Silk

Agenda

* Presentation times are subject to change

Day 1: Wednesday, July 27

Session I: Silk Production and Processing

Underwater Adhesive Silks of Caddisfly Larvae: Chemical and Structural Studies
Russell Stewart, PhD, University of Utah

Aquatic caddisfly (Trichoptera) larvae stitch together composite shelters with adhesive silk and materials adventitiously gathered from their environments. Caddisflies are closely related to terrestrial silk-spinning moths and butterflies (Lepidoptera). Understanding the molecular adaptation of caddisfly silk to aquatic environments may provide important insights for the molecular design of synthetic underwater adhesives. Elemental analysis, specific antibody probes, and tandem mass spectrometry revealed a repeating pattern of phosphorylated serines (pSX)4 blocks alternating with arginine-rich blocks in the H-fibroin protein.^[1] Our working hypothesis is that mutual charge neutralization by alternating phosphate and arginine blocks may lead to partial dehydration and liquid-liquid phase separation of silk in the silk gland. Reorganization of the electrostatic associations due to stress induced elongation during fiber spinning may lead to additional charge neutralization, dehydration, and insoluble nano-fibril formation. This model is highly analogous to models for silkworm and spider silk formation through staggered associations of alternating hydrophobic and hydrophilic blocks. The peptidyl phosphate of caddisfly silk may provide a built-in probe to test the stress-induced structural reorganization hypothesis. The presence of phosphoserine has been confirmed in species from all three sub-orders of caddisflies by ATR-FTIR and/or immunohistochemistry. The structure of the silk press, the site of a major structural reorganization of the silk protein, is being characterized.

1. Stewart RJ, Wang CS. 2010. Adaptation of caddisfly larval silks to aquatic habitats by phosphorylation of h-fibroin serines. Biomacromolecules 11(4): p. 969-74.

Initiation of Spider Silk Assembly
Thomas Scheibel, PhD, University of Bayreuth

It is well known that silk proteins form fibrillar structures at high protein concentrations. Hence, it is surprising that spider silk proteins are stored in a soluble form at high concentrations and can be transformed into extremely stable fibres on demand. Silk proteins are reminiscent of amphiphilic block copolymers containing stretches of polyalanine and glycine-rich polar elements forming a repetitive core domain flanked by highly conserved non-repetitive (NR) amino- and carboxy-terminal domains. The N-terminal domain comprises a secretion signal and is monomeric in solution, while the dimeric C-terminal domain is important for the control of solubility and fibre formation initiated by changes in ionic composition and mechanical stimuli. Recently, we solved the solution structure of both the N-terminal and the C-terminal domain of a spider dragline silk protein and provided evidence that the structural state of these domains is essential for switching between the storage and the assembly form of silk proteins in a controlled manner upon exposure to chemical and mechanical stimuli. In addition, the terminal domains also play a role in the alignment of secondary structural features formed by the repetitive sequence elements in the backbone of spider silk proteins, which is known to be important for the mechanical properties of the fibre.

From Flow to Fibres
Chris Holland, PhD, The University of Oxford

The processing history of a silk feedstock contributes significantly, if not primarily, towards a fibres' mechanical properties. Rheology has provided us with a tool to compare and contrast the mechanical properties of such feedstocks, be they natural, reconstituted/regenerated or recombinant. The differences between them are clear, yet the molecular basis for these disparities are yet to be fully uncovered.  Novel rheo-spectro/microscopic techniques to study silk feedstocks sheds light onto this core problem, as I will discuss 

Silks as Fusion Proteins and Carriers for New Functional Materials
David Kaplan, PhD, Tufts University

Spider and silkworm silks provide the basis for self-assemblying and robust material properties. As such these novel proteins from nature can provide an important starting point for new material systems that offer functional utility in many areas of high performance needs, from strong and tough systems, to optics and electronics platforms, to implants for regenerative medicine. Two emerging opportunities that build upon these features will be discussed. One is to exploit genetic control of silk sequence and chemistry to add new features to the protein. The challenge is to add these new capabilities without disrupting native material properties. Examples of recent success towards this challenge include metal binding domains, antimicrobial peptides, cell membrane targeting domains and the regulation of material morphology from self-assembled variants. The second approach is to utilize the native silk protein to entrain labile substrates, leading to remarkable stabilization of these compounds. This approach exploits the unique block copolymer nature and low water content of silk, resulting in the ability to sequester, stabilize and store a range of bioactive compounds with remarkable robustness. In both of the experimental approaches, the unique structure and chemistry of silks is exploited for the basic materials foundation, while new functions are added.

