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Hydrogels are three-dimensional macroscopic networks swollen by water such that the materials possess solid-like mechanical properties but are primarily solvent by weight. Biopolymers, in particular natural proteins and synthetic polypeptides, have received a great deal of attention as building blocks for hydrogels because of their biocompatibility, biodegradability, tunable mechanical properties, molecular recognition abilities and intelligent responses to external stimuli such as pH, ionic strength, temperature, light, ligand binding, and stress.1–6 These protein and polypeptide hydrogels have been widely investigated for a number of potential applications, particularly biomedical materials. For example, artificial protein hydrogels with engineered mechanical properties have been developed for applications in tissue engineering, such as vascular grafts, neural tissue regeneration, and scaffolds for controlling cell behavior and stem cell lineage.7–11 Proteins allow materials to communicate with biological systems, enabling cell adhesion at integrin-binding sites such as Arg-Gly-Asp12–14 or material remodeling through the incorporation of protease cleavage sites.15–17 In addition, stimuli-responsive protein gels have been explored as ligand-triggered actuators for micro-optics, biosensors, and controlled release vehicles for drug delivery.4, 18–20
Protein-based hydrogels are easily prepared through four broad synthetic strategies: (a) processing of natural proteins, (b) solid-phase peptide synthesis,21, 22 (c) biological protein expression,23, 24 and (d) N-carboxyanhydride (NCA) polypeptide synthesis.25–27 As the processing of natural proteins does not enable control over protein sequence, synthetic methods such as solid-phase peptide synthesis and biological protein expression have been developed to design materials with precise sequence control, polydispersity index of 1.0, and the ability to incorporate noncanonical amino acids into the material.28, 29 NCA polymerization provides a complementary approach to these methods, producing materials without monomer-level sequence control that resemble the structure of traditional homopolymers and block copolymers.
The viscoelastic or elastic properties of protein hydrogels generally arise from two classes of structures. In polymer gels, the material is constructed by chemically or physically crosslinking protein or polypeptide chains to form a gel owing to the interplay between swelling and elasticity of the protein chains. In contrast, fibrillar gels are formed by self-assembly of large fibrils that form effective crosslinks owing to branching defects in the fibrils or physical entanglement. The rigidity of the fibrils may significantly contribute to the observed mechanical properties of these gels. Several excellent reviews have extensively covered the physics of fibrillar gels, such as gelation mechanisms and hydrogel rheology.3, 30
In this mini review, we explore the hierarchical structures and intermolecular interactions present in engineered protein polymer hydrogels that enable new materials properties and present challenging questions in polymer physics. In four highlighted areas, the unique properties of protein hydrogels distinguish them from synthetic polymer materials. How do the physics of associating protein domains used as physical crosslinkers affect hydrogel mechanics and thermodynamics? How does the primary structure of minimal consensus sequences from natural elastin, resilin, and silk proteins impact structure, thermodynamics, and mechanics in protein gels? How do the force–extension curves of structured protein chains translate into bulk hydrogel mechanics? What types and magnitudes of responsive transitions may be developed based on protein binding or conformational changes? In each area, we review recent research progress and provide our perspective on important unmet challenges.
