Spidroins

The dragline silk is composed of the two spidroins MaSpI and MaSpII. A 2.4-kb segment from the 3 end of the original MoSpl-mRNA and a 2-kb segment from the... 

Fig. 11.1 Synthetic spidroin and spidroin-ELP plant expression cassettes.

Silk proteins (spidroins in spiders and fibroins in Lepidoptera insects) are assembled into well-defined nanofibrillar architectures (Craig and Riekel, 2002 Eby et al., 1999 Inoue et al., 2000b, 2001 Li et al., 1994 Putthanarat et al, 2000 Vollrath et al., 1996). Spidroins and fibroins are largely constructed from two chemically distinct repetitive motifs or blocks (Table I), an insoluble crystalline block and a soluble less-crystalline block (Craig, 2003 Fedic et al., 2002 Hayashi and Lewis, 2000 Hayashi et al., 1999). The crystalline blocks are composed of short side-chained amino acids in highly repetitive sequences that give rise to /1-sheet structures. 

Fig. 4. Time-induced conformational change of spider silk protein (spidroin) in solution. Solutions of silk proteins at 1% w/v in distilled water were monitored using circular dichroism. The graph shows a change in secondary structure with time. The silk proteins underwent a kinetically driven transition from a partially unfolded structure to a -sheet-rich structure (from Dicko et al., 2004c). ( ) after 0 days, (O) after 1 day, and (A) after 2 days. The conformational change appeared faster at 20°C compared to 5°C, suggesting a hydrophobically driven mechanism. (Copyright 2004 American Chemical Society.)...

Chen, X., Knight, D. P., Shao, Z. Z., and Vollrath, F. (2002). Conformation transition in silk protein films monitored by time-resolved Fourier transform infrared spectroscopy Effect of potassium ions on Nephila spidroin films. Biochemistry 41, 14944-14950. 

Dicko, C., Kenney, J. M., Knight, D., and Vollrath, F. (2004a). Transition to a beta-sheet-rich structure in spidroin in vitro The effects of pH and cations. Biochemistry 43, 14080-14087. 

Valluzzi, R., Szela, S., Avtges, P., Kirschner, D., and Kaplan, D. (1999). Methionine redox controlled crystallization of biosynthetic silk spidroin. J. Phys. Chem. B 103, 11382-11392. 

Yeast and bacterial systems often give low levels of expression of silks, and this has led to the development of production systems in tobacco and potato. Scheller et al. (2001) have shown that spider silk proteins can be produced in transgenic plants. They inserted synthetic spider silk protein (spidroin) genes into transgenic plants under the control of the CaMV35S promoter. Using this system they were able to demonstrate the accumulation of recombinant silk proteins to a level of at least 2% of total soluble protein in the endoplasmic reticulum of tobacco leaves, and potato tubers. 

From the viewpoint of zootaxa, the silkworm and the spider belong to insect and arachnid of arthropod, respectively. Their silk proteins (fibroin for silkworm silk and spidroin for spider major ampullate silk) do not have any genetic heritage in common and their amino acids sequence compositions are different too. However, the silkworm and spider employ a similar spinning process to produce silk. Furthermore, the silkworm silk and the major ampullate silk have a number of similar structural characteristics, both at the level of the secondary protein structure and the condensed silk morphology. Therefore, for the sake of convenience, they are discussed together in some parts of this text. 

Spider silk only has one protein monofilament, and the core-skin structure has been observed in some of them (Frische et al., 1998 Poza et al., 2002). It is thought that both the skin and the core are mainly composed of spidroins, which are differed from the primary structure (spidroin 1 and spidroin 2, >350 kDa calculated from mRNA) (Hinman and Lewis, 1992 Sponner et al., 2005a, b Xu and Lewis, 1990). 

To some extent, the properties of the protein are mainly determined by its primary structure (i.e., the amino acid sequence). The two kinds of structural protein, fibroin and spidroin have a distinct and highly repetitive primary structure, which results in specific secondary and tertiary... 

Table 1 The structure elements in fibroin and spidroin (Hakimi et al, 2007).

