Biomechanical Design of Elastic Protein Biomaterials - American

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Biomechanical design of elastic protein biomaterials: A balance of protein structure and conformational disorder Lisa D Muiznieks, and Fred W. Keeley ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00469 • Publication Date (Web): 10 Oct 2016 Downloaded from http://pubs.acs.org on October 15, 2016

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ACS Biomaterials Science & Engineering

Biomechanical design of elastic protein biomaterials: A balance of protein structure and conformational disorder

Lisa D. Muiznieks1* and Fred W. Keeley1,2

1 Molecular Structure and Function Program, Research Institute, The Hospital for Sick Children, 686 Bay St, Toronto ON Canada M5G 0A4 2 Department of Biochemistry and Department of Laboratory Medicine and Pathobiology, 1 King’s College Circle, University of Toronto, Toronto ON Canada M5S 1A8 * To whom correspondence should be addressed (email: [email protected])

Keywords: protein elasticity; elastic modulus; tensile strength; protein biomaterials; biomechanical design

1 Environment ACS Paragon Plus


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Abstract Elastic biomaterials are found across biology where they fulfill diverse loadbearing and energy storage and dissipation functions. This class of biomaterials is comprised of elastic proteins that provide materials with combinations of extensibility, stiffness, tensile strength, toughness, and viscoelastic properties. Differences in mechanical properties are due in large part to variations in the ratio of secondary structure and conformational disorder of constituent protein monomers, arising from differences in amino acid sequence. This natural diversity provides rich inspiration for the design of elastic biomaterials. Here we review the relationship between sequence, structure, disorder, and mechanical properties of elastic proteins from natural materials ranging from highly extensible and soft, to mechanically strong and tough. We describe molecular strategies as well as recombinant efforts to design materials with tailored mechanical properties, with the ultimate aim of rationally engineering biomaterials for advanced biomedical applications.


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1 Elastic proteins and engineering of elastic protein materials Elasticity is the ability of a material to deform under force and return to its original shape once the force is removed.1 Elastic proteins provide elasticity to biomaterials from a phylogenetically diverse range of species. Elastic biomaterials take many forms such as extensible fibres, latticed networks, compressible pads, hard bone and beak. Correspondingly, these materials exhibit a range of mechanical properties spanning orders of magnitude. Understanding the molecular basis of how elastic proteins function is a key step to the rational design of elastic protein materials for diverse biomedical applications such as implant coatings, drug delivery, suture thread and tissue engineering (overview in Fig. 1). In particular, the inability and/or inefficiency (scarring, loss of function) of the body to repair damaged tissue, e.g. in cases of burns, intervertebral disc degeneration, and heart disease, and the associated burden of care (cardiovascular disease alone accounted for ~30 % of deaths worldwide in 2013)2 are major driving forces behind the design of elastic biomaterials for wound healing and tissue engineering, including vascular conduits, heart valves, skin, cartilage (joints), ligaments, and bone. While biomaterials based on synthetic polymers are strong, their success for tissue repair is often limited due to an elicited immune response and/or poor material compliance.3 Natural polymers present a biocompatible alternative, with superior biological and cell interactive properties.3 To date, the design of elastic materials has largely focused on important facets of immunological and cellular compatibility. However, tissues have different native mechanical properties (Table 1) reflecting the differences in force (e.g. magnitude, direction, frequency) under which they must



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function.4 Moreover, substrate stiffness is as important as biochemical signaling in directing the lineage of stem cell differentiation.5 Thus, engineered biomaterials must also match the mechanical requirements of the tissue they are designed to augment or replace. One promising overarching strategy for biomechanical design is a bottom up approach based on an increasingly detailed understanding of sequence-structure-function relationships of elastic proteins across nature, including the contributions of specific regions of sequence to different mechanical properties.6-13 Clear from this growing body of work is that both structured and conformationally disordered regions of sequence make key and distinct contributions to mechanical properties. Thus, elucidating the underlying sequence and structural bases for the differences in mechanical properties between natural biomaterials will provide substantial opportunities for biomimetic design. This review will discuss the sequence and structural features of natural elastic protein materials with mechanical properties ranging from compliant, extensible and resilient, to mechanically strong and tough. Focus will be placed on current understanding of the contributions of structure and conformational disorder to mechanical properties. Strategies for rationally tuning the mechanical properties of elastic biomaterials, and efforts at mechanical design of synthetic and recombinant elastic proteins will be addressed.

2 Diversity of elastomeric protein biomaterials across nature Elastic protein biomaterials are optimized for diverse physiological functions, fine-tuned by evolution for properties such as extensibility and strength (Table 2). Diversity comes from across biology, manifest as convergent evolution to fulfill a variety


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of load-bearing and energy storage/ dissipation functions. The protein elastin imparts elasticity to vertebrate tissues such as blood vessels, lung and skin.14 The insect protein resilin displays near-perfect energy conservation enabling the catapult-like jumping of fleas, as well as the high frequency beating of insect wings.15 An elastic pad in the hinge of bivalve molluscs has excellent capacity for storing elastic energy that is used in the rapid opening and closing of scallop shells to facilitate locomotion.16 These elastic proteins are readily stretched/ compressed, are highly extensible, and recoil without losing much energy to heat, but are not particularly strong. A number of elastomeric proteins form fibrous networks that impart structural support. Elastic silk proteins are spun by spiders into tough and strong aerial filters and safety lines, and by silkworms into protective cocoons. The strength of silk rivals that of high tensile steel and the synthetic polymer Kevlar.12 Other natural protein-based biomaterials display mechanical gradients across their length, designed with properties of exceptional toughness, strength and/or hardness for survival in harsh environments and defense against predators.17,18 Separately, wheat gluten is an energy storage protein of wheat with incidental elasticity that is exploited in the making of bread dough.19 These natural, protein-based biomaterials provide rich inspiration for material design. They share basic features of self-assembly into insoluble polymers through the cross-linking of monomeric protein ‘building blocks’. Elastic protein monomers differ with respect to proportions and type of secondary structure and conformational disorder. Unraveling the contributions of protein sequence and structure to the mechanical properties of elastic materials will support biomimetic design through rational sequence mutation.



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3 Definition and measurement of mechanical properties Elastic mechanical properties are measured from stress-strain (force-deformation) profiles, commonly using tensile (stretching) or compressive cycles of loading/unloading, or rheological measurements using shear forces. Stress is the force on a material per cross-sectional area and is expressed in units of Pascals (Pa, N m-2). Physiological forces on tissues are frequently in the range of kPa to MPa. Strain is the change in length of a material upon application of a stress, relative to the initial length. Stress-strain profiles yield several mechanical properties that collectively describe the mechanical behaviour of a material (Fig. 2). Tensile strength (σmax; Pa) is the stress required to break a material. Failure strain (εmax; no units when reported as a decimal fraction, but often reported as percent change in length) is defined as the strain at break, e.g. a measure of maximum extensibility. Elastic modulus (Einit; Pa) is calculated as the tangent gradient of the stress-strain curve in the initial, linear region and describes material stiffness, i.e. how readily a material can be deformed with force. Stiffer materials have a higher elastic modulus. Note that stress-strain curves for biological materials are frequently J-shaped, corresponding to a softer initial ‘toe’ region and a stiffer response at higher strains. In some cases, the stress-strain relationship may be highly nonlinear, and an incremental elastic modulus can be used to describe material stiffness, calculated as the tangent to the curve at a given strain. Toughness (J m-3) is the energy to break, obtained as the area under the stress-strain to failure curve. Materials require a combination of strength and extensibility to be tough.


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Moreover, biological materials are viscoelastic, i.e. they exhibit deformation behaviour that is dependent on time and strain rate due to the presence of both viscous and elastic responses to force.20 The viscous response results in measurable hysteresis between successive load/unloading cycles, i.e. unloading follows a trajectory of lower stress. However, the original shape is recovered after removal of the force due to the elastic response. In contrast, plastic deformation is a non-recoverable change in shape. Hysteresis corresponds to energy loss, calculated as the percentage difference in area under the loading curve compared to the unloading curve. The complementary property is resilience, calculated as the percentage area under the unloading curve normalized by the area under the loading curve. The most resilient elastomers lose the least amount of energy to heat (have excellent recovery of elastic energy) due to a largely entropic driving force for recoil, e.g. the high resilience of resilin enables high frequency beating of insect wings without leading to overheating of the animal. In many cases in nature, low to moderate resilience is required for function to dampen recoil, such as for silk that forms the spider web capture spiral (Table 2). Stress relaxation is calculated as the percent reduction in stress over time when a material is held at constant strain, and is due to internal molecular rearrangement (viscous response), e.g. breaking/reforming or straining of bonds, typically recoverable once the stress is removed. Rheological measurements of materials under shear forces enable calculation of the shear modulus (G; Pa), comprised of an energy storage modulus (G´; Pa) and an energy loss modulus (G´´; Pa). This technique is frequently applied to colloidal suspensions and hydrogel materials and provides information about sol-gel transitions, and elastic and viscous behaviour, including shear thinning and thickening.21-23



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4 Relationship between sequence, structure (disorder) and elasticity: considerations for the design of elastic biomaterials The driving force for elastic recoil (f) is the sum of an entropic component (fs) and an internal energy (fe) component, i.e. f = fs + fe.24 Internal energy arises from backbone configurational distortions (bond strain), and secondary structure transitions and/or unfolding upon deformation, and is a component of structured proteins, e.g. keratin, hagfish threads, and fibrin.25,26 Thus, structured elastomers recoil on the basis of stored potential energy of stretched internal bonds (reviewed by Miserez and Guerette).25 In contrast, entropic (rubber-like) elasticity is derived from the long-range conformational disorder of amorphous materials.24 The driving force for entropic recoil is provided by a return to greater conformational entropy in the relaxed state compared to in the stretched state. Entropic elasticity is reviewed by Gosline et al.,1 and Rauscher and Pomès.24 This review focuses on the design of entropic elastomers. Entropic elastic proteins all require sufficient polymer chain length (i.e. distance between cross-links) and chain disorder (entropy), as well as cross-links so chains do not slide past one another under an applied force.26,27 As such, the monomer building blocks of entropic elastomeric proteins share basic features of sequence composition and modularity. These proteins are typically block copolymers, comprised of repetitive or semi-repetitive sequence blocks, e.g. An, AnBn, (AB)n, that correspond to disordered and structured regions (Fig. 3). Variation in the type of structure, and the proportion, length and arrangement of domains endows biomaterials with a broad spectrum of mechanical properties. Sequences that are responsible for conferring extensibility and resilient recoil are predominantly disordered,


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while sequence blocks that are more ordered tend to contribute strength, stiffness and/or toughness. Structured blocks frequently take, but are not limited to, the form of short βsheet regions (e.g. ~5-10 residues), arranged in an amorphous matrix of disordered segments.28 Elastic proteins are frequently of low amino acid complexity. Elastomeric sequence blocks contain motifs of ~3 to 15 residues with varying degrees of repetition and homology within and between domains and proteins (Fig. 3; Table 3). In particular, elastic sequence blocks are enriched in glycine (G) or proline and glycine (PG) residues. Indeed, a high (2P+G ≥ 60%) compositional threshold of proline and glycines is shown to inhibit the formation of extended secondary structure, i.e. α-helix and β-sheet.29 This is due to a high entropic penalty for the structural confinement of glycine residues, lacking a side chain, and because proline has a fixed (rigid) phi dihedral angle and lacks the amide hydrogen required for making backbone hydrogen bonds. Proline is typically evenly spaced throughout elastic sequences, and is often located at the boundary of ordered and disordered blocks. Remarkably, this relatively simple PG relationship holds across a wide range of elastomeric proteins,29 including those described in this review. Secondary structure formation is limited within elastomeric blocks to transiently populated local motifs, frequently type-II β-turns and non-hydrogen-bonded polyproline II helix (PPII).26,29,30 One common elastomeric sequence motif is the PG dipeptide in the form xPGx, where x is often G but never P. This sequence has a high propensity to form a β-turn, i.e. hydrogen bond formation between the carbonyl oxygen of residue i and the amide hydrogen of residue i+3, with PG at the corners of the turn.31 The dynamic



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equilibrium of β-turns, PPII, and coil in elastic proteins provides flexibility to elastic sequence blocks.29,30 Elastomeric domains of some proteins are hydrophilic, notably resilin and wheat gluten, often corresponding to a higher composition of glutamine and serine residues, whereas others are highly hydrophobic, e.g. elastin and silk fibroin. For disordered, hydrophobic elastic proteins (specifically elastin), the hydrophobic effect (i.e. increase in solvent entropy) also contributes to elastic recoil.32 The contributions from chain entropy and the hydrophobic effect most likely differ between different hydrophobic elastomeric proteins, depending on the amino acid sequence. Entropic elastomers generally contain few non-local intramolecular interactions. However, residues with reactive side chains (e.g. lysine, cysteine, tyrosine) are required at intervals for intermolecular cross-linking. Strategies employed for cross-linking vary considerably between materials, including covalent enzyme-mediated cross-links, disulfide bonds and ‘physical’ contacts, made from hydrophobic interactions and/or hydrogen-bonded secondary structures. From a design perspective, we review variations in the proportion and location of structured and disordered blocks, the length of polymer chains (i.e. distance between cross-links), as well as cross-link type, density and number that lead to alterations in mechanical properties. Generally, increasing cross-linking density makes materials stiffer. Conversely, materials can be made less stiff by increasing the length of polymer chain segments between cross-links. Pore size of materials can also be varied. This parameter is often investigated with respect to the infiltration and interaction of cells within a material. However, modulation of pore size may directly influence mechanical


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properties on the basis of a balance of applied mechanical and osmotic forces. Such ‘poroelasticity’ may have important effects on mechanical properties and should be a future consideration for biomaterial design.