Spider Silk Proteins: Production and Fibers
Randy Lewis, PhD, Utah State University

Spider silks have attracted substantial interest for millennia. Only recently has an understanding of the molecular basis for their impressive properties been developing. A comparison of the various different spider silk sequences will be made and used as a basis for creating "synthetic" spider silks. Methods to produce the spider silk proteins and spin them into fibers will be presented.

Evolutionary Biomechanics of Spider Silk
Todd Blackledge, PhD, University of Akron

Substantial progress has occurred over the last several years in the effort to synthesize high quality mimics of spider silk. However, most of this research focuses on a limited number of "model" species of orb-weaving spiders. The almost 400 million year history of spiders offers substantial insight into the evolution of the key features of silk genes and spinning morphology that explain the exceptional properties of spiders silks, as well suggesting new applications for silk. For instance, hypotheses about the functional implications of key features such as the MaSp2 protein can be tested by comparison to taxa lacking these features. Furthermore, relationships between web ecology and silk biomechanics can be used to predict which of the world's 41,000 species of spiders produce silk with unusual or desirable properties. Finally, exploring the mechanical function of whole webs reveals how silks serve not only models for materials-based biomimicry but also for engineering design.

Properties of Recombinant Spider Silk Films
Andrew Smith, PhD, University of Bayreuth

Spider silk is recognized as a high performance fibre with high tensile strength, and toughness. A part of these properties is recognized to come from the arrangement of nanocrystalline regions in the fibre. However, are these properties retained in the production of recombinant spider silk films which has a planar rather than linear structure? Recombinant spider silk proteins can solubilised in various organic solvents as well as aqueous buffers and then cast to form films. Films from different solvents present slightly different molecular structures to one another, additionally post-treatment of these films alters the underlying molecular structure towards more β-sheets. The differences in molecular structure have an impact on the physical properties of the films which can be seen in the strength and elasticity and hence the toughness of the films. Polarized FTIR spectroscopy highlights that there is no orientation of the β-sheet regions in recombinant spider silk films, and that only films from certain solvents are capable of re-orientating the β-sheet regions under stress.

Spider Silks: Insights from Genomics and Transcriptomics
Cheryl Hayashi, PhD, University of California, Riverside

Spider silks are made almost entirely of proteins, with the overwhelming majority of proteins encoded by the spidroin multi-gene family (spidroin is a contraction of "spider fibroin"). Spidroins are very large molecules (e.g., 250kDa). A spidroin monomer is thousands of amino acids in length, over 90% of which is constructed from tandemly-arrayed repeats. Paralogs of the spidroin gene family can dramatically differ from each other in the length and amino acid sequence of their constituent repeat units. Furthermore, attributes of the repeat units are functionally linked to the extraordinary mechanical properties of spider silks. Fine-tuned by natural selection, spider silks are remarkable, high performance materials that compare favorably with, and in some cases exceed, the best manmade fibers in terms of strength and toughness. In this talk, high-throughput sequencing of cDNA and traditional molecular genetics are incorporated into a phylogenetic perspective to gain insights into the evolutionary and functional genomics underlying spider silk.

Sericin as Industrial Glue and as Additive to Tissue Culture Media
Frantisek Sehnal, PhD, University of South Bohemia

