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.

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 ß-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.

Self-Assembly of Recombinant Hagfish Thread Keratins Amenable to a Strain-Induced α-Helix to ß-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 ß-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 ß-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 ß-sheet transition.

Biomimetic production of silk-like recombinant squid sucker ring teeth proteins.

The sucker ring teeth (SRT) of Humboldt squid exhibit mechanical properties that rival those of robust engineered synthetic polymers. Remarkably, these properties are achieved without a mineral phase or covalent cross-links. Instead, SRT are exclusively made of silk-like proteins called "suckerins", which assemble into nanoconfined ß-sheet reinforced supramolecular networks. In this study, three streamlined strategies for full-length recombinant suckerin protein production and purification were developed. Recombinant suckerin exhibited high solubility and colloidal stability in aqueous-based solvents. In addition, the colloidal suspensions exhibited a concentration-dependent conformational switch, from random coil to ß-sheet enriched structures. Our results demonstrate that recombinant suckerin can be produced in a facile manner in E. coli and processed from mild aqueous solutions into materials enriched in ß-sheets. We suggest that recombinant suckerin-based materials offer potential for a range of biomedical and engineering applications.

Phase transition-induced elasticity of α-helical bioelastomeric fibres and networks.

Natural elastomeric fibres play central structural and functional roles in a variety of tissues produced by many organisms from diverse Phyla. Most of these fibres feature amorphous structure and their long-range elastic response is well described within the framework of entropic (rubber-like) elasticity. Recently, it has been recognized that long-range reversible deformation can also occur in biomacromolecular fibres or networks that feature significant secondary structure and long-range order. Their elastomeric response is then associated with conformational changes of the backbone of the constitutive protein-based polymers. Under axially imposed loads, several groups of proteins whose structure is dominated by α-helical coiled-coil structures can undergo unfolding transitions and secondary structure transformations, for example from coiled-coil α-helices to ß-sheet strands. In contrast to rubber-like biopolymers, the retractive elastic force in these biomacromolecular materials is not dominated by a return to a maximum entropic state, but is mostly the result of variations in internal energy associated with the conformational changes. Here, a review of α-helix based elastomeric materials is presented that encompasses examples and experimental evidence across multiple length scales, from the molecular to the macroscopic scale. We begin by summarizing the basic thermodynamic formalism of thermoelasticity. While this formalism is well established for amorphous (entropically-dominated) fibres under tensile loading, its extension towards conformational (internal energy-dominated) elasticity is less known. Recent experimental evidence as well as corroborating computer simulations are then reviewed and discussed in the light of secondary structure and nano-scale features of these biopolymers. Comparisons are also drawn with physiologically important structural fibres that share common characteristics at the molecular and the nano-scale, including intermediate filament (IF) proteins from the cell cytoskeleton, myosins from motor proteins, and fibrin from blood clot. We conclude with a discussion on future directions and opportunities for these materials from a biomimetics engineering perspective.

Crowdsourced analysis of fungal growth and branching on microfluidic platforms.

Fungal hyphal growth and branching are essential traits that allow fungi to spread and proliferate in many environments. This sustained growth is essential for a myriad of applications in health, agriculture, and industry. However, comparisons between different fungi are difficult in the absence of standardized metrics. Here, we used a microfluidic device featuring four different maze patterns to compare the growth velocity and branching frequency of fourteen filamentous fungi. These measurements result from the collective work of several labs in the form of a competition named the "Fungus Olympics." The competing fungi included five ascomycete species (ten strains total), two basidiomycete species, and two zygomycete species. We found that growth velocity within a straight channel varied from 1 to 4 µm/min. We also found that the time to complete mazes when fungal hyphae branched or turned at various angles did not correlate with linear growth velocity. We discovered that fungi in our study used one of two distinct strategies to traverse mazes: high-frequency branching in which all possible paths were explored, and low-frequency branching in which only one or two paths were explored. While the high-frequency branching helped fungi escape mazes with sharp turns faster, the low-frequency turning had a significant advantage in mazes with shallower turns. Future work will more systematically examine these trends.

Artificial hagfish protein fibers with ultra-high and tunable stiffness.