PHYSICAL GELS WITH PROTEIN-ASSOCIATING DOMAINS
Protein-associating groups present the opportunity to achieve a unique level of structural control in physically associating polymer gels by precisely engineering the valency and relative chain orientation in physical network junctions. In many cases, these materials provide ideal model systems for comparison with theories exploring the effect of associative groups on both the polymer thermodynamics31, 32 and the mechanical properties.33–36 The most common associating protein sequence used in physical gels is the coiled-coil,37 an α-helical sequence that associates to form multimeric twisted helical bundles. Through changes in the protein sequence, the multiplicity of the network junction may be tuned from two to seven38–40 with well-defined chain orientation.1, 41, 42 Simple coiled-coil dimers display a heptameric repeat structure with associations between helices governed by interactions along a hydrophobic stripe and salt bridging [Fig. 1(a)].43, 44 Even changes of one or a few amino acids in the protein sequence enable tuning of the coiled-coil multiplicity and orientation within this quaternary structure.45, 46
The incorporation of coiled-coil domains into artificially engineered proteins provides a versatile method for the preparation of responsive physical hydrogels, as the association of the coiled-coil domains may be responsively switched with temperature, ionic strength, pH, or the presence of denaturing agents [Fig. 1(b)].37, 38 At typical gelation concentrations of 5–10 w/v %, the proteins form transparent, homogeneous gels.47 Solid-state NMR measurements show that the protein midblock is in a hydrated and flexible state and that the coiled-coil domains move as rigid bodies, consistent with aggregated structures.48 Small-angle X-ray scattering (SAXS) studies on a tetravalent coiled-coil sequence confirm the formation of cylindrical aggregates within the hydrogels, consistent with the dilute solution structure of the coiled-coil.49 Although, in some cases, the responsive transition from gel to sol correlates with the dilute solution coiled-coil unfolding transition measured by circular dichroism (CD),37 for other sequences the coiled-coil in the gel has a higher melting temperature than the coiled-coil in solution.50 Depending on the coiled-coil sequence used, multiple unfolding transitions may also be observed, suggestive of intermediate states between fully aggregated and fully disordered with a lower degree of aggregation.51 Cylindrical aggregates identified by SAXS for tetrameric coiled-coils persist above the dilute solution coiled-coil melting temperature, further indicating that the dissociation transition may be concentration dependent.
The mechanical properties of coiled-coil and other associating protein hydrogels show rheology typically for physically associating polymer gels (Fig. 2).39, 52 A plateau in the elastic modulus, G′, is observed at high frequencies, with a peak in the loss modulus, G″, and a crossover in G′ and G″ observed upon decreasing frequency. At low frequencies, terminal behavior with G′ = ω1.8 and G″ = ω0.9 is observed, close to ideal terminal relaxation behavior.53, 54 The plateau modulus is typically in the range of 100–5000 Pa and crossover frequencies that are often in the order of 0.01–0.1 Hz. Models32, 34, 36 suggest that variations in the associating group-binding energy could provide tunability over a wider range of timescales. Increasing strain results in yielding, typically at strains of 0.25–1.0. In nonlinear strain sweep rheology,55 these materials show strongly shear-thinning behavior in both G′ and G″, with a weak strain overshoot in G1″ (the first harmonic coefficient of G″) only.54 Lissajous figures of hydrogels with pentameric zippers show flat-topped curves characteristic of yielding behavior owing to a shear-banding mechanism. For coiled-coil associating domains, self-healing in these materials is extremely rapid, with nearly full recovery of the original linear elastic modulus in seconds after the cessation of shear observed for several different coiled-coil sequences.54, 56 The rate of response of these mechanical properties is governed in large part by the dynamics of the coiled-coil junctions. The junction exchange time correlates strongly with the stress–relaxation time of the hydrogels, and changes in environmental conditions such as pH affect both properties.57 Shear thinning and rapid self-healing properties make the materials ideal for injectable biomedical hydrogels, and many investigations have demonstrated the utility of the materials for this purpose.58–60
Besides affecting the responsive transitions of hydrogels, the associating group molecular weight61 and coiled-coil sequence impact the mechanical properties of gels by controlling the structure of network junctions. Consistent with the theory of gelation, an association number of >2 is required for gelation of telechelic polymers.38 Trimeric coiled-coils based on collagen52, 62 and fibrin-inspired63 triple helices have been used as crosslinking domains, and the linear mechanical behavior of these gels agrees well with the established models34–36 for physical gels.52 Changing the coiled-coil from tetrameric to pentameric results in an increase in the hydrogel modulus and a slowing of the gel erosion rate, corresponding to an increase in the fraction of elastically effective chains within the network.39 It is hypothesized that this effect is observed because tetrameric coiled-coils can form junctions with an integer number of loops. However, pentameric coiled-coils must form at least a single bridge per junction, resulting in an increase in the number of elastically effective chains. Such odd–even effects may be particularly important at lower gel concentrations. Effects of chain orientation could also have a large impact on this loop formation process, and the separate exploration of valency and chain orientation in network junctions represents a promising direction for future research.
