Alagacone Sriskantha

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.

Continuous Production of Flexible Fibers from Transgenically Produced Honeybee Silk Proteins

Flexible and solvent stable fibers are produced after concentrated recombinant honeybee protein solutions are extruded into a methanol bath, dried, drawn in aqueous methanol, then covalently cross-linked using dry heat. Proteins in solution are predominantly coiled coil. Significant levels of non-orientated ß-sheets form during drying or after coagulation in aqueous methanol. Drawing generally aligns the coiled coil component parallel with the fibre axis and ß-sheet component perpendicular to the fiber axis. The fibres are readily handled, stable in the strong protein denaturants, urea and guanidinium, and suitable for a range of applications such as weaving and knitting.

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.

Controlling the Molecular Structure and Physical Properties of Artificial Honeybee Silk by Heating or by Immersion in Solvents

Honeybee larvae produce silken cocoons that provide mechanical stability to the hive. The silk proteins are small and non-repetitive and therefore can be produced at large scale by fermentation in E. coli. The recombinant proteins can be fabricated into a range of forms; however the resultant material is soluble in water and requires a post production stabilizing treatment. In this study, we describe the structural and mechanical properties of sponges fabricated from artificial honeybee silk proteins that have been stabilized in aqueous methanol baths or by dry heating. Aqueous methanol treatment induces formation of ß-sheets, with the amount of ß-sheet dictated by methanol concentration. Formation of ß-sheets renders sponges insoluble in water and generates a reversibly compressible material. Dry heat treatments at 190°C produce a water insoluble material, that is stiffer than the methanol treated equivalent but without significant secondary structural changes. Honeybee silk proteins are particularly high in Lys, Ser, Thr, Glu and Asp. The properties of the heat treated material are attributed to generation of lysinoalanine, amide (isopeptide) and/or ester covalent cross-links. The unique ability to stabilize material by controlling secondary structure rearrangement and covalent cross-linking allows us to design recombinant silk materials with a wide range of properties.

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.

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.

Design of silk proteins with increased heme binding capacity and fabrication of silk-heme materials

In our previous studies, heme was bound into honeybee silk to generate materials that could function as nitric oxide sensors or as recoverable heterogeneous biocatalysts. In this study, we sought to increase the heme-binding capacity of the silk protein by firstly redesigning the heme binding site to contain histidine as the coordinating residue and secondly, by adding multiple histidine residues within the core of the coiled coil core region of the modified silk protein. We used detergent and a protein denaturant to confirm the importance of the helical structure of the silk for heme coordination. Aqueous methanol treatment, which was used to stabilize the materials, transformed the low-spin, six-coordinate heme to a five-coordinate high-spin complex, thus providing a vacant site for ligand binding. The optimal aqueous methanol treatment time that simultaneously maintains the helical protein structure and stabilizes the silk material without substantial leaching of heme from the system was determined.

Modification of Honeybee Silk by the Addition of Antimicrobial Agents

Honeybee silk proteins can be produced at high levels in recombinant systems, fabricated into materials, and are tolerant of amino acid modifications: properties that make them exciting templates for designing new functional materials. Here, we explore the properties of materials either made from silk-antimicrobial peptide (AMP) fusion proteins or silk containing entrapped AMPs or silver nanoparticles. Inclusion of AMP within the silk protein sequence did not affect our ability to express the proteins or process them into films. When AMP-silk proteins and Escherichia coli cells were coincubated in solution, a reduction in cell numbers was observed after degradation of the chimeric protein to release a truncated version of the AMP. In films, the AMP was retained in the silk with leaching rates of <1% per day. Films containing silver nanoparticles were antimicrobial, with the silk preventing aggregation of nanoparticles and slowing the rate of dissolution of the particles.

Did aculeate silk evolve as an antifouling material

Many of the challenges we currently face as an advanced society have been solved in unique ways by biological systems. One such challenge is developing strategies to avoid microbial infection. Social aculeates (wasps, bees and ants) mitigate the risk of infection to their colonies using a wide range of adaptations and mechanisms. These adaptations and mechanisms are reliant on intricate social structures and are energetically costly for the colony. It seems likely that these species must have had alternative and simpler mechanisms in place to ensure the maintenance of hygienic domicile conditions prior to the evolution of these complex behaviours. Features of the aculeate coiled-coil silk proteins are reminiscent of those of naturally occurring α-helical antimicrobial peptides (AMPs). In this study, we demonstrate that peptides derived from the aculeate silk proteins have antimicrobial activity. We reconstruct the predicted ancestral silk sequences of an aculeate ancestor that pre-dates the evolution of sociality and demonstrate that these ancestral sequences also contained peptides with antimicrobial properties. It is possible that the silks evolved as an antifouling material and facilitated the evolution of sociality. These materials serve as model materials for consideration in future biomaterial development.