5 Phase separation of elastomeric proteins Many elastic proteins undergo a liquid-liquid phase separation that is on-pathway for material assembly. This process is characterized by a stimulus-responsive, sharp increase in solution turbidity corresponding to the formation of protein-rich colloidal droplets in a protein-poor solution phase.33 In some cases, protein phase separation exhibits a temperature-dependence similar to lower or upper critical solution temperature (LCST/ UCST) behaviour observed for many polymers but uncommon for proteins. Phase separation of elastin (‘coacervation’, LCST), results in the self-association of disordered hydrophobic domains, concomitant with an increase in solvent entropy.33 The growth, stability, interactions, and reversibility of colloid-like elastin droplets are modulated by the sequence of the protein/peptide.34 Resilin (dual U/LCST)35 and recombinant abductin (UCST)36 also exhibit reversible temperature-dependent phase separation. For resilin, it is suggested that negatively charged residues partition at the droplet interface with water, while positively charged residues are located in the core.35 Recombinant block copolymers of Araneus diadematus dragline silk containing the Cterminal domain exhibit LCST behaviour.37 The mussel foot protein (mfp-1) that coats byssal threads also forms micro-droplet coacervates that protect against frictional wear,38 but as yet there is no evidence to suggest elastomeric byssal proteins Col-P or Col-D exhibit this behaviour as well. Colloidal nanoparticles form from silk fibroin39 and a



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single ensemble repeat of recombinant spider wrapping silk,40 predicted to be on-pathway for fibre formation, and from recombinant squid beak17 and sucker ring teeth proteins.41 In these cases, nanoparticle formation is induced by ionic strength, hydration state, buffer and/or processing conditions, and for silk fibroin, is suggested to arise from the amphiphilic nature of the block copolymers, similar to micelle formation. Phase separation of elastomeric proteins provides a unique avenue for material design, e.g. droplets/capsules and fast-setting hydrogels. However, for the design of elastic materials such as sheets, tubes and fibres, the question arises as to whether phase separation is a necessary design consideration. In the case of elastin at least, the same forces drive both phase separation and mechanical properties such as elastic recoil, specifically, a combination of increased solvent entropy (the hydrophobic effect) and increased polypeptide chain entropy (conformational disorder) (Rauscher and Pomès, pers. comm.). Thus, it is likely that sequence-structure effects on phase separation will also offer important insights into the mechanical design of a wide range of elastic materials.

6 Properties and design of highly disordered elastomeric protein biomaterials Materials from highly disordered elastomeric proteins exhibit a low modulus, are typically extensible by >150 %, and are highly resilient, i.e. lose minimal energy to heat upon loading/unloading. However, they are not very strong. Unifying features include extensive conformational disorder, and only limited or no regions of α-helix and β-sheet secondary structure. Moreover, these protein materials are only elastic when hydrated, and brittle when dry.32


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6.1 Elastin Elastin is an essential component of many vertebrate tissues. For example, elastin comprises up to 50 % dry weight of the aorta, where it dampens the pulsatile pressure of blood flow from the heart.14 Elastin is laid down in the extracellular matrix during early development and with few exceptions, does not appreciably turn over (half life ~70 years).42,43 Thus, networks of this elastic protein must withstand in excess of billions of cycles of extension and recoil over a lifetime. The elastin monomer, tropoelastin, is comprised of hydrophobic domains (~30 residues long in humans), alternating with cross-linking domains (Fig. 3; Table 3).14 Hydrophobic elastin domains contain ~80 % glycine, valine, proline, leucine and alanine residues, arranged commonly as VPG, GV and GGV, although with little domain homology. Cross-linking domains contain two or three strictly-spaced lysine residues on poly-A or PG-rich background that form the predominant elastin cross-links desmosine and isodesmosine (tetrafunctional) via the action of lysyl oxidases.44

6.1.1 A simplified sequence approach: (VPGVG)n-based block copolymers The elastin consensus sequence is classically described as (VPGVG)n. Early biophysical studies of synthetic (VPGVG)10-15 demonstrated temperature-dependent phase separation and fibre formation.45 Poly-(VPGVG) is highly disordered but not random, specifically, it lacks extended secondary structure in preference for local βturns.29 As β-turns are not made from all VPGV motifs concurrently, (VPGVG)n does not adopt a putative ‘β-spiral’ secondary structure as once proposed, but is flexible.



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Recombinant biology can be used to readily introduce mutations into the pentapeptide sequence, modulating biophysical properties. The temperature of phase separation, Tc, can be tuned by mutating position X of (VPGXG)n to any residue except proline, where increasing the hydrophobicity lowers Tc.46 Increasing the number of tandem repeats also lowers Tc. (VPGVG)n-based block copolymers have largely been designed and functionalized to form phase-separated particles, micelles, and hydrogels with applications in drug delivery, reviewed by MacEwan and Chilkoti,47 and RodriguezCabello et al.48 However, little mechanical data is associated with these studies. Mutations at position X´ of (VPX´VG)n modulate mechanical properties.49 Together, this functional tunability has led to the recombinant production of a large range of block copolymers, particularly of the form BAB, based on (VPX´XG)n sequence blocks.50 Endblocks (i.e. block B) introduce greater hydrophobicity (e.g. IPAVG, VPAVG), designed to phase separate at ambient temperature. Notably, (VPAVG)n features a G/A mutation that stabilizes type-I β-turn formation between V1(CO)-V4(NH), resulting in materials with thermoplastic behaviour, i.e. stiffening with increasing temperature, yet retaining the capacity for reversible deformation.51 The midblock sequence (i.e. block A) is designed to be more hydrophilic (e.g. VPGEG, VPGAG) to have a higher Tc than block B. Mechanical properties of (VPGVG)n-based block copolymer materials are commonly tuned by modifying the length, sequence and ratio of blocks, the solvent and temperature used for casting materials, preconditioning, and the type (e.g. structural/ physical, chemical) and density of cross-links (Table 4).51-57 For example, increasing the length of flexible, elastic sequence blocks can be employed to lower the stiffness and strength of materials and increase failure strain (e.g. stiffness B93-fold) of the stress-strain response.87

6.2 Resilin Resilin is a highly resilient elastic protein found in specific regions of the insect cuticle. The adult insect cuticle is laid down during the pupal stage and is largely not turned over.88 Thus, resilin must last the lifetime of the insect, as for elastin. Resilin is a component of the wing tendon of dragonflies and locusts, enabling high frequency (~25200 Hz) beating for flying and hovering.89,90 Resilin is also found in the tymbal organ of


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cicadas enabling ~4 kHz sound vibrations,91 and in the joints of fleas, providing energy storage upon compression for jumping.92 The resilin-rich dragonfly tendon has a stiffness of ~2 MPa, extensibility of 190 %, and resilience of 92 %.1 The resilin monomer of Drosophila melanogaster (57 kDa) is comprised of two elastomeric domains separated by one chitin-binding domain, corresponding to three exon products (Fig. 3).93 Exons I and III encode repetitive elastic domains of 323 and 235 residues, respectively. Exon I contains 18 copies of a 15-residue imperfect repeat (Table 3). Tensile testing of recombinant exon I from the fruit fly (rec1-resilin), reveals this domain has a low modulus (~26 kPa) and provides resilin with high resilience (>90 %) and extensibility (~300 %) (Table 6).88,94 Exon III contains 11 copies of a 13-residue imperfect repeat, and provides the energy storage capacity of resilin, but has lower resilience (65 %) than exon I.8,95 These elastic domains are separated by a non-repetitive, hydrophobic chitin-binding domain (62 residues) that anchors the protein to chitin in the cuticle.93,96 A recombinant putative resilin sequence from the mosquito Anopheles gambiae displays similar mechanical properties to rec1-resilin (Table 6).94 Structural analyses of elastomeric resilin domains and peptides are consistent with predominant disorder (little extended secondary structure) and the presence of transiently populated β-turns and PPII helix.97,98 Small-angle x-ray scattering studies reveal rec-1 resilin is intrinsically disordered, yet more compact than a chemically denatured protein.99 Recombinant full-length resilin and exon III alone exhibit a transition to βturns under high (240 °C) temperature that is not observed for exon I alone, suggesting the importance of β-turns in energy storage capacity of exon III.8



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Native resilin contains di- and tri-tyrosine cross-links, involving an estimated 25 % of tyrosines.100 This corresponds to a predicted cross-link spacing of ~40-60 residues.101,102 Cross-linking of recombinant resilin polypeptides can be controlled in vitro via tyrosines using the photoreactive linker [Ru(bpy)3]2+ or horseradish peroxidase, or by the recombinant insertion of other cross-linking residues, e.g. lysines. Efforts to investigate and tune the mechanical and functional properties of resilin materials include modulating cross-link density, design of hybrid block copolymers, and the addition of bioactive sequence motifs.23,103,104 A resilin-like polypeptide, RLP12, based on 12 copies of the 15-residue sequence motif from exon I, has been recombinantly functionalized with cell adhesion, proteolytic degradation, and/or heparin-binding motifs, in addition to alternate cross-linking sites (lysines and/or the replacement of tyrosines by methionine, phenylalanine).23,105,106 RLP12 hydrogels display functional versatility, as well as tunable gelation and mechanical properties (Table 6).23,105-108 Resilin-based materials show potential for the engineering of vocal chords, which function in a specialized highfrequency range.23 Moreover, muscle-mimetic biomaterials have been designed from block copolymers of resilin and globular GB1 domains (derived from the B1 immunoglobulin-binding domain of streptococcal protein G).103 These materials mimic the mechanical saw-tooth unfolding profile of muscle titin, and are highly resilient at low strains and dissipate energy at high strains.

6.3 Abductin Abductin is an elastic protein found in the hinge ligament of bivalve molluscs, e.g. scallops. Abductin stores elastic energy when compressed and works against the


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adductor muscle to open the shells once the muscle relaxes.16,109 The rapid opening and closing of the shells expels a dorsal jet of water from the scallop, resulting in propulsion.110 Abductin has a high composition of glycine (>60 %), methionine and phenylalanine residues (Table 3),16,111 and functions as an entropic elastomer with essentially no internal energy component to elastic recoil (i.e. fe≈0, so f≈fs).110 Mechanical properties of abductin are largely obtained from studies of the native hinge ligament of the mollusc, which is characterized by low stiffness and strength, as well as high resilience and extensibility, similar to elastin and resilin (Table 2). Synthetic abductin comprised of the elastomeric consensus sequence (FGGMGGGNAG)4 and related glycine-rich peptides, including GMGGG and GXGG, X=A, M, F, show predominant disorder in solution by circular dichroism and NMR in combination with type-II β-turns and distorted PPII helix.112,113 A recombinant construct corresponding to (FGGMGGGXAG)12, where residue X=N, K, exhibits reversible UCST behaviour characterized by viscoelastic, gel-like rheological properties, and an elastic storage modulus (G´) of ~100 Pa.36 Tensile mechanical data is currently unavailable for synthetic or recombinant materials, but the inclusion of lysine residues in these constructs for cross-linking will enable such future investigation.36

7 Properties and design of stiff, strong and tough elastic protein biomaterials One challenge of using elastomeric proteins that have a high composition of conformational disorder for many tissue engineering applications is their intrinsic lack of tensile strength and toughness. The following section profiles elastomeric proteins that are stiffer, stronger and/or tougher than those described above. A common feature of



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many strong and tough protein materials is the presence of short, but well-defined blocks of secondary structure in the protein monomers. One evolutionary ‘blueprint’ for strength utilises nano-confined β-sheet blocks, such as from poly-(GA) and Ala-rich sequence.9,114 Efforts to mimic and modulate mechanical properties from synthetic and recombinant polypeptide materials are described.