The specificity of coiled-coil association enables heterotelechelic protein polymers to be prepared where each endgroup associates into a different types of homomultimeric junction [Fig. 1(c)].64–66 Hydrogels with orthogonally associating tetrameric and pentameric coiled-coil domains demonstrate an increase in the plateau elastic modulus owing to an increase in the number of elastically effective chains.39 Heterotelechelic proteins have also been prepared where the two ends of the molecule are engineered to specifically associate into strong heterodimeric and heterotetrameric bundles using interactions between acidic and basic coiled-coil sequences,12 enabling gelation at lower concentrations than for association of the acidic sequence alone.
The effect of protein midblock molecular weight on gel properties depends strongly on the type of coiled-coil junction owing to an interplay between the number density of chains in the gel and the effect of midblock length on the fraction of polymer chains in loop or bridge conformations within the protein gels. For tetrameric junctions which have a strong propensity to form loops, increasing the midblock from approximately 100–200 amino acids in results in an increase in the gel modulus at a constant concentration owing to an increase in the fraction of elastically effective bridging chains.53 However, for pentameric junctions, the modulus decreases with the same change in midblock molecular weight at constant protein concentration in the gel.54 Owing to the coiled-coil junction structure, pentameric coiled-coils have a much stronger tendency to form bridges at all midblock molecular weights, and hence the decreasing chain density dominates and the modulus drops.
Several groups have engineered protein hydrogels56, 67 or protein–polymer hybrids56, 65, 68 that incorporate multiple coiled-coil domains spaced evenly along the main chain [Fig. 1(c)]. For gels with pentavalent coiled-coils, incorporating four coiled-coil sequences per protein results in gels similar to those from telechelic proteins with the corresponding coiled-coil sequence and flexible block length.54, 56 Synthetic polymers with coiled-coil side groups demonstrate that both a minimum coiled-coil length (four heptads for the specific sequence explored) and a minimum graft density are required to induce gelation [Fig. 1(c)].66, 69 Coiled-coil unfolding, which is intuitively expected to lead to disruption of crosslinks, may in fact lead to gel collapse,70 suggesting that the protein unfolding dramatically decreases the overall solubility of the polymer gel. Side-functional polymers prepared by “grafting through” acrylated coiled-coil domains71 or coupling of coiled-coils to four-arm poly(ethylene glycol) (PEG) polymers72 have similar properties to the telechelic proteins with the same tetrameric coiled-coil sequence,57 suggesting that the specific chain topology has a less important effect on the mechanical properties.
Drawbacks of many physically associating protein gels are their high erosion rate and weak mechanical properties. Erosion has been slowed by about two orders of magnitude using heterotelechelic proteins, when compared to homotelechelic proteins with the same coiled-coil sequences.73 This is hypothesized to be owing to reduced loop formation. Chemical crosslinking of the associating domains after self-assembly has been demonstrated as another way to limit junction relaxation and improve the robustness of the materials.13, 71, 74 Alternately, we have shown that incorporation of protein hydrogelators into the midblock of a triblock copolymer enables the formation of a protein gel within one nanodomain of a block copolymer, producing a double network-like structure where the coiled-coil association is completely complementary to block copolymer self-assembly driven by hydrophobic interactions.56 The resulting hydrogels show enhanced toughness, reduced creep compliance, and reduced erosion rate compared to the unmodified protein gels. This illustrates the advantage of selective association between proteins to produce materials with multiple orthogonally responsive functionalities.
