Paul A. Guerette

Silk Properties Determined by Gland-Specific Expression of a Spider Fibroin Gene Family

Spiders produce a variety of silks that range from Lycra-like elastic fibers to Kevlar-like superfibers. A gene family from the spider Araneus diadematus was found to encode silk-forming proteins (fibroins) with different proportions of amorphous glycine-rich domains and crystal domains built from poly(alanine) and poly(glycine-alanine) repeat motifs. Spiders produce silks of different composition by gland-specific expression of this gene family, which allows for a range of mechanical properties according to the crystal-forming potential of the constituent fibroins. These principles of fiber property control may be important in the development of genetically engineered structural proteins.

Accelerating the design of biomimetic materials by integrating RNA-seq with proteomics and materials science

Efforts to engineer new materials inspired by biological structures are hampered by the lack of genomic data from many model organisms studied in biomimetic research. Here we show that biomimetic engineering can be accelerated by integrating high-throughput RNA-seq with proteomics and advanced materials characterization. This approach can be applied to a broad range of systems, as we illustrate by investigating diverse high-performance biological materials involved in embryo protection, adhesion and predation. In one example, we rapidly engineer recombinant squid sucker ring teeth proteins into a range of structural and functional materials, including nanopatterned surfaces and photo-cross-linked films that exceed the mechanical properties of most natural and synthetic polymers. Integrating RNA-seq with proteomics and materials science facilitates the molecular characterization of natural materials and the effective translation of their molecular designs into a wide range of bio-inspired materials.

From Soft Self-Healing Gels to Stiff Films in Suckerin- Based Materials Through Modulation of Crosslink Density and β-Sheet Content

Suckerins are block-copolymer-like structural proteins constituting the building blocks of the strong squid sucker-ring teeth. Here, recombinant suckerin-19 is processed into biomaterials spanning a wide range of elasticity, from very soft hydrogels to stiff films with elastic modulus in the gigapascal range. The elasticity is controlled by the interplay between the β-sheet content and induced di-tyrosine crosslinking.

Infiltration of chitin by protein coacervates defines the squid beak mechanical gradient

The beak of the jumbo squid Dosidicus gigas is a fascinating example of how seamlessly nature builds with mechanically mismatched materials. A 200-fold stiffness gradient begins in the hydrated chitin of the soft beak base and gradually increases to maximum stiffness in the dehydrated distal rostrum. Here, we combined RNA-Seq and proteomics to show that the beak contains two protein families. One family consists of chitin-binding proteins (DgCBPs) that physically join chitin chains, whereas the other family comprises highly modular histidine-rich proteins (DgHBPs). We propose that DgHBPs play multiple key roles during beak bioprocessing, first by forming concentrated coacervate solutions that diffuse into the DgCBP-chitin scaffold, and second by inducing crosslinking via an abundant GHG sequence motif. These processes generate spatially controlled desolvation, resulting in the impressive biomechanical gradient. Our findings provide novel molecular-scale strategies for designing functional gradient materials.

Multi-scale thermal stability of a hard thermoplastic protein-based material

Although thermoplastic materials are mostly derived from petro-chemicals, it would be highly desirable, from a sustainability perspective, to produce them instead from renewable biopolymers. Unfortunately, biopolymers exhibiting thermoplastic behaviour and which preserve their mechanical properties post processing are essentially non-existent. The robust sucker ring teeth (SRT) from squid and cuttlefish are one notable exception of thermoplastic biopolymers. Here we describe thermoplastic processing of squid SRT via hot extrusion of fibres, demonstrating the potential suitability of these materials for large-scale thermal forming. Using high-resolution in situ X-ray diffraction and vibrational spectroscopy, we elucidate the molecular and nanoscale features responsible for this behaviour and show that SRT consist of semi-crystalline polymers, whereby heat-resistant, nanocrystalline β-sheets embedded within an amorphous matrix are organized into a hexagonally packed nanofibrillar lattice. This study provides key insights for the molecular design of biomimetic protein- and peptide-based thermoplastic structural biopolymers with potential biomedical and 3D printing applications.

Self-Assembly of Recombinant Hagfish Thread Keratins Amenable to a Strain-Induced α-Helix to β-Sheet Transition

Hagfish slime threads are assembled from protein-based bundles of intermediate filaments (IFs) that undergo a strain-induced α-helical coiled-coil to β-sheet transition. Draw processing of native fibers enables the creation of mechanically tuned materials, and under optimized conditions this process results in mechanical properties similar to spider dragline silk. In this study, we develop the foundation for the engineering of biomimetic recombinant hagfish thread keratin (TK)-based materials. The two protein constituents from the hagfish Eptatretus stoutii thread, named EsTKα and EsTKγ, were expressed in Escherichia coli and purified. Individual (rec)EsTKs and mixtures thereof were subjected to stepwise dialysis to evaluate their protein solubility, folding, and self-assembly propensities. Conditions were identified that resulted in the self-assembly of coiled-coil rich IF-like filaments, as determined by circular dichroism (CD) and transmission electron microscopy (TEM). Rheology experiments indicated that the concentrated filaments assembled into gel-like networks exhibiting a rheological response reminiscent to that of IFs. Notably, the self-assembled filaments underwent an α-helical coiled-coil to β-sheet transition when subjected to oscillatory shear, thus mimicking the critical characteristic responsible for mechanical strengthening of native hagfish threads. We propose that our data establish the foundation to create robust and tunable recombinant TK-based materials whose mechanical properties are controlled by a strain-induced α-helical coiled-coil to β-sheet transition.