7.1 Silk proteins (fibroins and spidroins) Silk is a fibrous product of spiders and silkworms,115 and is also produced by other insects such as honeybees.116 Silkworms spin a single type of silk, whereas orbweaving spiders have up to seven specialized abdominal glands for spinning multiple silk types. Silk fibroins and spidroins are typically large (200 to >350 kDa) protein monomers comprised of internal, multi-block ‘ensemble’ repeats that often contain smaller repetitive sequence motifs (Fig. 3).115 The large size and repetitive nature of silk proteins have made full sequence identification and recombinant production challenging. Moreover, it has proven difficult to recapitulate the mechanical properties of native silks in vitro.117 The exceptional mechanical properties of silks are attributed to a combination of sequence composition and fibre spinning, involving an increase in protein concentration, a drop in pH, and shear and elongation forces, promoting the folding and alignment of semi-crystalline phases.117,118 A sequence-structure approach modulating the block length, sequence and arrangement of various spider silks has been applied to investigate effects on mechanics, including attempts to rationally design materials with tailored mechanical properties, and will be addressed in this section. For silk reviews the reader is referred to Tokareva et al.,119 and Gosline et al.12


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7.1.1 Silkworm fibroin Silk fibroin is spun by the silkworm Bombyx mori and used to form a protective cocoon around the insect during its metamorphosis into a moth. Silk fibroin is a strong (>600 MPa) fibre (Table 2) comprised of heavy (~350 kDa) and light (25 kDa) chains, as well as the protein P25 (30 kDa) in a 6:6:1 ratio, and a glycoprotein coating (sericin).120 The silk fibroin heavy chain is comprised of twelve tandem blocks (typically ~300-525 residues each) rich in the consensus repeat motif (GAGAGS)n (~67 %) and other GA-rich sequences containing tyrosine (~33 %).121 These domains form β-sheet structures responsible for the high (~50 %) crystallinity of fibres (particularly from GAGAGS repeats), which are aligned with the fibre axis within a more disordered matrix.120-122 Separating these domains are short (~27 residues) highly homologous, yet non-repetitive, sequences that contain charged residues and a proline, and thus, likely break β-sheet blocks.121 Light chains are relatively elastic but contain no consensus sequence repeats, and are linked to heavy chains by a disulfide bond.120 Silkworms are readily farmed, making silk fibroin a commercially viable source of silk. Silk fibroin can be processed into different forms (e.g. fibres, sheets, gels), with degradation rates and bioactivity that can be controlled by β-sheet content, co-blending, surface modifications and the addition of bioactive molecules, while improvements to purification protocols, e.g. to remove the native sericin coating, have led to reduced silk antigenicity.123-128 The ability to control silk fibroin properties has led to its development for diverse applications such as suture thread,129 drug delivery,123 the potential for bone, nerve and ligament engineering,126-128 and biomedical sensors and optical devices,



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including cornea replacements.124,125 Recombinant block copolymers have been produced based on silk fibroin poly-(GA) or (GAGAGS)n and elastin (G(V/X)GVP)n motifs.130,131 Silk blocks form distorted β-sheet structures while elastin blocks remain disordered.131 The elastic modulus of these silk-elastin materials varies over 4-fold upon modifying the number and length of repeat blocks, and the residue in position X.132 Replacing elastin domain 30 by poly-(GA) in the context of full-length human tropoelastin results in a 60 % increase in elastic modulus and stress relaxation.76

7.1.2 Dragline silk Major ampullate (MA) spider silk forms the frame of orb webs, and the dragline on which the spider hangs. MA is the strongest silk (~1-4 GPa dry),1 and displays efficient dissipation of elastic energy, allowing spiders to control their dragline descent. It is also tough, enabling the web to absorb the impact of prey, and the fast drop of the spider on a dragline attached at an angle perpendicular to the frame.12 When wet, MA silk shrinks by ~45 % (supercontraction).133 Water swollen MA fibres display lower stiffness (up to three orders of magnitude, i.e. modulus of 10 MPa; fibres become rubber-like)

compared to dry fibres, lower strength (5-10 MPa), and increased extensibility (140 %).1,25 MA silk proteins are categorized into spidroin-1 (MaSp-1) and spidroin-2 (MaSp2) on the basis of sequence composition. MaSp-1 is rich in (GGX)n repeats that are notably deficient in proline residues (Table 3).134 Tripeptide repeat blocks contribute to fibre extensibility and are predicted to form a flexible 31-helix, also known as a polyglycine II helix,135 an extended structure with approximate 3-fold rotational


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symmetry (θ,ψ angles around -60°, +135°), and lacking intrachain hydrogen bonds.136-138 In contrast, MaSp-2 contains (GPGXX)n sequence repeats139 that form β-turns with P in position i+1, and display enhanced mobility and conformational disorder when wet.140 Short (~5-10 residue) poly-A stretches are interspersed between elastic sequence blocks of both spidroins and adopt semi-crystalline β-sheet secondary structure,138,141 corresponding to ~20-25 % fibre volume (Fig. 3).13 β-sheet blocks are of nanometer dimensions and align with the fibre axis, contributing to tensile strength.9,142 A lower proline content within elastic repeats correlates with less conformational disorder and stiffer materials, suggesting that changing the ratio of MaSp-1 and -2 in dragline silk may be an evolutionary strategy to expand its range of mechanical properties.143-145 Tandem MA ensemble repeats are flanked in the full-length monomer by non-repetitive N- and Cterminal domains (120

91

-

16

35

200

53

35

870

75

109

28

45

1200

120

13

90

6

10000

27-35

35

160

3 9750

10004000 500 687

200-270 86

35 -

150 376

7000 5000 0.27

600 115 -

18 23 80

200

-

-

1

50

850

-

100


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Table 3. Elastomeric protein consensus sequences. Numbers in brackets refer to the approximate proportions of elastomeric sequence and structured sequence per monomer, where feasible. Protein Abductin111 Col-P186 Col-D185 Flag156 Gluten HMW19 Lamprin228 MaSp-1134,229 MaSp-213,139 MiSp230 Resilin93,231

Silk fibroin heavy chain121 Histidine-rich squid beak proteins17 Suckerins114,175 Tropoelastin232

Consensus sequence/s GGFGGMGGG, FGGMGGGNAG GXGPG (GXX´)~150 (GXX´)~175 AAAX, GA, GGL (GPGGX)~43-63, X=A,S,Y,V (GGX)~6-12, X=A,S,T PGQGQQ, GYYPTSLQQ GGLGY (GGX)~6-10, X=Q,Y,A (A)4-10 (GPGXX)~9, XX=QQ, GY (A)~9 GGX, X=Y,Q (GA)n(A)n GGRPSDSYGAPGGGN, GYSGGRPGGQDLG, AQTPSSQYPAG GAGAGS, GAGAGY

Comments Elastic, disordered (85 %) Elastic, disordered (26 %) Collagen-like, triple helix (44 %) Collagen-like, triple helix (53 %) Silk-like, β-sheet (20 %) Elastic, disordered Elastic, putative 31-helix Elastic, disordered (74-84 %) Elastic Elastic, putative 31-helix β-sheet (~25 %) Elastic, disordered β-sheet (~20 %) Elastic, putative 31-helix β-sheet Elastic, disordered (90 %)

GAGFA, GHGLY, GHGXX´

β-sheet blocks (fibres ~50 % crystalline) α-helix in coacervate phase

GGLY, GGL, GGY, GYG AATAVSHTTHHA PGVXG, VPG, GGV (A)3-10KAAK

Elastic, likely disordered β-sheet; Blocks separated by Pro Elastic, disordered (80 %) Partially stable α-helix (20 %)



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Table 4. Mechanical properties of (VPGVG)n-based elastin materials. Physical crosslinks refer to structural or hydrophobic contacts only, no covalent links present. GA: glutaraldehyde. HMDI: hexamethylene diisocyanate, 5x or 10x molar ratio. The resilience of LysB10 materials was calculated as 52 % (Physical), 39% (GA). TFE: trifluoroethanol. Peptide

P2 C5 B9

LysB10 aE43 aE70 Name P2 C5 B9 LysB10

aE43 aE70

Molecular weight, kDa 72.0 134.1 165.3

Cross-links

Elastic modulus, MPa 16.65 0.05 0.03

Tensile strength, MPa 3.00 0.96 0.78

Failure strain, % 128 822 1084

Shear modulus, kPa 280 6.5 10.5

Physical56 Physical56 Physical (in water)56 Physical (in 35.26 2.28 250 57 TFE) 209 Physical54 0.53 2.88 430 54 GA 1.6 3.62 223 0.58 480 190 43.0 HMDI, x552 HMDI, x1052 0.93 420 310 69.8 HMDI, x552 0.40 570 130 Sequence B-(VPGVG)2-B, where B=[(I/V)PAVG]~80, I:V = 4:1 B-[VPGEG(VPGVG)4]30-B B-[VPGEG(VPGVG)4]48-B VPAVGKVPAVG(IPAVG)4(IPAVG)165IPAVGKAAKVPGAG[(VPGA G)2VPGEG(VPGAG)2]28VPAVGKAAKVPGAGVPAVG(IPAVG)4(IPA VG)165IPAVGKAAKA MMASMTGGQQMGRKTMG-[LD-CS5-(VPGIG)25VP]3-LEKAAKLE, CS5=cell binding sequence MMASMTGGQQMGRKTMG-[LD-CS5-(VPGIG)25VP]5-LEKAAKLE


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Table 5. Mechanical properties of recombinant ELPs. Materials were cross-linked with genipin except where stated. Tensile strength was extracted from raw stress-strain data from studies referenced below. Modulus and strength were converted to MPa as described previously.66,171 Resilience was measured using the fourth cycle of loading/unloading to 50 % strain. Stress relaxation was measured after 500 s at 50 % strain. PQQ: pyrroloquinoline quinone. x: human cross-linking domains 21/23. XL: cross-link. TE: tropoelastin. Ac: aciniform silk (A. trifasciata). Molecular Elastic Tensile Failure Resili Stress Modification compared to weight, modulus strength strain, ence, relaxation, kDa , MPa , MPa % % % 20-(x-24)2 20-(x-24)2 n.a.6 17.0 1.5 1.0 73 81 10 20-(x-24)2 PQQ cross17.2 0.25 0.19 86 80 links59 20-(x-24)4 Longer6 31.0 1.9 1.8 105 81 12 XL domains 16.9 1.3 1.2 91 70 23 20-(xU-24)2 more αhelical6 20-(8-12-24)2 Disordered XL 18.8 1.5 1.3 90 83 7 domains6 8–14|36 Disordered XL 12.7 5.3 3.1 68 71 12 domains; higher XL density6 20-x-24-x-30 Proline-poor; 14.9 1.4 1.0 74 81 6 64 shorter 14.9 1.5 0.7 50 82 8 20-x-30-x-24 Proline-poor; 64 shorter 20-x-60-x-24 Proline-poor64 16.8 1.4 1.2 88 84 8 30-(x-30)2 Proline-poor; 11.6 6.5 2.0 33 44 41 shorter64 30P-(x-30P)2 Shorter64 11.9 4.0 2.5 40 47 39 Proline-rich6 17.4 1.1 0.9 89 81 7 20-(x-20ch)2 20-x-24-x-L Contains 17.6 2.3 1.8 72 72 17 lamprin motif6 20-x-24-x-W34 Contains silk 15.8 1.7 1.5 92 85 13 171 motif Name Origin Sequence 20 human TE FPGFGVGVGGIPGVAGVPGVGGVPGVGGVPGVGIS x human TE PEAQAAAAAKAAKYGVGTPAAAAAKAAAKAAQF U x 23 mutant PEAQAAAAAKAAKYAAAAAAAAAAKAAAKAAQF 24 human TE GLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGV GVAPGVGVAPAIGP 8 human TE GAVVPQPGAGVKPGKVP 12 human TE GVGPFGGPQPGVPLGYPIKAPKLP Peptide