Stiff fibers are used as reinforcing phases in a wide range of high-performance composite materials. Silk is one of the most widely studied bio-fibers, but alternative materials with specific advantages are also being explored. Among these, native hagfish (Eptatretus stoutii) slime thread is an attractive protein-based polymer. These threads consist of coiled-coil intermediate filaments (IFs) as nano-scale building blocks, which can be transformed into extended ß-sheet-containing chains upon draw-processing, resulting in fibers with impressive mechanical performance. Here, we report artificial hagfish threads produced by recombinant protein expression, which were subsequently self-assembled into coiled-coil nanofilaments, concentrated, and processed into ß-sheet-rich fibers by a "picking-up" method. These artificial fibers experienced mechanical performance enhancement during draw-processing. We exploited the lysine content to covalently cross-link the draw-processed fibers and obtained moduli values (E) in tension as high as ∼20 GPa, which is stiffer than most reported artificial proteinaceous materials.

Modular peptides from the thermoplastic squid sucker ring teeth form amyloid-like cross-ß 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 SRT's 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 ß-sheet structure. The peptides spontaneously self-assembled into microfibers enriched with cross ß-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 ß-sheet-based fibers exhibit high protection factors that bear resemblance to amyloids. The high stability of the ß-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 ß-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 SRT's 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-ß structures.

From Soft Self-Healing Gels to Stiff Films in Suckerin-Based Materials Through Modulation of Crosslink Density and ß-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 ß-sheet content and induced di-tyrosine crosslinking.

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 ß-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.

Biomimetic self-assembly of recombinant marine snail egg capsule proteins into structural coiled-coil units.

The egg capsules of the marine snails from the Melongenidea family feature unique biomechanical properties, including large reversible elasticity combined with a relatively high stiffness and outstanding strain energy absorption, making it an attractive biomimetic model system for restorative and tissue engineering applications. The capsules' building blocks are proteins called egg capsule proteins (ECPs), which we recently sequenced. ECPs are predicted to contain relatively large coiled-coil domains, which are directly responsible for the high elasticity arising from the extension of α-helical coiled-coil domains into extended ß-sheet domains. In this work, de novo synthesized ECPs genes were cloned and expressed in a bacterial expression system. Following purification under denaturing conditions by strong ion-exchange chromatography, individual and paired mixtures of ECPs were self-assembled using a controlled dialysis protocol, resulting in the folding of ECPs into soluble coiled-coil units. Circular Dichroism (CD) spectroscopy of the fibrils suggested that ECPs self-assembled into heteromeric coiled-coil units. The enhancement of the α-helical coiled-coil content during pair assembly was confirmed by Fourier Transform Infrared (FTIR) spectroscopy. Transmission Electron Microscopy (TEM) imaging of covalently-fixed self-assembled units corroborated the formation of elongated intermediate filaments-like structures. Polymer statistical analysis of Atomic Force Microscopy (AFM) images of unfixed self-assembled fibrils suggested that the observed coiled-coils were made of dimeric subunits. This study establishes the key protein engineering and physicochemical parameters needed to assemble ECPs into building blocks that can be processed into biomaterials that mimic the unique biomechanical properties of marine snail egg capsules.

Testing hypotheses of adaptive variation in cricket ovipositor lengths.

We experimentally tested a series of hypotheses proposed by Masaki (1979, 1986) for the evolution of ovipositor length in crickets. Female crickets use the ovipositor to bury eggs in the soil, where it was hypothesized to protect their eggs from desiccation, cold and other disturbance. However, we found no effect of depth on the overwinter survival of eggs of three species of Nemobiinae. The probability of hatchlings reaching the soil surface was negatively correlated with depth documenting a significant cost to females laying eggs deep in the soil. Hatchling survival may be an important agent of selection on ovipositor length in habitats of different soil moistures. Hatchling survival in the soil was also correlated with body size, which may impose a constraint on egg-size fecundity trade-offs. Females of a bivoltine population of Allonemobius socius lay eggs at shallower depths when reared under summer compared to fall conditions and, therefore, may be able to respond to selection through behavioral plasticity when morphological adaptation is constrained by allometry.

Nanoconfined ß-sheets mechanically reinforce the supra-biomolecular network of robust squid Sucker Ring Teeth.

The predatory efficiency of squid and cuttlefish (superorder Decapodiformes) is enhanced by robust Sucker Ring Teeth (SRT) that perform grappling functions during prey capture. Here, we show that SRT are composed entirely of related structural "suckerin" proteins whose modular designs enable the formation of nanoconfined ß-sheet-reinforced polymer networks. Thirty-seven previously undiscovered suckerins were identified from transcriptomes assembled from three distantly related decapodiform cephalopods. Similarity in modular sequence design and exon­intron architecture suggests that suckerins are encoded by a multigene family. Phylogenetic analysis supports this view, revealing that suckerin genes originated in a common ancestor ~350 MYa and indicating that nanoconfined ß-sheet reinforcement is an ancient strategy to create robust bulk biomaterials. X-ray diffraction, nanomechanical, and micro-Raman spectroscopy measurements confirm that the modular design of the suckerins facilitates the formation of ß-sheets of precise nanoscale dimensions and enables their assembly into structurally robust supramolecular networks stabilized by cooperative hydrogen bonding. The suckerin gene family has likely played a key role in the evolutionary success of decapodiform cephalopods and provides a large molecular toolbox for biomimetic materials engineering.