Bombyx mori contains 3 sericin-encoding genes, of which Ser2 is very complex and variable. A Ser2 allele identified in the Daizo stock spans 12,5 kb and includes 13 exons, two of which are products of duplication. Two mRNAs (5.7 and 3.2 kb) are produced due to alternative splicing of the largest exon (designated 9a). The predicted proteins consist of 1740 and 882 amino acids, respectively, of which more than 17% is lysine and 15 % is serine. Both proteins (220 kDa and 130 kDa, respectively) are produced in the middle silk gland section and coat the silk fiber at the start of spinning when firm cocoon attachment to a substrate is constructed. Protein gel stored in the glands at this time has strongly adhesive properties. It is suggested that this adhesion is due to the presence of 50 copies of the highly conserved repetitive motif DSEKAKPNDRSPSHK (residues not conserved in all repeats are underlined). This repetitive sequence shows remarkable similarity (35% identity over 600 amino acids) with the mussel adhesive plaque protein (Mytilus edulis) and with repetitive sequence of the adhesive protein trans-sialidase from Trypanosoma cruzi. We propose that the strength and adhesiveness of sericin 2 and blue mussel adhesive protein are mediated by the distribution of specific amino acids. Unlike the blue mussel protein, which contains a large number of tyrosine residues, the sericin 2 proteins contain few tyrosines but are rich in the polar residues. The adhesive properties of sericin 2 can be exploited in the cell cultures. Most cell types require attachment to a surface and the nature of this substrate has a major effect on cell growth. Tissue culture plastics are treated to become hydrophilic and negatively charged. The performance of many cell types can be dramatically improved by coating dishes with positively charged polymers, (i.e., poly-D-lysine), or tethered adhesion molecules (AMs), including laminin and collagen. We found that crude sericin extracts can spread even over hydrophobic surfaces that cause other water solutes (including poly-D-lysine) to bead up into droplets. We have tested coating of plastic dishes with several natural and recombinant Bombyx sericins for their ability to support growth of Drosophila Cl8+ cells, which grow strictly in an anchorage-dependent manner. Our results show that the cells do not grow (and mostly die within 48 hrs) in untreated polystyrene plastic dishes, but survive after coating the dish surface with sericin proteins (either natural or recombinant). Several truncated recombinant sericin variants (deduced size A=33,5; B=30,2; GL1=67,6; and GL2=30,2 kDa, respectively) containing repeats of the short positively charged motif DTEKAKPNDRSPSHK were expressed in Escherichia coli and showed to affect cell adhesion.

Targeted Mutagenesis in Bombyx Mori
Michal Zurovec, PhD, University of South Bohemia

Recent progress in molecular biology has facilitated the use of virtually any organism to address key problems of applied and general biology. It opens a wide range of possibilities for genome manipulations of Bombyx mori. One of the crucial tools is the disruption of specific genes by targeted mutagenesis, allowing a complete removal or a modification of the gene function. The gene targeting methods used so far required elaborate procedures involving construction of transgenic animals and genetic crossings lasting three or more generations or laborious modifications of embryonic stem cells. The newly emerging gene knockout technology is based on zinc finger nucleases (ZFN), synthetic fusion proteins combining a customized zinc-finger DNA-binding domain with a non specific nuclease subunit. ZFNs specific for short sequence motifs within exons can now be designed and injected to embryos in the form of RNA. Such chimerical enzymes are able to cut the DNA at the selected 18 bp target sites. The ZFNs were used earlier for targeted gene disruption in several model organisms including D. melanogaster, zebrafish, mouse and C. elegans. We have established a ZFN-based gene targeting method in B. mori (Takasu et al. 2010). We used the advantage of sex-linked gene BmBLOS2. The mutation of this gene alters the epidermal color of the silkworm providing them with an easily scored phenotype. We prepared three different ZFNs attacking three different sites of BmBLOS2. We were able to receive mutants with one of these enzymes. Our results show that the ZFN mRNA microinjection into B. mori embryos is effective to induce somatic, as well as germline, mutations in a targeted gene by non-homologous end joining (NHEJ). The efficiency of the mutagenesis was relatively low and the frequency of germline mutants was only 0.28 percent. It became obvious that routine usage of this method with diverse genes would require preliminary activity testing of several ZFNs in each case. Such tests would allow preselecting the best active enzymes from the pool of designed candidates. In collaboration with other groups, we have recently adopted a simple yeast-based tests of ZFN activity, which allow identification of efficient nucleases before performing the laborious embryo microinjections and screenings. Our study is the first example of ZFN gene targeting in an insect other than Drosophila melanogaster. Possible applications of gene targeting in silkworm biotechnology will be discussed.