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8–14

human TE

36 30 30P 60

human TE human TE 30 mutant 30 mutant

20ch

chicken TE

L

lamprin

W34

Ac silk

GAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGARFPGV GVLPGVPTGAGVKPKAPGVGGAFAGIPGVGPFGGPQPG VPLGYPIKAPKLPGGYGLPYTTGKLPYGYGPGGVAGAA GKAGYPTGT GGACLGKACGRKRK GLVGAAGLGGLGVGGLGVPGVGGLG GLVGAAGLPGLGVPGLGVPGVGGLG GLVGAAGLGGLGVGGLGVPGVGGLGGLVGAAGLGGLG VGGLGVPGVGGLG GAGVPGVGVPGVGIGGVPGVPGVPGVPGVPGVPGVPGVP GVPGVPGVPGVPGVVPGVG GGLGYGGLGYGGLGVAGLGVAGLGYGGLGYPGAALGG VYTHHAALGGLGYPLG GAGYTGPSGPSTGPSGYPGPLGGGAPFGQSGFGG


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Table 6. Mechanical properties of recombinant resilin materials. Ru: photo-activated [Ru(bpy)3]2+/ ammonium persulfate. HRP: horseradish peroxidase/ hydrogen peroxide. Fenton: catalytic photo-Fenton reaction. THPP: [tris(hydroxymethyl) phosphino] propionic acid. ChBD: chitin-binding domain. Percentages indicate tyrosines or lysines cross-linked, where reported. Rec-1 resilin, An16, RLP12: tested in tension, hydrated. Exon I, Exon III, Full-length: tested by AFM nano-indentation after drying on mica or silica. Shear modulus (kPa) of RLP12 materials was calculated as 2.8 (THPP, 21%), 4.6 (THPP, 29%) and 6.9 (THPP, 39%).23 Peptide

Rec1resilin An16 RLP12

Molecular weight, kDa 28.5 18.5 27.5

Cross-links Ru, 19%88,94

Elastic modulus, MPa 0.026

Tensile strength, MPa 0.055

Ru, 14%94 THPP, 21%23 THPP, 29%23 THPP, 39%23 HRP95 Fenton95 None8,95 HRP95 None8,95 HRP96 None8,96

0.0057 0.014 0.024 0.034 9.8 2-10 0.63 2.55

0.071 0.041 0.069 0.073 -

Failure strain, % 250300 347 335 277 246 -

Resilience % 90-100 94 >90 >90 >90 93 94.5 90 86 63 96 94

Exon I

~30

Exon III

~23

Full-length

57

Name Rec1-resilin An16 RLP12

Sequence (GGRPSDSYGAPGGGN)17, first exon of the Drosophila CG15920 gene (AQTPSSQYGAP)16, Anopheles gambiae mosquito GGGGDQK[(GGRPSDSFGAPGGGNGGRPSDSFGAPGGGNGGRPSD SFGAPGGGNGGK)2GGGRGDSPG]2GGPQGIWGQGGRGGCKAAKR PKAAKDKQTKGEDLGDPMASMTGGQQMG Contains a cell adhesion site every 6th repeat, and C-terminal protease cleavage site followed by a polysaccharide binding site (underlined) (GGRPSDSYGAPGGGN)18 (GYSGGRPGGQDLG)11 (GGRPSDSYGAPGGGN)18-ChBD-(GYSGGRPGGQDLG)11

Exon I Exon III Full-length



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Table 7. Mechanical properties of recombinant silk protein materials. Fl: flagelliform silk. MA: major ampullate silk. Ac: aciniform silk. fhc: silk fibroin heavy chain. Some values estimated from graphs. Peptide

Molecular Elastic Tensile Failure Toughness, Ref weight, modulus, strength, strain, % MJ m-3 kPa GPa MPa 7 (G)32 54 56 132 62 7 (GY)12 60 47 45 18 7 (GF)12 59 136 37 36 7 (GFY)8 66 151 85 89 161 (Y1S8)20 62 2.5 96 80 62 161 (A1S8)20 58 4.6 136 80 94 163 (A2S8)14 78 5.3 315 32 72 169 W2 38.0 1.7 67 31 18 170 W4 57.1 2.4 115 37 34 154 N1L-(AQ)24-NR3 134 5 308 54 90 154 N1L-(AQ)12-NR3 76 4 370 110 189 154 (AQ)12-NR3 60 3 383 95 172 154 N1L-(AQ)12-NR3 76 3 251 82 111 154 (AQ)12 48 2 66 38 20 153 96-mer 284.9 21 508 15 153 64-mer 192.8 10 260 4.5 153 32-mer 100.7 8 200 3.5 153 16-mer 54.7 4 80 2.5 Module Origin Sequence G Fl, (GGX)n GGAGGSGGAGGSGGVGGSGGT Y Fl, (GPGGX)n (GPGGSGPGGY)4 F Fl, spacer TIIEDLDITIDGADGPITISEELTISGAGGS (Y1S8)20 MA+Fl [(GPGGY-GPGGS)2-GPSGPG-(A)8]20 (A1S8)20 MA+Fl [(GPGGA-GPGGA)2-GPSGPG-(A)8]20 (A2S8)14 MA+Fl+fhc [(GPGGA)8-GPSGPG-(A)8]14, flanked by N-, C-terminal domains of silk fibroin heavy chain W2 Ac Two tandem 200-residue repeats from A. trifasciata W4 Ac Four tandem 200-residue repeats from A. trifasciata Module N1L

AQ NR3

Sequence (MaSp-2, A. diadematus ADF-3) GQANTPWSSKANADAFINSFISAASNTGSFNMGGRITPSKLQALD MAFASSVAEIAASEGTTGVVNSRFISEIRSLIGMFAQASANDVYAA SAAAPSGVAYQAPAQAQISFTLRGQQPVS GPYGPGASAAAAAAGGYGPGSGQQ|(GPGQQ)5 GAASAAVSVGGYGPQSSSAPVASAAASRLSTNQAALSNTISSVVSQ VSASNPGLSGCDVLGQINYGASAQYTQMVGQSVAQALAG

Module Sequence (MaSp-1, N. clavipes) 16- to 96-mer (SGRGGLGGQGAGMAAAAAMGGAGQGGYGGLGSQGT)n=16,32,64,96


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References (1) Gosline, J.; Lillie, M.; Carrington, E.; Guerette, P.; Ortlepp, C.; Savage, K. Elastic proteins: biological roles and mechanical properties. Philos. Trans. R. Soc. Lond. B: Biol. Sci. 2002, 357, 121-132. (2) Roth, G. A.; Huffman, M. D.; Moran, A. E.; Feigin, V.; Mensah, G. A.; Naghavi, M.; Murray, C. J. Global and regional patterns in cardiovascular mortality from 1990 to 2013. Circulation 2015, 132, 1667-1678. (3) Wise, S. G.; Yeo, G. C.; Hiob, M. A.; Rnjak-Kovacina, J.; Kaplan, D. L.; Ng, M. K.; Weiss, A. S. Tropoelastin: a versatile, bioactive assembly module. Acta Biomater. 2014, 10, 1532-1541. (4) Muiznieks, L. D.; Keeley, F. W. Molecular assembly and mechanical properties of the extracellular matrix: A fibrous protein perspective. Biochim. Biophys. Acta 2013, 1832, 866-875. (5) Murphy, W. L.; McDevitt, T. C.; Engler, A. J. Materials as stem cell regulators. Nat. Mater. 2014, 13, 547-557. (6) Miao, M.; Sitarz, E.; Bellingham, C. M.; Won, E.; Muiznieks, L. D.; Keeley, F. W. Sequence and domain arrangements influence mechanical properties of elastin-like polymeric elastomers. Biopolymers 2013, 99, 392-407. (7) Adrianos, S. L.; Teule, F.; Hinman, M. B.; Jones, J. A.; Weber, W. S.; Yarger, J. L.; Lewis, R. V. Nephila clavipes Flagelliform silk-like GGX motifs contribute to extensibility and spacer motifs contribute to strength in synthetic spider silk fibers. Biomacromolecules 2013, 14, 1751-1760. (8) Qin, G.; Hu, X.; Cebe, P.; Kaplan, D. L. Mechanism of resilin elasticity. Nat. Commun. 2012, 3, 1003. (9) Nova, A.; Keten, S.; Pugno, N. M.; Redaelli, A.; Buehler, M. J. Molecular and nanostructural mechanisms of deformation, strength and toughness of spider silk fibrils. Nano Lett. 2010, 10, 2626-2634. (10) Brooks, A. E.; Nelson, S. R.; Jones, J. A.; Koenig, C.; Hinman, M.; Stricker, S.; Lewis, R. V. Distinct contributions of model MaSp1 and MaSp2 like peptides to the mechanical properties of synthetic major ampullate silk fibers as revealed in silico. Nanotechnol. Sci. Appl. 2008, 1, 9-16. (11) Blackledge, T. A.; Hayashi, C. Y. Silken toolkits: biomechanics of silk fibers spun by the orb web spider Argiope argentata (Fabricius 1775). J. Exp. Biol. 2006, 209, 24522461. (12) Gosline, J. M.; Guerette, P. A.; Ortlepp, C. S.; Savage, K. N. The mechanical design of spider silks: from fibroin sequence to mechanical function. J. Exp. Biol. 1999, 202, 3295-3303. (13) Guerette, P. A.; Ginzinger, D. G.; Weber, B. H.; Gosline, J. M. Silk properties determined by gland-specific expression of a spider fibroin gene family. Science 1996, 272, 112-115. (14) Keeley, F. W. In Evolution of the extracellular matrix, Biology of the extracellular matrix; Keeley, F. W.; Mecham, R. P., Eds.; Springer-Verlag: Berlin, Heidelberg, 2013, p 73-119. (15) Bennet-Clark, H. The first description of resilin. J. Exp. Biol. 2007, 210, 3879-3881.



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(16) Kelly, R. E.; Rice, R. V. Abductin: a rubber-like protein from the internal triangular hinge ligament of pecten. Science 1967, 155, 208-210. (17) Tan, Y.; Hoon, S.; Guerette, P. A.; Wei, W.; Ghadban, A.; Hao, C.; Miserez, A.; Waite, J. H. Infiltration of chitin by protein coacervates defines the squid beak mechanical gradient. Nat. Chem. Biol. 2015, 11, 488-495. (18) Bell, E.; Gosline, J. Mechanical design of mussel byssus: material yield enhances attachment strength. J. Exp. Biol. 1996, 199, 1005-1017. (19) Shewry, P. R.; Halford, N. G.; Belton, P. S.; Tatham, A. S. The structure and properties of gluten: an elastic protein from wheat grain. Philos. Trans. R. Soc. Lond. B: Biol. Sci. 2002, 357, 133-142. (20) Lillie, M. A.; Piscitelli, M. A.; Vogl, A. W.; Gosline, J. M.; Shadwick, R. E. Cardiovascular design in fin whales: high-stiffness arteries protect against adverse pressure gradients at depth. J. Exp. Biol. 2013, 216, 2548-2563. (21) Ding, D.; Guerette, P. A.; Fu, J.; Zhang, L.; Irvine, S. A.; Miserez, A. From Soft Self-Healing Gels to Stiff Films in Suckerin-Based Materials Through Modulation of Crosslink Density and beta-Sheet Content. Adv. Mater. 2015, 27, 3953-3961. (22) Cirulis, J. T.; Keeley, F. W.; James, D. F. Viscoelastic properties and gelation of an elastin-like polypeptide. J. Rheol. 2009, 53, 1215-1228. (23) Li, L.; Teller, S.; Clifton, R. J.; Jia, X.; Kiick, K. L. Tunable mechanical stability and deformation response of a resilin-based elastomer. Biomacromolecules 2011, 12, 23022310. (24) Rauscher, S.; Pomès, R. Structural disorder and protein elasticity. Adv. Exp. Med. Biol. 2012, 725, 159-183. (25) Miserez, A.; Guerette, P. A. Phase transition-induced elasticity of alpha-helical bioelastomeric fibres and networks. Chem. Soc. Rev. 2013, 42, 1973-1995. (26) Tatham, A. S.; Shewry, P. R. Elastomeric proteins: biological roles, structures and mechanisms. Trends Biochem. Sci. 2000, 25, 567-571. (27) Mark, J. E. Rubber elasticity. J. Chem. Educ. 1981, 58, 898-903. (28) Keten, S.; Xu, Z.; Ihle, B.; Buehler, M. J. Nanoconfinement controls stiffness, strength and mechanical toughness of beta-sheet crystals in silk. Nat. Mater. 2010, 9, 359-367. (29) Rauscher, S.; Baud, S.; Miao, M.; Keeley, F. W.; Pomès, R. Proline and glycine control protein self-organization into elastomeric or amyloid fibrils. Structure 2006, 14, 1667-1676. (30) Bochicchio, B.; Pepe, A.; Tamburro, A. M. Investigating by CD the molecular mechanism of elasticity of elastomeric proteins. Chirality 2008, 20, 985-994. (31) Hutchinson, E. G.; Thornton, J. M. A revised set of potentials for beta-turn formation in proteins. Protein Sci. 1994, 3, 2207-2216. (32) Gosline, J. M. The physical properties of elastic tissue. Int. Rev. Connect. Tissue Res. 1976, 7, 211-249. (33) Yeo, G. C.; Keeley, F. W.; Weiss, A. S. Coacervation of tropoelastin. Adv. Colloid Interface Sci. 2011, 167, 94-103. (34) Muiznieks, L. D.; Cirulis, J. T.; van der Horst, A.; Reinhardt, D. P.; Wuite, G. J.; Pomès, R.; Keeley, F. W. Modulated growth, stability and interactions of liquid-like coacervate assemblies of elastin. Matrix Biol. 2014, 36, 39-50.


