Holly E. Trueman

An Unlikely Silk: The Composite Material of Green Lacewing Cocoons

Spiders routinely produce multiple types of silk; however, common wisdom has held that insect species produce one type of silk each. This work reports that the green lacewing ( Mallada signata, Neuroptera) produces two distinct classes of silk. We identified and sequenced the gene that encodes the major protein component of the larval lacewing cocoon silk and demonstrated that it is unrelated to the adult lacewing egg-stalk silk. The cocoon silk protein is 49 kDa in size and is alanine rich (>40%), and it contains an alpha-helical secondary structure. The final instar lacewing larvae spin protein fibers of approximately 2 microm diameter to construct a loosely woven cocoon. In a second stage of cocoon construction, the insects lay down an inner wall of lipids that uses the fibers as a scaffold. We propose that the silk protein fibers provide the mechanical strength of the composite lacewing cocoon whereas the lipid layer provides a barrier to water loss during pupation.

Fifty years later: The sequence, structure and function of lacewing cross-beta silk

Classic studies of protein structure in the 1950s and 1960s demonstrated that green lacewing egg stalk silk possesses a rare native cross-beta sheet conformation. We have identified and sequenced the silk genes expressed by adult females of a green lacewing species. The two encoded silk proteins are 109 and 67 kDa in size and rich in serine, glycine and alanine. Over 70% of each protein sequence consists of highly repetitive regions with 16-residue periodicity. The repetitive sequences can be fitted to an elegant cross-beta sheet structural model with protein chains folded into regular 8-residue long beta strands. This model is supported by wide-angle X-ray scattering data and tensile testing from both our work and the original papers. We suggest that the silk proteins assemble into stacked beta sheet crystallites bound together by a network of cystine cross-links. This hierarchical structure gives the lacewing silk high lateral stiffness nearly threefold that of silkworm silk, enabling the egg stalks to effectively suspend eggs and protect them from predators.

Cross-linking in the silks of bees, ants and hornets

Silk production is integral to the construction of nests or cocoons for many Aculeata, stinging Hymenopterans such as ants, bees and wasps. Here we report the sequences of new aculeate silk proteins and compare cross-linking among nine native silks from three bee species (Apis mellifera, Bombus terrestris and Megachile rotundata), three ant species (Myrmecia forficata, Oecophylla smaragdina and Harpegnathos saltator) and three hornets (Vespa analis, Vespa simillima and Vespa mandarinia). The well studied silks of spiders and silkworms are comprised of large proteins that are cross-linked and stabilized predominantly by intra and intermolecular beta sheet structure. In contrast, the aculeate silks are comprised of relatively small proteins that contain central coiled coil domains and comparatively reduced amounts of beta sheet structure. The hornet silks, which have the most beta sheet structure and the greatest amount of amino acid sequence outside the coiled-coil domains, dissolve in concentrated LiBr solution and appear to be stabilized predominantly by beta sheet structure like the classic silks. In contrast, the ant and bee silks, which have less beta sheet and less sequence outside the coiled-coil domains, could not be dissolved in LiBr and appear to be predominantly stabilized by covalent cross-linking. The iso-peptide cross-linker, ε-(γ-glutamyl)-lysine that is produced by transglutaminase enzymes, was demonstrated to be present in all silks by mass spectrometry, but at greater levels in silks of ants and bees. The bee silks and ant cocoons, but not the Oecophylla nest silks, appeared to be further stabilized by tanning reactions.

Micromolar biosensing of nitric oxide using myoglobin immobilized in a synthetic silk film.

In this work we investigate the use of coiled-coil silk proteins, produced in recombinant Escherichia coli, as a new material for immobilizing biosensors. Myoglobin was embedded in transparent honeybee silk protein films. Immobilized myoglobin maintained a high affinity for nitric oxide (KD NO=52 µM) and good sensitivity with a limit of detection of 5 µM. The immobilized myoglobin-silk protein film was stable and could be stored as a dry film at room temperature for at least 60 days. The effect of immobilization on the structure of myoglobin was fully investigated using UV/visible, Fourier Transform Infrared and Raman spectroscopy, which indicated a weakening in the strength of the iron-histidine bond. This study demonstrates that recombinant coiled-coil silk proteins provide a safe and environmentally friendly alternative to sol-gels for stabilizing heme proteins for use as optical biosensors.

A new class of animal collagen masquerading as an insect silk

Collagen is ubiquitous throughout the animal kingdom, where it comprises some 28 diverse molecules that form the extracellular matrix within organisms. In the 1960s, an extracorporeal animal collagen that forms the cocoon of a small group of hymenopteran insects was postulated. Here we categorically demonstrate that the larvae of a sawfly species produce silk from three small collagen proteins. The native proteins do not contain hydroxyproline, a post translational modification normally considered characteristic of animal collagens. The function of the proteins as silks explains their unusual collagen features. Recombinant proteins could be produced in standard bacterial expression systems and assembled into stable collagen molecules, opening the door to manufacture a new class of artificial collagen materials.