Day 2: Thursday, July 28

Session II: Structure–Function Relationships in Silk

Variability in Silks
Fritz Vollrath, PhD, Oxford University

It has been amply demonstrated that the mechanical properties of a specific spider's silk can show considerable variability that can be attributed to spinning conditions.  There is some evidence that chemical composition might also show some variability.   It is not surprising that silkworm silks are no different as I shall explore.  Such variability in Bombyx silks might have implication beyond the fibre and carry forward into reconstituted silk feedstocks.

Variability and Control of Mechanical Properties of Silk Fibers
Gustavo Guinea, PhD, Universidad Politécnica de Madrid, Spain

The exceptional mechanical properties of silk fibers and the possibility of producing artificial fibers endowed with similar outstanding properties can be alleged as the main justification for the intensive research on these materials. The detailed mechanical characterization of silk fibers presents a number of peculiarities compared with other materials, mostly related to their small cross-sectional area and the necessity of measuring very low forces, which require the development of specific experimental procedures. In addition to the instrumental difficulty, the analysis of silk fibers was traditionally hampered by the extreme intrinsic variability of the material.

The development of procedures to control variability, so that fibers with reproducible tensile properties can be obtained, is one of the major aims of our research. This effort resulted in the implementation of several techniques, which are essentially based on a basic property of spider silk: supercontraction. Supercontraction was initially defined by a significant reduction of the length of the fiber when immersed in water or in environments with high relative humidity. It was later found that supercontraction is the most evident manifestation of a fundamental trait of spider silk: the existence of a ground state. Any fiber can revert to this ground state independently from its previous loading history through a simple supercontraction process. In turn, the existence of a ground state allows tuning the properties of the natural material. As shown in this work, we have been able to transfer this desirable property of the natural material to bioinspired fibers, opening the possibility to adapt the tensile behaviour of these fibers to their intended uses.

Similarities and Differences in the Microstructure of Natural and Bioinspires Silk Fibers
José Pérez-Rigueiro, PhD, Universidad Politécnica de Madrid, Spain

The detailed characterization of the microstructure of natural silks represents one of the major challenges in the field, and a basic requirement for the production of high performance bioinspired fibers. The absence of a technique which cover the whole range of microstructural scales, from the organization of the molecules to the macroscopic performance of silk, forces the combined use of several characterization procedures that render partial information on the material. The combination of these data, albeit incomplete, starts to offer an approximate picture of the microstructural organization of silk fibers.

Following this rationale, we present here our latest findings on the microstructural characterization of natural and bioinspired silks as obtained from atomic force microscopy, Raman spectroscopy and X-ray diffraction. Our previous work to obtain fibers with reproducible mechanical properties allowed the microstructural characterization of fibers, discarding the variability usually associated with these materials, and to complement Raman spectroscopy and X-ray diffraction data recorded by other authors. The development of a robust procedure for the observation of silk fibers with atomic force microscopy has allowed, for the first time, the analysis of the fibers through this technique with a nanometer resolution. The correlation of the processing conditions, microstructural data and mechanical properties allows defining some conditions required for the production of high performance bioinspired fibers, whose properties resemble those of the natural materials.

Emergence of Complex Structures in Silks
Cedric Dicko, PhD, Lund University

At the heart of silk function and diversity are complex structures. The assembly and interactions of these complex structures can impact not only silks' mechanical properties but also their chemical functionality. The definition and description, however, of these complex structures is not trivial. In the present contribution, starting from silks' diversity and mechanical shortcomings, I will describe how assembly of complex structures and its control can be use as a guide to understand the central role played by anisotropy and anisotropy enabling environment in biological materials in general.

The particular case of concentration will be illustrated using acoustically levitated silk droplets monitored by small angle x-ray scattering (SAXS). Detailed analysis shows that the two current views, involving liquid crystallinity or micellar assembly rather than being conflicting can be reconciled by the present study. It is now conceivable that oriented liquid crystal like domains exist within larger micellar assemblies. The results will be discussed in light of conformation, interactions during assembly and ultimately functionality of the final fibre.

Modelling Structure-Property Relations in Silks
David Porter, PhD, Oxford University  

A model framework for structure property relations in silk and other proteins has been developed, based upon the management of stored and dissipated energy at the molecular level for stiffness and toughness, respectively.  We show that the full property profile of any silk can be predicted, based upon the simple structural features of order-disorder fractions and hydration of the disordered fraction.  We suggest that the unique combination of stiffness and toughness in native silks is due to the specific inter-peptide group hydrogen bonding, which originates at the instability transition during spinning.