A distinguishing characteristic of protein hydrogels is the ability to utilize selective protein associations to prepare two-component materials that gel upon mixing. Although the physical properties of these materials are similar to one-component hydrogels, they offer processing advantages by enabling easy encapsulation of cargo such as cells during gelation by mixing two components.59 Most of the associating groups used to prepare two-component gels are heterodimeric, necessitating the incorporation of more than two associating groups per polymer chain to achieve a degree of functionality high enough for gelation. Heterodimeric associating pairs have included heparin oligomers and heparin-binding coiled-coils attached to the ends of four arm PEG,60, 75, 76 WW domains and proline-rich domains incorporated along the backbone of engineered proteins,59, 77 and heterodimeric coiled-coils in the polymer backbone78 or incorporated as side chains.79, 80 Mixing difunctional biotin proteins with streptavidin, which naturally tetramerizes to form network junctions81 and a recently reported “dock and lock” mechanism based on homodimerizing RIIa subunits of c-AMP-dependent kinase A and A-kinase anchoring protein58 further illustrate the versatility of two-component physical gels driven by selective protein associations.
AMORPHOUS ARTIFICIALLY ENGINEERED PROTEIN GELS
The elasticity, extensibility, and resilience of natural elastin and resilin have inspired the design of protein-based hydrogels based on consensus sequences derived from these proteins that endow artificially engineered protein polymers with similar elastic properties. Elastin, which is found in elastic tissues (i.e., lungs, skin, and blood vessels), has a consensus pentapeptide sequence VPGXG, where X can be any amino acid residue except proline. This sequence has been the most characterized building block of elastin-like polypeptides (ELPs),82, 83 but other consensus sequences of ELPs such as VPGG, VPAXG, IPAXG, and APGVGV provide the ability to vary the mechanical properties of the materials.84, 85 Resilin, well-known for its extremely high resilience, is a structural component directly related to the mechanics of insect flight,86 jumping,87 walking,88 and phonation.89 For resilin-like polypeptides (RLPs), a 15-residue repeat sequence GGRPSDSYGAPGGGN derived from the exon I region of proresilin is the most widely used repeat motif.90 Intermolecular interactions usually do not lead to the formation of any higher ordered structure in elastin or resilin; instead, the internal chain dynamics and the interaction between water and elastin/resilin molecules determine the protein elasticity and resilience.
One of the most important characteristics of ELPs is their thermoresponsive behavior in aqueous solutions. At low temperature, ELPs are soluble in water, with the protein adopting a random coil configuration. Raising the temperature decreases the solubility of ELPs, and the proteins start to aggregate into fibrillar structures91 at the inverse transition temperature Tt. This phase transition is referred to as the inverse temperature transition and is manifested by abrupt changes in turbidity,92 rheological behavior,93 and a latent heat of transition.94 The inverse temperature transition resembles the low critical solution temperature (LCST) transition of many synthetic polymers, such as poly(N-isopropyl acrylamide).95 Upon thermal transition, water molecules are released from the solvation shell of the ELPs, and the entropy gain of water molecules results in a decrease in free energy that drives the phase separation and chain collapse.96
However, the change in coil configuration across the inverse transition of ELPs is fundamentally different than the LCST behavior of synthetic polymers.97 Synthetic polymers undergo a coil–globule transition where the collapsed polymer globules are structureless. In contrast, the pentapeptide units of ELPs form β-turns (Fig. 3) and further organize into an ordered β-spiral structure. CD experiments confirm that an individual pentapeptide is the structural unit that creates the β-turn structure and thus the inverse temperature transition.98 The rigid β-spiral structures can assemble into fibers driven by intermolecular hydrophobic interactions and hydrogen bonding, resulting in structured aggregates above the inverse transition temperature. This conformational change was examined by stretching ELP molecules by atomic force microscope (AFM)-based single-molecule force spectroscopy (SMFS). The effective Kuhn segment length of an ELP polymer increases from 0.33 ± 0.03 nm at 11 °C to 0.41 ± 0.03 nm at 42 °C, evidencing a structure deviation from random coil at high temperature.99 The elasticity is still entropic in origin as it originates from a decrease in the conformational entropy upon chain extension.96
