The processing of water soluble high molecular weight silk proteins into water insoluble fibers in both spider and silkworm involves many factors including disulfide bond formation, cation interactions, glycosylation and perhaps other chemical or physical steps (Kaplan et al., 1992a,b). Initially, some degree of self-organization or assembly drives the formation of the crystalline repeats in the protein fibers. In the silkworm, changes in physiological conditions such as pH and salt concentrations in the gland accompany the processing and presumably help maintain solubility despite increasing protein concentration during passage through the various regions. Physical shear generated during spinning the soluble silk appears in a large part responsible for conversion to the insoluble silk fiber in the natural spinning process (Ilzuka 1985a,b; Magoshi et al., 1985, 1994). In the spider and silkworm there are three distinct regions to the glands and two sets of these organs feeding into one final thread (Magoshi et al., 1985, 1994; Tillinghast and Townley, 1994). In the silkworm the protein concentration is approximately 20~30% in the middle region of the gland where the fibroin is stored and sericin is synthesized, and significantly higher in the
anterior region of the gland where spinning is initiated (Magoshi et al., 1985, 1994). In the spider the dragline protein is synthesized in the pair of major ampullate glands. Depending on environmental conditions and needs, the amino acid composition of the silk can vary considerably, not only between individual spiders but also for the same spider on different days (Work and Young, 1987; Vollrath, 1999) [35], which raises questions about the genomic sequences and organization of the genes encoding these proteins. Compared to the silkworm, the major ampullate gland in the spider is smaller and there is no sericin contribution in the middle region of the gland. The process leads to the formation of a lyotropic liquid crystalline phase prior to spinning in both the spider and the silkworm, and in many of the different glands of the spider responsible for the different silks (Kerkam et al., 1991; Viney et al., 1994). The formation of this liquid crystalline phase is characterized by axial alignment and interaction of polymer chains in various stages of registry with each other while remaining soluble in aqueous medium. This is one method that allows the spider to maintain a relatively high concentration of the protein in aqueous solution prior to spinning without resulting in the formation of insoluble -sheets.
Solubilization of spun spider silks is difficult. Most solvents used to solubilize globular proteins will not suffice. The highly organized fibrous structure of silk and the extensive hydrogen bonding and van der Waals interactions lead to the exclusion of water from the intersheet regions of the -sheets after spinning. Silk fibers are insoluble in water, dilute acids and alkali, chaotropic agents such as urea and guanidine hydrochloride and most organic solvents (Lombardi and Kaplan, 1990; Mello et al., 1994). The silks are also resistant to most proteolytic enzymes, with chymotrypsin an exception for silkworm fibroin. Spider silk can be solubilized by immersion of the fibers in very high concentration salt solutions such as lithium bromide, lithium thiocyanate or calcium chloride and other calcium salts. Also high concentrations of propionic acidrhydrochloric acid mixtures and formic acid can be used (Mello et al., 1994). After solubilization, dialysis into chaotropic agents, water or buffers can
be carried out; however, rapid reprecipitation or gelation is a common result. This aggregation is mostly due to the formation of beta-sheet conformations. Solid state 13C-NMR and
2H-NMR studies of N. clavipes dragline silk indicate that the crystalline fractions are composed primarily of alanine rich-sequences, including highly oriented regions (Simmons et al., 1994, 1996). The crystalline polyalanine regions are not affected by water while the other domains are plasticized leading to supercontraction and shrinkage of the major ampullate dragline silk from N. clavipes (Jelinski et al., 1999).
Thin films have been prepared from silk proteins (Muller et al., 1993) and a silk conformation (silk III)(secondary structure with a diffraction pattern that matches a polyglycine 31-helical structure) have been observed depending on the conditions used to prepare the silk protein (Valluzzi et al., 1996). These hexagonally packed threefold helices were observed by electron diffraction analysis of the thin films after transfer to transmission electron microscopy grids. Cholesteric liquid crystalline phases have been observed with the protein based on observations in situ after cryogenic quenching and microtoming of actively spinning N. clavipes and B. mori, followed by TEM, electron diffraction and AFM characterization (Willcox et al., 1996) [36]. Correlations between primary silk primary sequence and self-assembly at air-water and organic solvent- water interfaces are providing insight into the processing options for these types of proteinbased materials to ‘direct’ the assembly in a desired path of secondary and higher-order structure formation (Valluzzi et al., 1999a,b; Wilson et al., 2000).
Unlike man-made fibers, the silks of spiders are spun from aqueous solutions and at atmospheric pressure in a process still poorly understood. The molecular mechanism of this process involves the conversion of a highly concentrated, predominantly disordered silk protein (spidroin) into Ӫ-sheet-rich structures.