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(35) Dutta, N. K.; Truong, M. Y.; Mayavan, S.; Choudhury, N. R.; Elvin, C. M.; Kim, M.; Knott, R.; Nairn, K. M.; Hill, A. J. A genetically engineered protein responsive to multiple stimuli. Angew. Chem., Int. Ed. Engl. 2011, 50, 4428-4431. (36) Su, R. S.; Renner, J. N.; Liu, J. C. Synthesis and characterization of recombinant abductin-based proteins. Biomacromolecules 2013, 14, 4301-4308. (37) Exler, J. H.; Hummerich, D.; Scheibel, T. The amphiphilic properties of spider silks are important for spinning. Angew. Chem., Int. Ed. Engl. 2007, 46, 3559-3562. (38) Miller, D. R.; Das, S.; Huang, K. Y.; Han, S.; Israelachvili, J. N.; Waite, J. H. Mussel Coating Protein-Derived Complex Coacervates Mitigate Frictional Surface Damage. ACS Biomater. Sci. Eng. 2015, 1, 1121-1128. (39) Jin, H. J.; Kaplan, D. L. Mechanism of silk processing in insects and spiders. Nature 2003, 424, 1057-1061. (40) Xu, L.; Tremblay, M. L.; Orrell, K. E.; Leclerc, J.; Meng, Q.; Liu, X. Q.; Rainey, J. K. Nanoparticle self-assembly by a highly stable recombinant spider wrapping silk protein subunit. FEBS Lett. 2013, 587, 3273-3280. (41) Ding, D.; Guerette, P. A.; Hoon, S.; Kong, K. W.; Cornvik, T.; Nilsson, M.; Kumar, A.; Lescar, J.; Miserez, A. Biomimetic production of silk-like recombinant squid sucker ring teeth proteins. Biomacromolecules 2014, 15, 3278-3289. (42) Davis, E. C. Stability of elastin in the developing mouse aorta: a quantitative radioautographic study. Histochemistry 1993, 100, 17-26. (43) Shapiro, S. D.; Endicott, S. K.; Province, M. A.; Pierce, J. A.; Campbell, E. J. Marked longevity of human lung parenchymal elastic fibers deduced from prevalence of D-aspartate and nuclear weapons-related radiocarbon. J. Clin. Invest. 1991, 87, 18281834. (44) Vrhovski, B.; Weiss, A. S. Biochemistry of tropoelastin. Eur. J. Biochem. 1998, 258, 1-18. (45) Urry, D. W.; Long, M. M.; Cox, B. A.; Ohnishi, T.; Mitchell, L. W.; Jacobs, M. The synthetic polypentapeptide of tropoelastin coacervates and forms filamentous aggregates. Biochem. Biophys. Acta 1974, 371, 597 - 602. (46) Urry, D. W.; Luan, C. H.; Parker, T. M.; Gowda, D. C.; Prasad, K. U.; Reid, M. C.; Safavy, A. Temperature of Polypeptide Inverse Temperature Transition Depends on Mean Residue Hydrophobicity. J. Am. Chem. Soc. 1991, 113, 4346-4348. (47) MacEwan, S. R.; Chilkoti, A. Applications of elastin-like polypeptides in drug delivery. J. Controlled Release 2014, 190, 314-330. (48) Rodriguez-Cabello, J. C.; Arias, F. J.; Rodrigo, M. A.; Girotti, A. Elastin-like polypeptides in drug delivery. Adv. Drug Deliv. Rev. 2016, 97, 85-100. (49) Wright, E. R.; McMillan, R. A.; Cooper, A.; Apkarian, R. P.; Conticello, V. P. Thermoplastic elastomer hydrogels via self-assembly of an elastin-mimetic triblock polypeptide. Adv. Funct. Mater. 2002, 12, 149-154. (50) Wright, E. R.; Conticello, V. P. Self-assembly of block copolymers derived from elastin-mimetic polypeptide sequences. Adv. Drug Deliv. Rev. 2002, 54, 1057-1073. (51) Wu, X.; Sallach, R. E.; Caves, J. M.; Conticello, V. P.; Chaikof, E. L. Deformation responses of a physically cross-linked high molecular weight elastin-like protein polymer. Biomacromolecules 2008, 9, 1787-1794. (52) Nowatzki, P. J.; Tirrell, D. A. Physical properties of artificial extracellular matrix protein films prepared by isocyanate crosslinking. Biomaterials 2004, 25, 1261-1267.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(53) Di Zio, K.; Tirrell, D. A. Mechanical properties of artificial protein matrices engineered for control of cell and tissue behavior. Macromolecules 2003, 36, 1553-1558. (54) Sallach, R. E.; Cui, W.; Wen, J.; Martinez, A.; Conticello, V. P.; Chaikof, E. L. Elastin-mimetic protein polymers capable of physical and chemical crosslinking. Biomaterials 2009, 30, 409-422. (55) Wu, X.; Sallach, R.; Haller, C. A.; Caves, J. A.; Nagapudi, K.; Conticello, V. P.; Levenston, M. E.; Chaikof, E. L. Alterations in physical cross-linking modulate mechanical properties of two-phase protein polymer networks. Biomacromolecules 2005, 6, 3037-3044. (56) Nagapudi, K.; Brinkman, W. T.; Thomas, B. S.; Park, J. O.; Srinivasarao, M.; Wright, E.; Conticello, V. P.; Chaikof, E. L. Viscoelastic and mechanical behavior of recombinant protein elastomers. Biomaterials 2005, 26, 4695-4706. (57) Nagapudi, K.; Brinkman, W. T.; Leisen, J.; Thomas, B. S.; Wright, E. R.; Haller, C. A.; Wu, X.; Apkarian, R. P.; Conticello, V. P.; Chaikof, E. L. Protein-Based Thermoplastic Elastomers. Macromolecules 2005, 38, 345-354. (58) Martinez, A. W.; Caves, J. M.; Ravi, S.; Li, W.; Chaikof, E. L. Effects of crosslinking on the mechanical properties, drug release and cytocompatibility of protein polymers. Acta Biomater. 2014, 10, 26-33. (59) Bellingham, C. M.; Lillie, M. A.; Gosline, J. M.; Wright, G. M.; Starcher, B. C.; Bailey, A. J.; Woodhouse, K. A.; Keeley, F. W. Recombinant human elastin polypeptides self-assemble into biomaterials with elastin-like properties. Biopolymers 2003, 70, 445455. (60) Bellingham, C. M.; Woodhouse, K. A.; Robson, P.; Rothstein, S. J.; Keeley, F. W. Self-aggregation characteristics of recombinantly expressed human elastin polypeptides. Biochim. Biophys. Acta 2001, 1550, 6-19. (61) Tamburro, A. M.; Bochicchio, B.; Pepe, A. Dissection of human tropoelastin: exonby-exon chemical synthesis and related conformational studies. Biochemistry 2003, 42, 13347-13362. (62) Muiznieks, L. D.; Weiss, A. S.; Keeley, F. W. Structural disorder and dynamics of elastin. Biochem. Cell Biol. 2010, 88, 239-250. (63) Reichheld, S. E.; Muiznieks, L. D.; Stahl, R.; Simonetti, K.; Sharpe, S.; Keeley, F. W. Conformational transitions of the cross-linking domains of elastin during selfassembly. J. Biol. Chem. 2014, 289, 10057-10068. (64) Muiznieks, L. D.; Reichheld, S. E.; Sitarz, E. E.; Miao, M.; Keeley, F. W. Prolinepoor hydrophobic domains modulate the assembly and material properties of polymeric elastin. Biopolymers 2015, 103, 563-573. (65) He, D.; Miao, M.; Sitarz, E. E.; Muiznieks, L. D.; Reichheld, S.; Stahl, R. J.; Keeley, F. W.; Parkinson, J. Polymorphisms in the human tropoelastin gene modify in vitro selfassembly and mechanical properties of elastin-like polypeptides. PLoS One 2012, 7, e46130. (66) Vieth, S.; Bellingham, C. M.; Keeley, F. W.; Hodge, S. M.; Rousseau, D. Microstructural and tensile properties of elastin-based polypeptides crosslinked with genipin and pyrroloquinoline quinone. Biopolymers 2007, 85, 199-206. (67) Miao, M.; Cirulis, J. T.; Lee, S.; Keeley, F. W. Structural determinants of crosslinking and hydrophobic domains for self-assembly of elastin-like polypeptides. Biochemistry 2005, 44, 14367-14375.