Modular peptides from the thermoplastic squid sucker ring teeth form amyloid-like cross-β supramolecular networks

The hard sucker ring teeth (SRT) from decapodiforme cephalopods, which are located inside the sucker cups lining the arms and tentacles of these species, have recently emerged as a unique model structure for biomimetic structural biopolymers. SRT are entirely composed of modular, block co-polymer-like proteins that self-assemble into a large supramolecular network. In order to unveil the molecular principles behind SRTs self-assembly and robustness, we describe a combinatorial screening assay that maps the molecular-scale interactions between the most abundant modular peptide blocks of suckerin proteins. By selecting prominent interaction hotspots from this assay, we identified four peptides that exhibited the strongest homo-peptidic interactions, and conducted further in-depth biophysical characterizations complemented by molecular dynamic (MD) simulations to investigate the nature of these interactions. Circular Dichroism (CD) revealed conformations that transitioned from semi-extended poly-proline II (PII) towards β-sheet structure. The peptides spontaneously self-assembled into microfibers enriched with cross β-structures, as evidenced by Fourier-Transform Infrared Spectroscopy (FTIR) and Congo red staining. Nuclear Magnetic Resonance (NMR) experiments identified the residues involved in the hydrogen-bonded network and demonstrated that these self-assembled β-sheet-based fibers exhibit high protection factors that bear resemblance to amyloids. The high stability of the β-sheet network and an amyloid-like model of fibril assembly were supported by MD simulations. The work sheds light on how Nature has evolved modular sequence design for the self-assembly of mechanically robust functional materials, and expands our biomolecular toolkit to prepare load-bearing biomaterials from protein-based block co-polymers and self-assembled peptides. STATEMENT OF SIGNIFICANCE The sucker ring teeth (SRT) located on the arms and tentacles of cephalopods represent as a very promising protein-based biopolymer with the potential to rival silk in biomedical and engineering applications. SRT are made of modular, block co-polymer like proteins (suckerins), which assemble into a semicrystalline polymer reinforced by nano-confined β-sheets, resulting in a supramolecular network with mechanical properties that match those of the strongest engineering polymers. In this study, we aimed to understand the molecular mechanisms behind SRTs self-assembly and robustness. The most abundant modular peptidic blocks of suckerin proteins were studied by various spectroscopic methods, which demonstrate that SRT peptides form amyloid-like cross-β structures.

Squid Suckerin Biomimetic Peptides Form Amyloid-like Crystals with Robust Mechanical Properties

We present the self-assembly of fibers formed from a peptide sequence (A1H1) derived from suckerin proteins of squid sucker ring teeth (SRT). SRT are protein-only biopolymers with an unconventional set of physicochemical and mechanical properties including high elastic modulus coupled with thermoplastic behavior. We have identified a conserved peptide building block from suckerins that possess the ability to assemble into materials with similar mechanical properties as the native SRT. A1H1 displays amphiphilic characteristics and self-assembles from the bottom-up into mm-scale fibers initiated by the addition of a polar aprotic solvent. A1H1 fibers are thermally resistant up to 239 °C, coupled with an elastic modulus of ∼7.7 GPa, which can be explained by the tight packing of β-sheet-enriched crystalline building blocks as identified by wide-angle X-ray scattering (WAXS), with intersheet and interstrand distances of 5.37 and 4.38 Å, respectively. A compact packing of the peptides at their Ala-rich terminals within the fibers was confirmed from molecular dynamics simulations, and we propose a hierarchical model of fiber assembly of the mature peptide fiber.

Functional design of spider's silk

The orb-web weaving spiders produce a broad range of high performance structural fibers (i.e. silks) with mechanical properties that are superbly matched to their function. Our interest in these materials stems both from an interest in the biology of the spiders and the design of their webs and also from a desire to discover principles of mechanical design of protein-based structural materials that can guide the development of novel bio-engineered materials. All spiders produce silks, but the orb-web weaving spiders are unique in their ability to produce seven different silks, each from distinct gland/spinneret complexes. Considering the wide diversity of spider species, there is likely to be an enormous range of material properties available in spider silk. However, at present, we only have information on two species of spiders, and only two of their seven silks have been studied in any detail. These are: (1) the silk produced by the major ampullate gland, which forms the safety-line or dragline of the spider and also is used to form the frame of its orb-web, and (2) the viscid silk produced by the flagelliform gland, which forms the glue-covered catching spiral of the web. In this paper we describe several aspects of the mechanical design of the dragline and viscid silks produced by the spider Araneus diadematus.