable potential for the study of single natural silk fibers.

Regenerative potential of silk conduits in repair of peripheral nerve injury in adult rats

Various attempts have been made to develop artificial conduits for nerve repair, but with limited success. We describe here conduits made from Bombyx mori regenerated silk protein, and containing luminal fibres of Spidrex(®), a silk-based biomaterial with properties similar to those of spider silk. Assessment in vitro demonstrated that Spidrex(®) fibres support neurite outgrowth. For evaluation in vivo, silk conduits 10 mm in length and containing 0, 100, 200 or 300 luminal Spidrex(®) fibres, were implanted to bridge an 8 mm gap in the rat sciatic nerve. At 4 weeks, conduits containing 200 luminal Spidrex(®) fibres (PN200) supported 62% and 59% as much axon growth as autologous nerve graft controls at mid-conduit and distal nerve respectively. Furthermore, Spidrex(®) conduits displayed similar Schwann cell support and macrophage response to controls. At 12 weeks, animals implanted with PN200 conduits showed similar numbers of myelinated axons (81%) to controls, similar gastrocnemius muscle innervation, and similar hindpaw stance assessed by Catwalk footprint analysis. Plantar skin innervation was 73% of that of controls. PN200 Spidrex(®) conduits were also effective at bridging longer (11 and 13 mm) gaps. Our results show that Spidrex(®) conduits promote excellent axonal regeneration and function recovery, and may have potential for clinical application.

Wet-Spinning of Regenerated Silk Fiber from Aqueous Silk Fibroin Solution: Discussion of Spinning Parameters

Regenerated silk fibroin (RSF) fibers were obtained by extruding a concentrated aqueous silk fibroin solution into an ammonium sulfate coagulation bath. A custom-made simplified industrial-type wet-spinning device with continuous mechanical postdraw was used. The effect of dope concentration, coagulation bath, extrusion rate, and postdraw treatment on the morphology of RSF fiber was examined. The results showed that although RSF fiber could be formed with dope concentration between 13 and 19% (w/w), the ones spun from 15% RSF solution showed the most regular morphology being dense and homogeneous in cross-section with a smooth surface and a uniform cylindrical shape. Though it had little effect on morphology, postdraw treatment especially under steam, significantly improved the mechanical properties of the RSF fibers.

Copper in the silk formation process of Bombyx mori silkworm

Evidence is presented here that cupric ions play a part in the natural spinning of Bombyx mori silk. Proton induced X‐ray emission studies revealed that the copper content increased from the posterior part to the anterior part of silk gland, and then further increased in the silk fiber. Spectrophotometric analysis demonstrated that cupric ions formed coordination complexes with silk fibroin chains while Raman spectroscopy indicated that they induced a conformation transition from random coil/helix to β‐sheet. Taken together these findings indicate that copper could play a role in the natural spinning process in silkworms.