Page 50 of 64

Page 51 of 64

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(68) Hrabchak, C.; Rouleau, J.; Moss, I.; Woodhouse, K.; Akens, M.; Bellingham, C.; Keeley, F.; Dennis, M.; Yee, A. Assessment of biocompatibility and initial evaluation of genipin cross-linked elastin-like polypeptides in the treatment of an osteochondral knee defect in rabbits. Acta Biomater. 2010, 6, 2108-2115. (69) Martin, S. L.; Vrhovski, B.; Weiss, A. S. Total synthesis and expression in Escherichia coli of a gene encoding human tropoelastin. Gene 1995, 154, 159-166. (70) Baldock, C.; Oberhauser, A. F.; Ma, L.; Lammie, D.; Siegler, V.; Mithieux, S. M.; Tu, Y.; Chow, J. Y.; Suleman, F.; Malfois, M.; Rogers, S.; Guo, L.; Irving, T. C.; Wess, T. J.; Weiss, A. S. Shape of tropoelastin, the highly extensible protein that controls human tissue elasticity. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 4322-4327. (71) Muiznieks, L. D.; Weiss, A. S. Flexibility in the solution structure of human tropoelastin. Biochemistry 2007, 46, 8196-8205. (72) Pometun, M. S.; Chekmenev, E. Y.; Wittebort, R. J. Quantitative observation of backbone disorder in native elastin. J. Biol. Chem. 2004, 279, 7982-7987. (73) Perry, A.; Stypa, M. P.; Foster, J. A.; Kumashiro, K. K. Observation of the glycines in elastin using 13C and 15N solid-state NMR spectroscopy and isotopoc labeling. J. Am. Chem. Soc. 2002, 124, 6832 - 6833. (74) Torchia, D. A.; Piez, K. A. Mobility of elastin chains as determined by 13C nuclear magnetic resonance. J. Mol. Biol. 1973, 76, 419 - 424. (75) Yeo, G. C.; Tarakanova, A.; Baldock, C.; Wise, S. G.; Buehler, M. J.; Weiss, A. S. Subtle balance of tropoelastin molecular shape and flexibility regulates dynamics and hierarchical assembly. Sci. Adv. 2016, 2, e1501145. (76) Muiznieks, L. D.; Miao, M.; Sitarz, E. E.; Keeley, F. W. Contribution of domain 30 of tropoelastin to elastic fiber formation and material elasticity. Biopolymers 2016, 105, 267-275. (77) Tu, Y.; Mithieux, S. M.; Annabi, N.; Boughton, E. A.; Weiss, A. S. Synthetic elastin hydrogels that are coblended with heparin display substantial swelling, increased porosity, and improved cell penetration. J. Biomed. Mater. Res. A 2010, 95, 1215-1222. (78) Toonkool, P.; Jensen, S. A.; Maxwell, A. L.; Weiss, A. S. Hydrophobic domains of human tropoelastin interact in a context-dependent manner. J. Biol. Chem. 2001, 276, 44575-44580. (79) Mithieux, S. M.; Rasko, J. E.; Weiss, A. S. Synthetic elastin hydrogels derived from massive elastic assemblies of self-organized human protein monomers. Biomaterials 2004, 25, 4921-4927. (80) Annabi, N.; Mithieux, S. M.; Weiss, A. S.; Dehghani, F. Cross-linked open-pore elastic hydrogels based on tropoelastin, elastin and high pressure CO2. Biomaterials 2010, 31, 1655-1665. (81) Mithieux, S. M.; Tu, Y.; Korkmaz, E.; Braet, F.; Weiss, A. S. In situ polymerization of tropoelastin in the absence of chemical cross-linking. Biomaterials 2009, 30, 431-435. (82) Yeo, G. C.; Aghaei-Ghareh-Bolagh, B.; Brackenreg, E. P.; Hiob, M. A.; Lee, P.; Weiss, A. S. Fabricated Elastin. Adv. Healthc. Mater. 2015, 4, 2530-2556. (83) Hu, X.; Wang, X.; Rnjak, J.; Weiss, A. S.; Kaplan, D. L. Biomaterials derived from silk-tropoelastin protein systems. Biomaterials 2010, 31, 8121-8131. (84) Liu, H.; Wise, S. G.; Rnjak-Kovacina, J.; Kaplan, D. L.; Bilek, M. M.; Weiss, A. S.; Fei, J.; Bao, S. Biocompatibility of silk-tropoelastin protein polymers. Biomaterials 2014, 35, 5138-5147.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(85) Rnjak-Kovacina, J.; Wise, S. G.; Li, Z.; Maitz, P. K.; Young, C. J.; Wang, Y.; Weiss, A. S. Electrospun synthetic human elastin:collagen composite scaffolds for dermal tissue engineering. Acta Biomater. 2012, 8, 3714-3722. (86) Wise, S. G.; Byrom, M. J.; Waterhouse, A.; Bannon, P. G.; Weiss, A. S.; Ng, M. K. A multilayered synthetic human elastin/polycaprolactone hybrid vascular graft with tailored mechanical properties. Acta Biomater. 2011, 7, 295-303. (87) Wise, S. G.; Liu, H.; Yeo, G. C.; Michael, P. L.; Chan, A. H.; Ngo, A. K.; Bilek, M. M.; Bao, S.; Weiss, A. S. Blended Polyurethane and Tropoelastin as a Novel Class of Biologically Interactive Elastomer. Tissue Eng., Part A 2016, 22, 524-533. (88) Elvin, C. M.; Carr, A. G.; Huson, M. G.; Maxwell, J. M.; Pearson, R. D.; Vuocolo, T.; Liyou, N. E.; Wong, D. C.; Merritt, D. J.; Dixon, N. E. Synthesis and properties of crosslinked recombinant pro-resilin. Nature 2005, 437, 999-1002. (89) Weis-Fogh, T. A rubber-like protein in insect cuticle. J. Exp. Biol. 1960, 37, 889907. (90) Chen, J.; Chen, J.; Chou, Y. On the natural frequencies and mode shapes of dragonfly wings. J. Sound Vib. 2008, 313, 643-654. (91) Bennet-Clark, H. Tymbal mechanics and the control of song frequency in the cicada Cyclochila australasiae. J. Exp. Biol. 1997, 200, 1681-1694. (92) Bennet-Clark, H. C.; Lucey, E. C. The jump of the flea: a study of the energetics and a model of the mechanism. J. Exp. Biol. 1967, 47, 59-67. (93) Ardell, D. H.; Andersen, S. O. Tentative identification of a resilin gene in Drosophila melanogaster. Insect Biochem. Mol. Biol. 2001, 31, 965-970. (94) Lyons, R. E.; Nairn, K. M.; Huson, M. G.; Kim, M.; Dumsday, G.; Elvin, C. M. Comparisons of recombinant resilin-like proteins: repetitive domains are sufficient to confer resilin-like properties. Biomacromolecules 2009, 10, 3009-3014. (95) Qin, G.; Rivkin, A.; Lapidot, S.; Hu, X.; Preis, I.; Arinus, S. B.; Dgany, O.; Shoseyov, O.; Kaplan, D. L. Recombinant exon-encoded resilins for elastomeric biomaterials. Biomaterials 2011, 32, 9231-9243. (96) Qin, G.; Lapidot, S.; Numata, K.; Hu, X.; Meirovitch, S.; Dekel, M.; Podoler, I.; Shoseyov, O.; Kaplan, D. L. Expression, cross-linking, and characterization of recombinant chitin binding resilin. Biomacromolecules 2009, 10, 3227-3234. (97) Nairn, K. M.; Lyons, R. E.; Mulder, R. J.; Mudie, S. T.; Cookson, D. J.; Lesieur, E.; Kim, M.; Lau, D.; Scholes, F. H.; Elvin, C. M. A synthetic resilin is largely unstructured. Biophys. J. 2008, 95, 3358-3365. (98) Tamburro, A. M.; Panariello, S.; Santopietro, V.; Bracalello, A.; Bochicchio, B.; Pepe, A. Molecular and supramolecular structural studies on significant repetitive sequences of resilin. ChemBioChem 2010, 11, 83-93. (99) Balu, R.; Knott, R.; Cowieson, N. P.; Elvin, C. M.; Hill, A. J.; Choudhury, N. R.; Dutta, N. K. Structural ensembles reveal intrinsic disorder for the multi-stimuli responsive bio-mimetic protein Rec1-resilin. Sci. Rep. 2015, 5, 10896. (100) Andersen, S. O. The Cross-Links in Resilin Identified as Dityrosine and Trityrosine. Biochim. Biophys. Acta 1964, 93, 213-215. (101) Neville, A. C. Biology of the arthropod cuticle; Springer-Verlag: Berlin, Heidelberg, 1975. (102) Andersen, S. O.; Weis-Fogh, T. Resilin. A rubber-like protein in arthropod cuticle. Adv. Insect Physiol. 1964, 2, 1-65.


Page 52 of 64

Page 53 of 64

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(103) Lv, S.; Dudek, D. M.; Cao, Y.; Balamurali, M. M.; Gosline, J.; Li, H. Designed biomaterials to mimic the mechanical properties of muscles. Nature 2010, 465, 69-73. (104) Bracalello, A.; Santopietro, V.; Vassalli, M.; Marletta, G.; Del Gaudio, R.; Bochicchio, B.; Pepe, A. Design and production of a chimeric resilin-, elastin-, and collagen-like engineered polypeptide. Biomacromolecules 2011, 12, 2957-2965. (105) Charati, M. B.; Ifkovits, J. L.; Burdick, J. A.; Linhardt, J. G.; Kiick, K. L. Hydrophilic elastomeric biomaterials based on resilin-like polypeptides. Soft Matter 2009, 5, 3412-3416. (106) Li, L.; Mahara, A.; Tong, Z.; Levenson, E. A.; McGann, C. L.; Jia, X.; Yamaoka, T.; Kiick, K. L. Recombinant Resilin-Based Bioelastomers for Regenerative Medicine Applications. Adv. Healthc. Mater. 2016, 5, 266-275. (107) Li, L.; Kiick, K. L. Transient dynamic mechanical properties of resilin-based elastomeric hydrogels. Front. Chem. 2014, 2, 21. (108) Li, L.; Tong, Z.; Jia, X.; Kiick, K. L. Resilin-Like Polypeptide Hydrogels Engineered for Versatile Biological Functions. Soft Matter 2013, 9, 665-673. (109) Alexander, R. M. Rubber-like properties of the inner hinge-ligament of Pectinidae. J. Exp. Biol. 1966, 44, 119-130. (110) Denny, M.; Miller, L. Jet propulsion in the cold: Mechanics of swimming in the Antarctic scallop Adamussium colbecki. J. Exp. Biol. 2006, 209, 4503-4514. (111) Cao, Q.; Wang, Y.; Bayley, H. Sequence of abductin, the molluscan 'rubber' protein. Curr. Biol. 1997, 7, R677-678. (112) Bochicchio, B.; Pepe, A.; Tamburro, A. M. Circular dichroism studies on repeating polypeptide sequences of abductin. Chirality 2005, 17, 364-372. (113) Bochicchio, B.; Jimenez-Oronoz, F.; Pepe, A.; Blanco, M.; Sandberg, L. B.; Tamburro, A. M. Synthesis of and structural studies on repeating sequences of abductin. Macromol. Biosci. 2005, 5, 502-511. (114) Guerette, P. A.; Hoon, S.; Ding, D.; Amini, S.; Masic, A.; Ravi, V.; Venkatesh, B.; Weaver, J. C.; Miserez, A. Nanoconfined beta-sheets mechanically reinforce the suprabiomolecular network of robust squid Sucker Ring Teeth. ACS Nano 2014, 8, 7170-7179. (115) Omenetto, F. G.; Kaplan, D. L. New opportunities for an ancient material. Science 2010, 329, 528-531. (116) Weisman, S.; Haritos, V. S.; Church, J. S.; Huson, M. G.; Mudie, S. T.; Rodgers, A. J.; Dumsday, G. J.; Sutherland, T. D. Honeybee silk: recombinant protein production, assembly and fiber spinning. Biomaterials 2010, 31, 2695-2700. (117) Vollrath, F.; Knight, D. P. Liquid crystalline spinning of spider silk. Nature 2001, 410, 541-548. (118) Dicko, C.; Kenney, J. M.; Vollrath, F. Beta-silks: enhancing and controlling aggregation. Adv. Protein Chem. 2006, 73, 17-53. (119) Tokareva, O.; Jacobsen, M.; Buehler, M.; Wong, J.; Kaplan, D. L. Structurefunction-property-design interplay in biopolymers: spider silk. Acta Biomater. 2014, 10, 1612-1626. (120) Hardy, J. G.; Römer, L. M.; Scheibel, T. R. Polymeric materials based on silk proteins. Polymer 2008, 49, 4309-4327. (121) Zhou, C. Z.; Confalonieri, F.; Medina, N.; Zivanovic, Y.; Esnault, C.; Yang, T.; Jacquet, M.; Janin, J.; Duguet, M.; Perasso, R.; Li, Z. G. Fine organization of Bombyx mori fibroin heavy chain gene. Nucleic Acids Res. 2000, 28, 2413-2419.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(122) Hu, X.; Kaplan, D.; Cebe, P. Determining Beta-Sheet Crystallinity in Fibrous Proteins by Thermal Analysis and Infrared Spectroscopy. Macromolecules 2006, 39, 6161-6170. (123) Coburn, J. M.; Na, E.; Kaplan, D. L. Modulation of vincristine and doxorubicin binding and release from silk films. J. Controlled Release 2015, 220, 229-238. (124) Lawrence, B. D.; Cronin-Golomb, M.; Georgakoudi, I.; Kaplan, D. L.; Omenetto, F. G. Bioactive silk protein biomaterial systems for optical devices. Biomacromolecules 2008, 9, 1214-1220. (125) Lawrence, B. D.; Marchant, J. K.; Pindrus, M. A.; Omenetto, F. G.; Kaplan, D. L. Silk film biomaterials for cornea tissue engineering. Biomaterials 2009, 30, 1299-1308. (126) Hopkins, A. M.; De Laporte, L.; Tortelli, F.; Spedden, E.; Staii, C.; Atherton, T. J.; Hubbell, J. A.; Kaplan, D. L. Silk Hydrogels as Soft Substrates for Neural Tissue Engineering. Adv. Funct. Mater. 2013, 23, 5140-5149. (127) Li, C.; Vepari, C.; Jin, H. J.; Kim, H. J.; Kaplan, D. L. Electrospun silk-BMP-2 scaffolds for bone tissue engineering. Biomaterials 2006, 27, 3115-3124. (128) Altman, G. H.; Horan, R. L.; Lu, H. H.; Moreau, J.; Martin, I.; Richmond, J. C.; Kaplan, D. L. Silk matrix for tissue engineered anterior cruciate ligaments. Biomaterials 2002, 23, 4131-4141. (129) Altman, G. H.; Diaz, F.; Jakuba, C.; Calabro, T.; Horan, R. L.; Chen, J.; Lu, H.; Richmond, J.; Kaplan, D. L. Silk-based biomaterials. Biomaterials 2003, 24, 401-416. (130) Xia, X.-X.; Xu, Q.; Hu, X.; Qin, G.; Kaplan, D. L. Tunable self-assembly of genetically engineered silk--elastin-like protein polymers. Biomacromolecules 2011, 12, 3844-3850. (131) Ohgo, K.; Kurano, T. L.; Kumashiro, K. K.; Asakura, T. Structure of the model peptides of Bombyx mori silk-elastin like protein studied with solid state NMR. Biomacromolecules 2004, 5, 744-750. (132) Wang, Q.; Xia, X.; Huang, W.; Lin, Y.; Xu, Q.; Kaplan, D. L. High Throughput Screening of Dynamic Silk-Elastin-Like Protein Biomaterials. Adv. Funct. Mater. 2014, 24, 4303-4310. (133) Work, R. W. Dimensions, birefringence and force–elongation behaviour of major and minor ampullate silk fibres from orb-web spinning spiders. Text. Res. J. 1977, 47, 650-662. (134) Xu, M.; Lewis, R. V. Structure of a protein superfiber: spider dragline silk. Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 7120-7124. (135) Bykov, S.; Asher, S. Raman studies of solution polyglycine conformations. J. Phys. Chem. B 2010, 114, 6636-6641. (136) van Beek, J. D.; Hess, S.; Vollrath, F.; Meier, B. H. The molecular structure of spider dragline silk: folding and orientation of the protein backbone. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 10266-10271. (137) Holland, G. P.; Creager, M. S.; Jenkins, J. E.; Lewis, R. V.; Yarger, J. L. Determining secondary structure in spider dragline silk by carbon-carbon correlation solid-state NMR spectroscopy. J. Am. Chem. Soc. 2008, 130, 9871-9877. (138) Kümmerlen, J.; van Beek, J. D.; Vollrath, F.; Meier, B. H. Local structure in spider dragline silk investigated by two-dimensional spin-diffusion nuclear magnetic resonance. Macromolecules 1996, 29, 2920-2928.