Fibroin (Bombyx mori) (ExPASy http //www.expasy.ch/) Spidroin (Nephila clavipe) (Shao et at, 2003)... 

Figure 3 The structural modules as a result from certain amino acid motifs (a) in spidroin (Hayashi et al., 1999 Hinman et al., 2000). The interaction of two different p-sheet forms poly(Ala) (b) and poly(Gly-Ala) (c) (Hayashi et al., 1999). Poly(Ala) has a tighter structure.

C- and N-terminal regions of spidroin are highly conserved among the spider silk proteins and play an important role in the assembly of spidroin (Huemmerich et al., 2004 Motriuk-Smith et al., 2005). 

In addition, one hypothesis for the secondary structure in spidroin suggests that there are amorphous phases, highly oriented crystals, and oriented noncrystalline phases coexisting (Grubb et al., 1997). This structure model has been used to explain the super-contraction of dragline (Liu et al., 2005b). 

Although the amino acid sequence as well as the secondary structure of fibroin differs from those of spidroin, the fibers spun from these proteins, that is, silkworm silk and spider silk have comparable mechanical properties. These may be attributed to the structural characteristics, both at the molecular and filament level. The superior mechanical properties of silk-based materials, such as films, coatings, scaffolds, and fibers produced using reconstituted or recombinant silk proteins, are determined by their condensed structures. 

Super-contraction, the chaperonage of the special structure of spidroin, is indeed an obstacle to the use of native spider dragline silk, especially in bioapplication (usually wet condition). Recently, it was found that the intrinsic properties of silk fibroin are much better than the data listed in Table 2. The inferior properties are generated by the spinning habit of... 

The attractive properties of silk fibers as a natural, sustainable product have inspired researchers to look for options to fabricate such fibers without the use of worms or spiders. Furthermore, these natural polymers, silk proteins (both fibroin and spidroin), allow for adjustable mechanical properties, thermal resistance (Drummy et al., 2005 Motta et al., 2002), as well as biomedical compatibility (Vepari and Kaplan, 2007). 

Regenerated spidroin and fibroin dissolved in various solvents are used as spinning dope while the coagulation baths are mainly alcohol (Table 4). In addition certain fiber post-treatments, such as drawing, are used as well. 

Table 4 Examples of wet spinning processes using regenerated fibroin, spidroin, and recombinant spidroin, with different solvents and coagulation baths (Zhou et al., 2006a)...

The wet spinning of regenerated spidroin was reported in the early 1990s by Jelinski et al. They dissolved spider silk in hexafluoroisopro-panol (HFIP) at a concentration of 2.5 wt% to produce an artificial fiber using water, methanol, isopropanol, and acetone as coagulation bath. The reconstituted silk could only be shaped in acetone but the structure... 

Figure 7 The morphologies of regenerated spidroin silks, (a) The silk formed in acetone and subsequently stretched (Seidel et al, 2000). (b) Silk drawn out of solution into air (Shao et al, 2003). (c) Silk produced from the recombinant ADF-3 (Lazaris et al, 2002).

It is well known that well-ordered (3-chitin (a polysaccharide) associated with a less ordered protein in the (3-sheet conformation is the main component of nacreous organic matrix in shell. The amino acid sequence of such proteins is very similar to those of silk fibroins. Indeed, the amino acid sequence of a major protein from the nacreous shell layer of the pearl oyster resembles that of spidroin (Sudo et al., 1997 Weiner and Traub, 1980). The question of whether silk-like proteins play an important role in shell formation is raised. When Falini et al. (1996) did the experiment with the proteins from the shell, they assembled a substrate in vitro that contained (3-chitin and natural silk fibroin and concluded that the silk fibroin may influence ion diffusion or the accessibility to the chi tin surface or both. Furthermore, cryo-TEM study of the structure of the Atrina shell nacreous organic matrix without dehydration... 

The role of the individual silk-like protein played is unclear, and whether the silk-like protein may dominate the crystallization of calcium carbonate or not is still unknown. To provide experimental insights into the interaction of minerals and proteins, a model system containing RSF or spidroin as templates may be used for the crystallization of calcium carbonate. 

---