Integrative and comparative analysis of coiled-coil based marine snail egg cases - a model for biomimetic elastomers.

Integrative and comparative analyses of biomaterials systems offer the potential to reveal conserved elements that are essential for mechanical function. The approach also affords the opportunity to identify variation in designs at multiple length scales, enabling the delineation of a range of parameters for creating precisely tuned biomimetic materials. We investigated the molecular design and structural hierarchy of elastomeric egg capsules from the marine snail Pugilina cochlidium (family Melongenidae) and compared these data with all available published studies in order to infer the structure-property relationships of the egg case from the molecular to the macroscopic scale. While mechanical similarities had previously been observed for two other marine melongenid snails, Busycotypus canaliculatus and Busycon carica, B. canaliculatus was the only species for which detailed molecular and nanostructural data were available. Egg capsules from P. cochlidium were found to exhibit mechanical properties and shock absorbing potential that was similar to B. canaliculatus. The two species also displayed similarity in hierarchical fibril bundling and a sub-micron staggering of 100-105 nm within filaments, as shown by atomic force microscopy and small angle X-ray diffraction. In situ Raman micro spectroscopy indicated that P. cochlidium egg cases undergo a stress-induced coiled-coil to extended ß-strand structural transformation that is very similar to that of B. canaliculatus. These observations supported the view that these structural and hierarchical elements are essential for egg case function. Comparative analysis of the primary amino acid sequences and structural predictions for all known egg case proteins suggested that while the proteins all contain sequences prone to adopt α-helical structures, the predicted location of coiled-coil domains and stutter perturbations varied within and between species. Despite these differences, mixtures of denatured native egg case proteins readily re-folded in citrate-phosphate assembly buffer into α-helix rich, coiled-coil based oligomers, as determined by attenuated total reflection Fourier transform infrared spectroscopy, circular dichroism and MALDI-TOF. It is concluded that both conserved and divergent designs in marine snail egg cases offer inspiration for the engineering of biomimetic elastomeric materials with a unique capability for mechanical energy absorption.

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.

Supercontraction stress in spider webs.

Silk produced from the major ampullate (MA) gland supercontracts when wet, and in this paper, we investigate the consequences of high humidity and of the added load of water droplets condensing from saturated air on the mechanical integrity of the spiders' orb web. We measured the development of the supercontraction stress (sigma(sc)) with time when fixed lengths of MA silk from Nephila clavipes and Argiope aurantia were exposed to increasing humidity. Supercontraction generated stresses of about 50 MPa, and extension of these samples to stresses between 150 and 1100 MPa show a time dependent relaxation over 1000 s to approximately 75% of the initial tension but show no indication of failure. We conclude that supercontraction can maintain tension in webs and does not limit the ability of the web to support loads in excess of the supercontraction stress.

Elastic proteins: biological roles and mechanical properties.

The term 'elastic protein' applies to many structural proteins with diverse functions and mechanical properties so there is room for confusion about its meaning. Elastic implies the property of elasticity, or the ability to deform reversibly without loss of energy; so elastic proteins should have high resilience. Another meaning for elastic is 'stretchy', or the ability to be deformed to large strains with little force. Thus, elastic proteins should have low stiffness. The combination of high resilience, large strains and low stiffness is characteristic of rubber-like proteins (e.g. resilin and elastin) that function in the storage of elastic-strain energy. Other elastic proteins play very different roles and have very different properties. Collagen fibres provide exceptional energy storage capacity but are not very stretchy. Mussel byssus threads and spider dragline silks are also elastic proteins because, in spite of their considerable strength and stiffness, they are remarkably stretchy. The combination of strength and extensibility, together with low resilience, gives these materials an impressive resistance to fracture (i.e. toughness), a property that allows mussels to survive crashing waves and spiders to build exquisite aerial filters. Given this range of properties and functions, it is probable that elastic proteins will provide a wealth of chemical structures and elastic mechanisms that can be exploited in novel structural materials through biotechnology.