An Australian webspinner species makes the finest known insect silk fibers

Aposthonia gurneyi, an Australian webspinner species, is a primitive insect that constructs and lives in a silken tunnel which screens it from the attentions of predators. The insect spins silk threads from many tiny spines on its forelegs to weave a filmy sheet. We found that the webspinner silk fibers have a mean diameter of only 65 nm, an order of magnitude smaller than any previously reported insect silk. The purpose of such fine silk may be to reduce the metabolic cost of building the extensive tunnels. At the molecular level, the A. gurneyi silk has a predominantly beta-sheet protein structure. The most abundant clone in a cDNA library produced from the webspinner silk glands encoded a protein with extensive glycine-serine repeat regions. The GSGSGS repeat motif of the A. gurneyi silk protein is similar to the well-known GAGAGS repeat motif found in the heavy fibroin of silkworm silk, which also has beta-sheet structure. As the webspinner silk gene is unrelated to the silk gene of the phylogenetically distant silkworm, this is a striking example of convergent evolution.

Convergently-evolved structural anomalies in the coiled coil domains of insect silk proteins.

The use of coiled coil proteins as the basis of silk materials is an engineering solution that has evolved convergently in at least five insect lineages-the stinging hymenopterans (ants, bees, hornets), argid sawflies, fleas, lacewings, and praying mantises-and persisted throughout large radiations of these insect families. These coiled coil silk proteins share a characteristic distinct from other coiled coil proteins, in that they are fabricated into solid materials after accumulating as highly concentrated solutions within dedicated glands. Here, we relate the amino acid sequences of these proteins to the secondary and tertiary structural information available from biophysical methods such as X-ray scattering, nuclear magnetic resonance and Raman spectroscopy. We investigate conserved and convergently evolved features within these proteins and compare these to the features of classic coiled coil proteins including tropomyosin and leucine zippers. Our analysis finds that the coiled coil domains of insect silk proteins have several common structural anomalies including a high prevalence of alanine residues in core positions. These atypical features of the coiled coil fibrous proteins - which likely produce deviations from canonical coiled-coil structure - likely exist due to selection pressures related to the process of silk fabrication and the final function of the proteins.

Harnessing disorder: onychophorans use highly unstructured proteins, not silks, for prey capture

Onychophora are ancient, carnivorous soft-bodied invertebrates which capture their prey in slime that originates from dedicated glands located on either side of the head. While the biochemical composition of the slime is known, its unusual nature and the mechanism of ensnaring thread formation have remained elusive. We have examined gene expression in the slime gland from an Australian onychophoran, Euperipatoides rowelli, and matched expressed sequence tags to separated proteins from the slime. The analysis revealed three categories of protein present: unique high-molecular-weight proline-rich proteins, and smaller concentrations of lectins and small peptides, the latter two likely to act as protease inhibitors and antimicrobial agents. The predominant proline-rich proteins (200 kDa+) are composed of tandem repeated motifs and distinguished by an unusually high proline and charged residue content. Unlike the highly structured proteins such as silks used for prey capture by spiders and insects, these proteins lack ordered secondary structure over their entire length. We propose that on expulsion of slime from the gland onto prey, evaporative water loss triggers a glass transition change in the protein solution, resulting in adhesive and enmeshing thread formation, assisted by cross-linking of complementary charged and hydrophobic regions of the protein. Euperipatoides rowelli has developed an entirely new method of capturing prey by harnessing disordered proteins rather than structured, silk-like proteins.

The other prey-capture silk: Fibres made by glow-worms (Diptera: Keroplatidae) comprise cross-β-sheet crystallites in an abundant amorphous fraction.

Glow-worms (larvae of dipteran genus Arachnocampa) are restricted to moist habitats where they capture flying prey using snares composed of highly extensible silk fibres and sticky mucus droplets. Little is known about the composition or structure of glow-worm snares, or the extent of possible convergence between glow-worm and arachnid capture silks. We characterised Arachnocampa richardsae silk and mucus using X-ray scattering, Fourier transform infrared spectroscopy and amino acid analysis. Silk but not mucus contained crystallites of the cross-β-sheet type, which occur in unrelated insect silks but have not been reported previously in fibres used for prey capture. Mucus proteins were rich in Gly (28.5%) and existed in predominantly a random coil structure, typical of many adhesive proteins. In contrast, the silk fibres were unusually rich in charged and polar residues, particularly Lys (18.1%), which we propose is related to their use in a highly hydrated state. Comparison of X-ray scattering, infrared spectroscopy and amino acid analysis data suggests that silk fibres contain a high fraction of disordered protein. We suggest that in the native hydrated state, silk fibres are capable of extension via deformation of both disordered regions and cross-β-sheet crystallites, and that high extensibility is an adaptation promoting successful prey capture. This study illustrates the rich variety of protein motifs that are available for recruitment into biopolymers, and how convergently evolved materials can nevertheless be based on fundamentally different protein structures.

Stabilization of viruses by encapsulation in silk proteins.

Viruses are important for a range of modern day applications. However, their utility is limited by their susceptibility to temperature degradation. In this study, we report a simple system to compare the ability of different dried protein films to stabilize viruses against exposure to elevated temperatures. Films from each of three different silks, silkworm, honeybee silk and hornet silk, stabilized entrapped viruses at 37 °C better than films of albumin from bovine serum (BSA) and all four proteins provided substantially more stabilization than no protein controls. A comparison of the molecular structure of the silks and BSA films showed no correlation between the ability of the proteins to stabilize the virus and the secondary structure of the protein in the films. The mechanism of stabilization is discussed and a hypothesis is suggested to explain the superior performance of the silk proteins.