The inverse transition temperature of ELPs is accurately predicted using a group contribution theory developed by Urry based on the hydrophobicity of the guest residue X in the pentapeptide repeat VPGXG (Fig. 4).100 Hydrophilic or charged guest residues increase Tt, whereas hydrophobic residues decrease it. The change of inverse temperature is a linear function of mole fraction of the guest residue, providing simple design rules for ELPs with a desired Tt. Adjusting the pH can increase the degree of ionization of charged residues, raising Tt (Fig. 4).101 Adding salts usually lowers the solubility of ELPs91, 102 according to the Hofmeister series, providing the opportunity to use ELP tags in nonchromatography protein purification by using inverse transition cycling.103 Similar to synthetic polymers exhibiting LCST and consistent with the Flory–Huggins theory of polymer solutions, ELPs with a larger molecular weight have a lower Tt.104
Compared with elastin, resilin contains more hydrophilic amino acid residues, indicating that the elasticity mechanism of resilin does not originate from hydrophobic interactions.105 An early structure-mechanics study reveals that the network strand of resilin between two crosslinks behaves as a random coil, in agreement with classic rubber elasticity theory.106 The unstructured feature of resilin is further confirmed by using NMR to study the confirmation of a synthetic RLP derived from Anopheles gambiae proresilin sequence.107 SAXS experiments show a power-law exponent of two in the high q regime, also consistent with a Gaussian random coil chain configuration. It is hypothesized that the high content of Gly and Pro in resilin might be responsible for the highly disordered structure.108 However, recent studies suggest that other chain structures may be important to the properties of resilin. Kaplan and coworkers thoroughly examined a full-length resilin in Drosophila melanogaster.109 The unstructured soft domain exon I displayed comparable elastic modulus and resilience to the full-length resilin. The hard domain exon III undergoes a structural transition from coil to β-turn structure during an energy input process; the return to random coil structure releases energy from exon III to exon I and may provide the mechanism behind resilin's superelasticity.
As both elastin and resilin homopolymers form hydrated elastic polymer chains, chemical crosslinks are required to incorporate them into protein hydrogels. The modulus is controlled by the crosslink density, the molecular weight of the network strand, or the density of elastically effective chains, according to the affine network or phantom network theories.110 Inspired by the fact that natural elastin and resilin are both three-dimensional chemically crosslinked networks,111, 112 a wide variety of crosslinking strategies to prepare ELP-based and RLP-based hydrogels have been investigated. To prepare chemical ELP hydrogels, lysine residues are crosslinked by reagents that specifically reacting with amines, such as β-[tris(hydroxymethyl)phosphino]propionic acid113 and glutaraldehyde.114 In addition, noncanonical amino acid p-azidophenylalanine has been incorporated into the ELP sequence to allow photocrosslinking upon the photolysis of azide groups, generating nitrenes that lead to crosslinking by CH and CC insertion reactions.115 ELP hydrogels can also be rapidly formed from disulfide crosslinks under mild oxidative conditions using cysteine residues incorporated in the ELP sequence.116 To prepare chemical RLP hydrogels, Ru(II) catalyst is used to photocrosslink tyrosine residues.117 The nonspecificity of such chemical crosslinking reactions usually results in inhomogeneous networks which contain network imperfections. To combine the mechanical properties of natural elastin and resilin with biological functionality, motifs for cell adhesion, matrix degradation, and polysaccharide binding have been incorporated into the ELP/RLP-based hydrogels.118–121 High elasticity and resilience are generally not affected by the added biological functional motifs.