Page 54 of 64

Page 55 of 64

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(139) Hinman, M. B.; Lewis, R. V. Isolation of a clone encoding a second dragline silk fibroin. Nephila clavipes dragline silk is a two-protein fiber. J. Biol. Chem. 1992, 267, 19320-19324. (140) Jenkins, J. E.; Creager, M. S.; Butler, E. B.; Lewis, R. V.; Yarger, J. L.; Holland, G. P. Solid-state NMR evidence for elastin-like beta-turn structure in spider dragline silk. Chem. Commun. (Cambridge, U. K.) 2012, 46, 6714-6716. (141) Simmons, A. H.; Michal, C. A.; Jelinski, L. W. Molecular orientation and twocomponent nature of the crystalline fraction of spider dragline silk. Science 1996, 271, 84-87. (142) Rousseau, M. E.; Lefevre, T.; Beaulieu, L.; Asakura, T.; Pezolet, M. Study of protein conformation and orientation in silkworm and spider silk fibers using Raman microspectroscopy. Biomacromolecules 2004, 5, 2247-2257. (143) Savage, K. N.; Gosline, J. M. The role of proline in the elastic mechanism of hydrated spider silks. J. Exp. Biol. 2008, 211, 1948-1957. (144) Liu, Y.; Sponner, A.; Porter, D.; Vollrath, F. Proline and processing of spider silks. Biomacromolecules 2008, 9, 116-121. (145) Creager, M. S.; Jenkins, J. E.; Thagard-Yeaman, L. A.; Brooks, A. E.; Jones, J. A.; Lewis, R. V.; Holland, G. P.; Yarger, J. L. Solid-state NMR comparison of various spiders' dragline silk fiber. Biomacromolecules 2012, 11, 2039-2043. (146) Gaines, W. A.; Sehorn, M. G.; Marcotte, W. R., Jr. Spidroin N-terminal domain promotes a pH-dependent association of silk proteins during self-assembly. J. Biol. Chem. 2010, 285, 40745-40753. (147) Huemmerich, D.; Helsen, C. W.; Quedzuweit, S.; Oschmann, J.; Rudolph, R.; Scheibel, T. Primary structure elements of spider dragline silks and their contribution to protein solubility. Biochemistry 2004, 43, 13604-13612. (148) Hagn, F.; Eisoldt, L.; Hardy, J. G.; Vendrely, C.; Coles, M.; Scheibel, T.; Kessler, H. A conserved spider silk domain acts as a molecular switch that controls fibre assembly. Nature 2010, 465, 239-242. (149) Rabotyagova, O. S.; Cebe, P.; Kaplan, D. L. Role of polyalanine domains in betasheet formation in spider silk block copolymers. Macromol. Biosci. 2010, 10, 49-59. (150) Stark, M.; Grip, S.; Rising, A.; Hedhammar, M.; Engstrom, W.; Hjalm, G.; Johansson, J. Macroscopic fibers self-assembled from recombinant miniature spider silk proteins. Biomacromolecules 2007, 8, 1695-1701. (151) Brooks, A. E.; Stricker, S. M.; Joshi, S. B.; Kamerzell, T. J.; Middaugh, C. R.; Lewis, R. V. Properties of synthetic spider silk fibers based on Argiope aurantia MaSp2. Biomacromolecules 2008, 9, 1506-1510. (152) Grip, S.; Johansson, J.; Hedhammar, M. Engineered disulfides improve mechanical properties of recombinant spider silk. Protein Sci. 2009, 18, 1012-1022. (153) Xia, X. X.; Qian, Z. G.; Ki, C. S.; Park, Y. H.; Kaplan, D. L.; Lee, S. Y. Nativesized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 14059-14063. (154) Heidebrecht, A.; Eisoldt, L.; Diehl, J.; Schmidt, A.; Geffers, M.; Lang, G.; Scheibel, T. Biomimetic fibers made of recombinant spidroins with the same toughness as natural spider silk. Adv. Mater. 2015, 27, 2189-2194. (155) Cranford, S. W.; Tarakanova, A.; Pugno, N. M.; Buehler, M. J. Nonlinear material behaviour of spider silk yields robust webs. Nature 2012, 482, 72-76.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(156) Hayashi, C. Y.; Lewis, R. V. Evidence from flagelliform silk cDNA for the structural basis of elasticity and modular nature of spider silks. J. Mol. Biol. 1998, 275, 773-784. (157) Hayashi, C. Y.; Lewis, R. V. Molecular architecture and evolution of a modular spider silk protein gene. Science 2000, 287, 1477-1479. (158) Zhou, Y.; Wu, S.; Conticello, V. P. Genetically directed synthesis and spectroscopic analysis of a protein polymer derived from a flagelliform silk sequence. Biomacromolecules 2001, 2, 111-125. (159) Ohgo, K.; Kawase, T.; Ashida, J.; Asakura, T. Solid-state NMR analysis of a peptide (Gly-Pro-Gly-Gly-Ala)6-Gly derived from a flagelliform silk sequence of Nephila clavipes. Biomacromolecules 2006, 7, 1210-1214. (160) Rousseau, M. E.; Lefevre, T.; Pezolet, M. Conformation and orientation of proteins in various types of silk fibers produced by Nephila clavipes spiders. Biomacromolecules 2009, 10, 2945-2953. (161) Teule, F.; Addison, B.; Cooper, A. R.; Ayon, J.; Henning, R. W.; Benmore, C. J.; Holland, G. P.; Yarger, J. L.; Lewis, R. V. Combining flagelliform and dragline spider silk motifs to produce tunable synthetic biopolymer fibers. Biopolymers 2012, 97, 418431. (162) Teulé, F.; Furin, W. A.; Cooper, A. R.; Duncan, J. R.; Lewis, R. V. Modifications of spider silk sequences in an attempt to control the mechanical properties of the synthetic fibers. J. Mater. Sci. 2007, 42, 8974-8985. (163) Teule, F.; Miao, Y. G.; Sohn, B. H.; Kim, Y. S.; Hull, J. J.; Fraser, M. J., Jr.; Lewis, R. V.; Jarvis, D. L. Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 923-928. (164) Hayashi, C. Y.; Blackledge, T. A.; Lewis, R. V. Molecular and mechanical characterization of aciniform silk: uniformity of iterated sequence modules in a novel member of the spider silk fibroin gene family. Mol. Biol. Evol. 2004, 21, 1950-1959. (165) Chaw, R. C.; Zhao, Y.; Wei, J.; Ayoub, N. A.; Allen, R.; Atrushi, K.; Hayashi, C. Y. Intragenic homogenization and multiple copies of prey-wrapping silk genes in Argiope garden spiders. BMC Evol. Biol. 2014, 14, 31. (166) Vasanthavada, K.; Hu, X.; Falick, A. M.; La Mattina, C.; Moore, A. M.; Jones, P. R.; Yee, R.; Reza, R.; Tuton, T.; Vierra, C. Aciniform spidroin, a constituent of egg case sacs and wrapping silk fibers from the black widow spider Latrodectus hesperus. J. Biol. Chem. 2007, 282, 35088-35097. (167) Ayoub, N. A.; Garb, J. E.; Kuelbs, A.; Hayashi, C. Y. Ancient properties of spider silks revealed by the complete gene sequence of the prey-wrapping silk protein (AcSp1). Mol. Biol. Evol. 2013, 30, 589-601. (168) Xu, L.; Tremblay, M. L.; Meng, Q.; Liu, X. Q.; Rainey, J. K. 1H, 13C and 15N NMR assignments of the aciniform spidroin (AcSp1) repetitive domain of Argiope trifasciata wrapping silk. Biomol. NMR Assign. 2012, 6, 147-151. (169) Tremblay, M. L.; Xu, L.; Lefevre, T.; Sarker, M.; Orrell, K. E.; Leclerc, J.; Meng, Q.; Pezolet, M.; Auger, M.; Liu, X. Q.; Rainey, J. K. Spider wrapping silk fibre architecture arising from its modular soluble protein precursor. Sci. Rep. 2015, 5, 11502. (170) Xu, L.; Rainey, J. K.; Meng, Q.; Liu, X. Q. Recombinant minimalist spider wrapping silk proteins capable of native-like fiber formation. PLoS One 2012, 7, e50227.


