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368 Biomimetics: Biologically Inspired Technologies

form of silk that is insoluble in water) are: 0.94 nm (interchain), 0.697 nm (fiber axis), 0.92 nm
(intersheet). These unit cell dimensions are consistent with a crystalline structure in which the
protein chains run antiparallel with interchain hydrogen bonds perpendicular to the chain axis
between carbonyl and amine groups, and van der Waal forces stabilizing the intersheet interactions
(based on the predominance of short side chain amino acids such as glycine, alanine, and serine in
b-sheet regions). Generally, silkworm fibroin in cocoons contains a higher content of crystallinity
(b-sheet content) than spider dragline silks such as from N. clavipes.
As more protein sequence data from various spiders and silkworms has been elucidated, it is
clear that these families of silk proteins are similar but also encompass a range of sequence
variations that reflect their functional properties. Silkworm fibroin is the protein that forms the
structural aspects of the fibers. These fibers are encased in a family of glue-like sericin proteins. The
primary sequence of amino acids of these proteins is found to be highly repeated, thus, small
regions of sequence chemistry in the protein chain are found elsewhere in other regions of the
chains. This design feature is critical to the function of this group of proteins as structural materials.
This design feature also allows the sequences of these large proteins to be represented in relatively
short sequences in terms of polymer design. This has led to the option of forming synthetic genetic
variants to represent the larger proteins, a useful laboratory technique to enhance the ability to
understand these proteins in a simplified form. These shorter genetic pieces can be polymerized
(multimerized) into longer genes to explore sequence and size relationships. These powerful tools
in molecular biology facilitate direct insight into the role of sequence chemistry, protein block sizes
and distributions, and protein polymer chain length on materials structure and function. Native and
synthetic silk clones have been generated in a variety of heterologous expression systems, including
bacteria, yeast, insect cells, plants, and mammalian cells (Wong and Kaplan, 2002).
Mechanism — Silk proteins are hydrophobic based on the predominance of glycine and alanine
amino acids. The chains self-assemble into insoluble b-sheets. Spiders and silkworm keep these
hydrophobic proteins soluble in water during their processing into fibers at concentrations up to 30
weight percent. The formation of liquid crystalline phases during silk processing has been reported
as part of this process (Vollrath and Knight, 2001). This process is accomplished in vivo without
premature crystallization into the insoluble b-sheets. Premature crystallization would be cata-
strophic for the animal as it would clog the spinning device. In recent studies, micelle and gel
states were identified that suggest these are important steps governing chain silk protein interactions
toward organized silk structures. These features can be duplicated in part using regenerated
silkworm silk and the control of water removal from aqueous solutions of these proteins via
osmotic stress (Jin and Kaplan, 2003). These ‘‘soft’’ micelles consist of flexible molecules and
the structures can grow and change shape in response to changes in protein concentration. This is
typical behavior of amphiphilic flexible surfactants that assemble into small spherical micelles and
evolve morphologically into cylinders and lamellar-layered structures. Lyotropic liquid crystalline
phase behavior can help explain the assembly of silk protein polymer chains into domains of high
concentration (Vollrath and Knight, 2001), with orientation driven by micellar behavior and water
efflux through channels in these structures.
Based on the micellar behavior and subsequent morphological features generated during silk
processing, critical design rules (chemistry of the amino acid sequence and blocks or regions of the
sequence chemistry) to match the processing environment have been described (Bini et al., 2004).
These design rules demonstrate a modified triblock design for these proteins that also contrasts in
important ways with traditional synthetic triblock co-polymers. In silks, the large (dominating in
size and chemical influence) internal hydrophobic blocks (crystallizable domains which promote
intra and interchain folding) are interrupted with very short hydrophilic blocks or spacers, puta-
tively to control water content in micellar states to prevent premature crystallization into b-sheets.
The large N- and C-terminal hydrophilic blocks interact with water and define micellar partitioning.
All protein sequences in silks adopt these general block ‘‘design rules’’ (Bini et al., 2004)
presumably to match the limits of the all aqueous processing environment. There is, however,