Structural Characterization of Spider Silk and the Silk Producing Process with Magnetic Resonance Methods
Gregory Holland, PhD, Arizona State University

Our research group has been focused on characterizing the structure and dynamics of the spider silk proteins, MaSp1 and MaSp2. Multi-dimensional, multi-nuclear solid-state NMR is implemented to characterize the secondary structure of the proteins in the silk fiber while, high-resolution magic angle spinning (HR-MAS) is being applied to study intact excised spider glands to elucidate the structure of the proteins prior to fiber formation. Additionally, we have been developing magnetic resonance imaging (MRI) with localized spectroscopy as a tool to interrogate the silk producing process in situ on live spiders. This presentation will be focused on applying these techniques to understand the structure-function relationship in various spider silks and elucidate the molecular level mechanisms and chemistries for converting the protein-rich fluid in the gland to a fiber with unparalleled mechanical properties.

Session III: Applications and Scale Up

Biospinning of Silk Fibers with Integrated Drugs
Michael Rheinnecker, PhD, Spintech

Biospinning enables the industrial production of endless filaments out of native silk protein solutions. Biospun fibers are compatible with standard textile machines and were manufactured into a series of prototypes of knitted and woven cardiovascular devices. Biospinning is also been used to manufacture out of silk/drug solutions endless silk fibers with pharmaceutical functionality. The features of biospun fibers will be discussed and compared to standard silk fiber.

Spidersilk Production in E. Coli for Industrial Applications
Alex Leimer, PhD, AmSilk

Natural spider silk has been recognized as a high performance material for some time and several methods have been applied to produce spider silk or derivatives thereof in transgenic animals, plants, yeast or bacteria. Despite the demonstrated properties of the material and proven utility, there are no spider silk products on the market. This has mainly been due to the lack of a cost efficient production method supplying amounts required for typical applications development. Our experience has shown that most applications require from 100g to several kg of material to be effective.

AMSilk is developing industrially scalable production processes for its Spidersilk based on E. Coli expression systems. The process delivers material of reproducible quality in a final format that can be used for transformation into any inter-stage form such as beads, coatings, films or other forms. The process is deisgned to meet regulatory criteria in the future with improved batch to batch consistency. Production capacity of the proteins is currently at the kg scale and expanding into the 100+ kg scale per campaign by the end of 2011. These materials will be used for application development at AMSilk and select partners.

AMSilk's Spidersilk has been shown to be non-toxic and non-immunogenic. The good biocompatibility provides the basis for the development of high value products in the medical device and drug delivery sectors. First applications range from implant coatings to silk beads as drug delivery particles. Data will be shown as to the utility of Spidersilk in specific applications. Future applications, depending on the development of a spinning process, will include textiles or fiber-based products.

Day 3: Friday, July 29

Session III: Applications and Scale Up (continued)

A New Silk Road for High Technology
Fiorenzo Omenetto, PhD, Tufts University

The use of silk as a material for technological applications has been introduced over the past few years. Silk is now finding new applications as a useful biocompatible material platform with utility in photonics and electronics, ranging from nanoscale optical lattices to metamaterials. We will overview how purified silkworm silk can be reassembled, among other things, in a multitude of high quality, micro- and nanostructured optical and optoelectronic elements largely or entirely composed of this organic, biocompatible and implantable protein matrix truly opening a new silk road that brings together the biological and high-tech worlds.

Engineered Silk Interfaces
Vladmir Tsukruk, PhD, Georgia Institute of Technology

We will briefly overview recent results from our research group on understanding engineered organic-inorganic interfaces of silk nanomaterials.  Particularly, the formation of mixed and segregated silk I and silk II secondary structures within first 5-100 nm of silk film was observed under different drying and vapor treatment conditions, metal ion adsorption and nanoparticles reduction, and molecular surface areas available for adsorbed molecules.  Molecular conformation, vertical and lateral segregations, surface morphologies, as well as optical and mechanical properties in relation to the interfacial interactions are discussed.

* More abstracts to follow.

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