Spidroins, the major silk proteins making up the spider’s dragline silk, originate in two distinct tissue layers (A and B) in the spider’s major ampullate gland. Earlier studies indicate
that the lumen in the tail and ampulla A zone portions are slightly alkaline or neutral whereas the B zone and duct are acidic (see Fig. 5~7). The origin of this pH gradient in the ampulla is not clear, nor has the gradient been fully quantified, though several hypotheses have been put forward. The secretion of acidic polysaccharides in the B zone and the presence of a high concentration of tyrosine residues in the A zone and tail might account for variation in pH (see Fig.5). A high activity of phosphatases in epithelial cells suggests that pH may be influenced by the secretion of phosphate ions into the lumen of the gland. More recently, the amino acid composition and the partial DNA sequences of spider silk proteins indicate the presence of residues such as histidine, arginine, glutamate/glutamine, and aspartate/asparagine that could influence the pH.
Fig 5: Spinning pathway of spider.
Flow chart summarizing the correlation between pH and the structure along the spinning pathway (the shape of the B-zone box represents the narrowing of the B-zone in the gland). (from [37])
Fig 6: Drawing of dissected major ampullate gland and associated strucutures taken from a light micrograph.
a, A-zone; b, B-zone; f, funnel; 1,2,3; first, second and third limbs of duct; m, duct levator muscle; v, valve; vm, valve tensor muscle; t, terminal tubule; s, spigot, Scale bar 1mm. (from [38])
Fig 7: The proposed stages in lamellar liquid crystalline assembly of the nano fibrils of the major ampullate (dragline) silk thread in orb web spiders (see text) showing.
The proposed stages in lamellar liquid crystalline assembly of the nano fibrils of the major ampullate (dragline) silk thread in orb web spiders (see text) showing: (i) a diagrammatic of the gland and duct (upper part of illustration); nematic discotic units; (ii) rod-shaped molecules of spidroin; (iii) partly unwound molecules; (iv) early stage of formation of the solid fibre and (v) fully formed fibre (see text). The lumen of the gland has been represented as much wider in proportion to length with only a small number of bi-layer discs (top left) and the epithelium, (e), only on one side of the duct and gland. The dotted lines represent the molecular director field.
This lies at right angles to the slow axis of polarization as a result of the assembly of the compactly wound, rod-shaped molecules of spidroin (ii) into bi-layered discs of the nematic discotic phase (i). These are present as an escaped nematic texture in the gland proper and first half of the duct (upper half of figure). Funnel (f ), draw down taper (d), spigot (s), thread (t), glycine-rich segments (g), polyalanine segments (a), mobile segments (m), bcrystallites (b), nano fibrils (n). Based on Knight & Vollrath (1999), Vollrath & Knight (2001), Knight &
Vollrath (2001a,b) and Chen et al. (2001). (From Phil. Trans. Soc. Lond. B (2002) 357, 155~163)
Fig 8: A spider's dragline spinneret.
A spider's dragline spinneret. The top part shows original drawings of the histology of the spinneret (lumen, L);
the bottom part outlines its spinning function, which entails drawing the liquid crystalline dope solution produced in the gland through a tapering s-shaped duct, thereby converting it into an elastic thread. Dope production occurs in two zones: the A-zone of the gland (see panel A) secretes spidroin, the protein forming the core of the thread, while the B-zone (see panel B) secretes the thread's thin coat. The secretory vesicles of the A-zone contain short, narrow filaments; most of those of the B-zone contain hexagonal columnar liquid crystals.
The greatly thickened cuticle of the funnel (in panel V) anchors the extensible duct to the less mobile sac, perhaps to prevent shearing of the dope when the spider wiggles. The duct itself has a thin cuticle, which acts as a dialysis membrane and may allow water and sodium ions out of the lumen, and potassium ions, surfactants and lubricants into the lumen to facilitate thread formation (see panel X). The epithelium of the s-shaped duct progressively increases in height from the funnel to the valve, suggesting increased pumping of water and ions as
the dope is drawn through the duct (panels C and D show the histology of epithelium in the first and second limbs, respectively). The drawdown process is mainly internal and starts in the third limb of the duct (see panel X), approximately 4mm from the exit spigot. Just before and after the start of the drawdown taper, single flask-shaped gland cells, such as seen in inset E, may contribute an extra coating to the thread. The `valve', shown in panel Y in the bottom of the figure, is not a restriction nozzle, but a clamp for gripping the thread. It may also act as a `ratchet' or `pump', to restart spinning after internal rupture of a filament. The section of duct (see inset F) after the valve appears to be highly specialized for water pumping, having tall cells with numerous mitochondria and apical microvilli and a large surface area provided by apical infoldings of the plasma
membrane. Finally, the thread is gripped by the flexible and elastic lips of the spigot (inset G), through which it passes to the outside world (see panel Z). The spigot strips off the last of the aqueous phase surrounding the thread, thus helping to retain water in the spider, and also places the thread under tension for the final air-drawing step. (from [34])