Page 56 of 64

Page 57 of 64

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(171) Muiznieks, L. D.; Keeley, F. W. Phase separation and mechanical properties of an elastomeric biomaterial from spider wrapping silk and elastin block copolymers. Biopolymers 2016, 105, 693-703. (172) Miserez, A.; Weaver, J. C.; Pedersen, P. B.; Schneeberk, T.; Hanlon, R. T.; Kisailus, D.; Birkedal, H. Microstructural and Biochemical Characterization of the Nanoporous Sucker Rings from Dosidicus gigas. Adv. Mater. 2009, 21, 401-406. (173) Park, S.; Wang, D. H.; Zhang, D.; Romberg, E.; Arola, D. Mechanical properties of human enamel as a function of age and location in the tooth. J. Mater. Sci.: Mater. Med. 2008, 19, 2317-2324. (174) Latza, V.; Guerette, P. A.; Ding, D.; Amini, S.; Kumar, A.; Schmidt, I.; Keating, S.; Oxman, N.; Weaver, J. C.; Fratzl, P.; Miserez, A.; Masic, A. Multi-scale thermal stability of a hard thermoplastic protein-based material. Nat. Commun. 2015, 6, 8313. (175) Guerette, P. A.; Hoon, S.; Seow, Y.; Raida, M.; Masic, A.; Wong, F. T.; Ho, V. H.; Kong, K. W.; Demirel, M. C.; Pena-Francesch, A.; Amini, S.; Tay, G. Z.; Ding, D.; Miserez, A. Accelerating the design of biomimetic materials by integrating RNA-seq with proteomics and materials science. Nat. Biotechnol. 2013, 31, 908-915. (176) Robson, P.; Wright, G. M.; Sitarz, E.; Maiti, A.; Rawat, M.; Youson, J. H.; Keeley, F. W. Characterization of lamprin, an unusual matrix protein from lamprey cartilage. Implications for evolution, structure, and assembly of elastin and other fibrillar proteins. J. Biol. Chem. 1993, 268, 1440-1447. (177) Bai, X.; Mamidala, P.; Rajarapu, S. P.; Jones, S. C.; Mittapalli, O. Transcriptomics of the bed bug (Cimex lectularius). PLoS One 2011, 6, e16336. (178) Tamburro, A. M.; Pepe, A.; Bochicchio, B.; Quaglino, D.; Ronchetti, I. P. Supramolecular amyloid-like assembly of the polypeptide sequence coded by exon 30 of human tropoelastin. J. Biol. Chem. 2005, 280, 2682-2690. (179) Flamia, R.; Zhdan, P. A.; Martino, M.; Castle, J. E.; Tamburro, A. M. AFM study of the elastin-like biopolymer poly(ValGlyGlyValGly). Biomacromolecules 2004, 5, 1511-1518. (180) Kumashiro, K. K.; Kurano, T. L.; Niemczura, W. P.; Martino, M.; Tamburro, A. M. 13C CPMAS NMR studies of the elastin-like polypeptide (LGGVG)n. Biopolymers 2003, 70, 221 - 226. (181) Bochicchio, B.; Pepe, A.; Tamburro, A. M. On (GGLGY) synthetic repeating sequences of lamprin and analogous sequences. Matrix Biol. 2001, 20, 243-250. (182) Coyne, K. J.; Waite, J. H. In search of molecular dovetails in mussel byssus: from the threads to the stem. J. Exp. Biol. 2000, 203, 1425-1431. (183) Lu, H. H.; Thomopoulos, S. Functional attachment of soft tissues to bone: development, healing, and tissue engineering. Annu. Rev. Biomed. Eng. 2013, 15, 201226. (184) Waite, J. H.; Vaccaro, E.; Sun, C.; Lucas, J. M. Elastomeric gradients: a hedge against stress concentration in marine holdfasts? Philos. Trans. R. Soc. Lond. B: Biol. Sci. 2002, 357, 143-153. (185) Qin, X. X.; Coyne, K. J.; Waite, J. H. Tough tendons. Mussel byssus has collagen with silk-like domains. J. Biol. Chem. 1997, 272, 32623-32627. (186) Coyne, K. J.; Qin, X. X.; Waite, J. H. Extensible collagen in mussel byssus: a natural block copolymer. Science 1997, 277, 1830-1832.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(187) Waite, J. H.; Qin, X. X.; Coyne, K. J. The peculiar collagens of mussel byssus. Matrix Biol. 1998, 17, 93-106. (188) Qin, X.; Waite, J. H. Exotic collagen gradients in the byssus of the mussel Mytilus edulis. J. Exp. Biol. 1995, 198, 633-644. (189) Hagenau, A.; Scheidt, H. A.; Serpell, L.; Huster, D.; Scheibel, T. Structural analysis of proteinaceous components in Byssal threads of the mussel Mytilus galloprovincialis. Macromol. Biosci. 2009, 9, 162-168. (190) Hagenau, A.; Suhre, M. H.; Scheibel, T. R. Nature as a blueprint for polymer material concepts: Protein fiber-reinforced composites as holdfasts of mussels. Prog. Polym. Sci. 2014, 39, 1564-1583. (191) Hagenau, A.; Papadopoulos, P.; Kremer, F.; Scheibel, T. Mussel collagen molecules with silk-like domains as load-bearing elements in distal byssal threads. J. Struct. Biol. 2011, 175, 339-347. (192) Qin, Z.; Buehler, M. J. Impact tolerance in mussel thread networks by heterogeneous material distribution. Nat. Commun. 2013, 4, 2187. (193) Claussen, K. U.; Lintz, E. S.; Giesa, R.; Schmidt, H. W.; Scheibel, T. Protein gradient films of fibroin and gelatine. Macromol. Biosci. 2013, 13, 1396-1403. (194) Miserez, A.; Schneberk, T.; Sun, C.; Zok, F. W.; Waite, J. H. The transition from stiff to compliant materials in squid beaks. Science 2008, 319, 1816-1819. (195) Miserez, A.; Rubin, D.; Waite, J. H. Cross-linking chemistry of squid beak. J. Biol. Chem. 2010, 285, 38115-38124. (196) Shoulders, M. D.; Raines, R. T. Collagen structure and stability. Annu. Rev. Biochem. 2009, 78, 929-958. (197) Fratzl, P.; Misof, K.; Zizak, I.; Rapp, G.; Amenitsch, H.; Bernstorff, S. Fibrillar structure and mechanical properties of collagen. J. Struct. Biol. 1998, 122, 119-122. (198) Depalle, B.; Qin, Z.; Shefelbine, S. J.; Buehler, M. J. Influence of cross-link structure, density and mechanical properties in the mesoscale deformation mechanisms of collagen fibrils. J. Mech. Behav. Biomed. Mater. 2015, 52, 1-13. (199) Vogl, A. W.; Lillie, M. A.; Piscitelli, M. A.; Goldbogen, J. A.; Pyenson, N. D.; Shadwick, R. E. Stretchy nerves are an essential component of the extreme feeding mechanism of rorqual whales. Curr. Biol. 2015, 25, R360-361. (200) Dennett, D. C. Darwin's Dangerous Idea; Simon and Schuster: New York, 1996. (201) Rho, J. Y.; Ashman, R. B.; Turner, C. H. Young's modulus of trabecular and cortical bone material: ultrasonic and microtensile measurements. J. Biomech. 1993, 26, 111-119. (202) Almeida-Silveira, M. I.; Lambertz, D.; Perot, C.; Goubel, F. Changes in stiffness induced by hindlimb suspension in rat Achilles tendon. Eur. J. Appl. Physiol. 2000, 81, 252-257. (203) Butler, D. L.; Guan, Y.; Kay, M. D.; Cummings, J. F.; Feder, S. M.; Levy, M. S. Location-dependent variations in the material properties of the anterior cruciate ligament. J. Biomech. 1992, 25, 511-518. (204) Akizuki, S.; Mow, V. C.; Muller, F.; Pita, J. C.; Howell, D. S.; Manicourt, D. H. Tensile properties of human knee joint cartilage: I. Influence of ionic conditions, weight bearing, and fibrillation on the tensile modulus. J. Orthop. Res. 1986, 4, 379-392.


Page 58 of 64

Page 59 of 64

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(205) Roth, V.; Mow, V. C. The intrinsic tensile behavior of the matrix of bovine articular cartilage and its variation with age. J. Bone Joint Surg. Am. 1980, 62, 11021117. (206) Balguid, A.; Rubbens, M. P.; Mol, A.; Bank, R. A.; Bogers, A. J.; van Kats, J. P.; de Mol, B. A.; Baaijens, F. P.; Bouten, C. V. The role of collagen cross-links in biomechanical behavior of human aortic heart valve leaflets–relevance for tissue engineering. Tissue Eng. 2007, 13, 1501-1511. (207) Lillie, M. A.; Gosline, J. M. The viscoelastic basis for the tensile strength of elastin. Int. J. Biol. Macromol. 2002, 30, 119-127. (208) Sodian, R.; Hoerstrup, S. P.; Sperling, J. S.; Daebritz, S.; Martin, D. P.; Moran, A. M.; Kim, B. S.; Schoen, F. J.; Vacanti, J. P.; Mayer, J. E., Jr. Early in vivo experience with tissue-engineered trileaflet heart valves. Circulation 2000, 102, III22-29. (209) Ni Annaidh, A.; Bruyere, K.; Destrade, M.; Gilchrist, M. D.; Ottenio, M. Characterization of the anisotropic mechanical properties of excised human skin. J. Mech. Behav. Biomed. Mater. 2012, 5, 139-148. (210) Shergold, O. A.; Fleck, N. A.; Radford, D. The uniaxial stress versus strain response of pig skin and silicone rubber at low and high strain rates. Int. J. Impact Eng. 2006, 32, 1384-1402. (211) Morrow, D. A.; Haut Donahue, T. L.; Odegard, G. M.; Kaufman, K. R. Transversely isotropic tensile material properties of skeletal muscle tissue. J. Mech. Behav. Biomed. Mater. 2009, 3, 124-129. (212) Iatridis, J. C.; MaClean, J. J.; Ryan, D. A. Mechanical damage to the intervertebral disc annulus fibrosus subjected to tensile loading. J. Biomech. 2005, 38, 557-565. (213) Nerurkar, N. L.; Elliott, D. M.; Mauck, R. L. Mechanical design criteria for intervertebral disc tissue engineering. J. Biomech. 2010, 43, 1017-1030. (214) Manoogian, S. J.; Bisplinghoff, J. A.; Kemper, A. R.; Duma, S. M. Dynamic material properties of the pregnant human uterus. J. Biomech. 2012, 45, 1724-1727. (215) Dahms, S. E.; Piechota, H. J.; Dahiya, R.; Lue, T. F.; Tanagho, E. A. Composition and biomechanical properties of the bladder acellular matrix graft: comparative analysis in rat, pig and human. Br. J. Urol. 1998, 82, 411-419. (216) Kemper, A. R.; Santago, A. C.; Stitzel, J. D.; Sparks, J. L.; Duma, S. M. Biomechanical response of human spleen in tensile loading. J. Biomech. 2012, 45, 348355. (217) Kemper, A. R.; Santago, A. C.; Stitzel, J. D.; Sparks, J. L.; Duma, S. M. Biomechanical response of human liver in tensile loading. Ann. Adv. Automot. Med. 2010, 54, 15-26. (218) Min, Y. B.; Titze, I. R.; Alipour-Haghighi, F. Stress-strain response of the human vocal ligament. Ann. Otol., Rhinol., Laryngol. 1995, 104, 563-569. (219) Umale, S.; Deck, C.; Bourdet, N.; Dhumane, P.; Soler, L.; Marescaux, J.; Willinger, R. Experimental mechanical characterization of abdominal organs: liver, kidney & spleen. J. Mech. Behav. Biomed. Mater. 2013, 17, 22-33. (220) Yuan, H.; Kononov, S.; Cavalcante, F. S.; Lutchen, K. R.; Ingenito, E. P.; Suki, B. Effects of collagenase and elastase on the mechanical properties of lung tissue strips. J. Appl. Physiol. (1985) 2000, 89, 3-14.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(221) Senior, R. M.; Bielefeld, D. R.; Abensohn, M. K. The effects of proteolytic enzymes on the tensile strength of human lung. Am. Rev. Respir. Dis. 1975, 111, 184188. (222) Alkhouli, N.; Mansfield, J.; Green, E.; Bell, J.; Knight, B.; Liversedge, N.; Tham, J. C.; Welbourn, R.; Shore, A. C.; Kos, K.; Winlove, C. P. The mechanical properties of human adipose tissues and their relationships to the structure and composition of the extracellular matrix. Am. J. Physiol. Endocrinol. Metab. 2013, 305, E1427-1435. (223) Sommer, G.; Eder, M.; Kovacs, L.; Pathak, H.; Bonitz, L.; Mueller, C.; Regitnig, P.; Holzapfel, G. A. Multiaxial mechanical properties and constitutive modeling of human adipose tissue: a basis for preoperative simulations in plastic and reconstructive surgery. Acta Biomater. 2013, 9, 9036-9048. (224) Denny, M. The physical properties of spider’s silk and their role in the design of orb-webs. J. Exp. Biol. 1976, 65, 483-506. (225) Teule, F.; Addison, B.; Cooper, A. R.; Ayon, J.; Henning, R. W.; Benmore, C. J.; Holland, G. P.; Yarger, J. L.; Lewis, R. V. Combining flagelliform and dragline spider silk motifs to produce tunable synthetic biopolymer fibers. Biopolymers 2011, 97, 418431. (226) Reddy, N.; Yang, Y. Novel protein fibers from wheat gluten. Biomacromolecules 2007, 8, 638-643. (227) Green, E. M.; Winlove, C. P. The Structure and Mechanical Properties of the Proteins of Lamprey Cartilage. Biopolymers 2014, 103, 187-202. (228) Sherratt, M. J.; Baldock, C.; Haston, J. L.; Holmes, D. F.; Jones, C. J.; Shuttleworth, C. A.; Wess, T. J.; Kielty, C. M. Fibrillin microfibrils are stiff reinforcing fibres in compliant tissues. J. Mol. Biol. 2003, 332, 183-193. (229) Ayoub, N. A.; Garb, J. E.; Tinghitella, R. M.; Collin, M. A.; Hayashi, C. Y. Blueprint for a high-performance biomaterial: full-length spider dragline silk genes. PLoS One 2007, 2, e514. (230) Colgin, M. A.; Lewis, R. V. Spider minor ampullate silk proteins contain new repetitive sequences and highly conserved non-silk-like "spacer regions". Protein Sci. 1998, 7, 667-672. (231) Lyons, R. E.; Lesieur, E.; Kim, M.; Wong, D. C.; Huson, M. G.; Nairn, K. M.; Brownlee, A. G.; Pearson, R. D.; Elvin, C. M. Design and facile production of recombinant resilin-like polypeptides: gene construction and a rapid protein purification method. Protein Eng., Des. Sel. 2007, 20, 25-32. (232) Indik, Z.; Yeh, H.; Ornstein-Goldstein, N.; Sheppard, P.; Anderson, N.; Rosenbloom, J. C.; Peltonen, L.; Rosenbloom, J. Alternative splicing of human elastin mRNA indicated by sequence analysis of cloned genomic and complementary DNA. Proc. Natl. Acad. Sci. U. S. A. 1987, 84, 5680-5684.


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