Inspired by the ability of triblock copolymers to form thermoplastic elastomers, artificially engineered protein block copolymer hydrogels based on ELP blocks have been engineered. These protein block copolymers undergo microphase separation in response to environment stimuli (such as temperature and pH) and can self-assemble into different nanostructures.85 BAB triblock copolymers are the most widely used molecular architecture in ELP-based block copolymer hydrogels, where A is a hydrophilic and elastic block and B is usually a thermally responsive hydrophobic block with plastic mechanical properties. A change from elastic to plastic mechanical response of the endblocks is imparted by changing the third amino acid in the VPGXG sequence from Gly to Ala, resulting in a change of the β-turn structure changes from type II to type I.80, 122 Above the inverse transition temperature, aggregation of endblocks establishes physical crosslinks owing to the formation of block copolymer nanodomains, whereas the highly solvated hydrophilic midblocks form bridges connecting the aggregated domains. The self-assembled nanostructure and the mechanical properties can be precisely controlled by tuning composition, the relative length of two blocks93 and the molecular architecture (diblock or triblock).123 Crosslinking the hydrophobic blocks in ELPs can stabilize the self-assembled nanostructures and effectively modulate the hydration level of ELP block copolymer hydrogels.124
ARTIFICIALLY ENGINEERED PROTEIN GELS WITH CRYSTALLINE DOMAINS
The exceptional mechanical strength, toughness, and extensibility of silk have motivated the development of silk-inspired polypeptide gels with crystalline domains. It is noteworthy that the molecular architecture of silk proteins resembles the design of synthetic polyurethane elastomers.125–127 In both silks and polyurethanes, semicrystalline regions establish crosslinks and provide the modulus and toughness, whereas amorphous regions allow stress distribution and homogenization to provide elasticity.128, 129 The hydrophobic interactions and hydrogen bonding among Ala-rich and Ala-Gly-rich repeat sequences in silk drive the formation of β-sheets.130 The aggregation of β-sheets then forms hard semicrystalline nanodomains, packing in a weakly ordered lamellar pattern.131, 132 A computational examination reveals that the modulus and toughness of spider silk fibers increase with increasing length of the lamellar nanocrystals,133 and the reorientation of weakly aligned nanocrystals in silks can lead to strain-hardening phenomenon.134 In addition to the high modulus and toughness, silks are an interesting type of material because of their moderate extensibility and elasticity; this combination of properties currently cannot be replicated with synthetic polymers.135 Silk elasticity derives from the presence of disordered domains that are mainly composed of 31-helices, β-turn spirals, and weakly ordered β-sheets.130 As determined by solid-state two-dimensional NMR, loosely conserved Gly-rich domains form the 31-helices, where GGX (X can be Glu, Tyr, and Leu) is the repeat unit.136 The helical structures are partially embedded inside the nonperiodic β-sheets.137, 138 The β-turn spirals forming GPGXX segments,139 resembling the characteristic structure in ELPs, provide mechanical extensibility.140
In contrast to elastin or resilin, natural silks exhibit their mechanical properties in air instead of in aqueous environment. The limited water solubility of silk, induced by the high percent crystallinity, can be a problem when the silk-like polypeptide motifs are used for hydrogel materials. To address this limitation, Kaplan and coworkers controlled the crystallinity of silk-derived polypeptides by introducing charged amino acid residues adjacent to the Ala-rich sequence to interrupt β-sheet formation, which can be achieved by oxidizing methionine to sulfoxides141, 142 or phosphorylating serine.143 The self-assembly of β-sheets is triggered by selectively reducing the sulfoxide or enzymatic dephosphorylation. An alternative approach to increase the water solubility is to construct silk–elastin-like polypeptides (SELPs). The SELPs have combined properties of silk and elastin: thermoresponsiveness, elasticity, and high mechanical strength. Similarly, incorporating semicrystalline blocks in thermoplastic elastomers can increase the mechanical strength and toughness.144, 145 The β-sheet forming silk-like sequence (GAGAGS) and the elastin-like sequence (VPGXG) are generally chosen as the building blocks in the SELPs.146–149 By changing the composition of the block copolymers, SELPs can undergo an irreversible sol–gel transition with increasing temperature. Unlike ELP block copolymers, SELP hydrogels can be solid at low temperature and are insensitive to pH, possibly owing to the highly hydrophobic crystalline domain based on strong H-bonded β-sheets.149, 150 There continues to be interest in controlling the self-assembly process of SELPs either by refining the molecular architecture or by using external stimulus to direct the assembly process.151, 152
HYDROGELS WITH STRETCHABLE PROTEIN TERTIARY STRUCTURES
Fundamentally, the physical properties of a polymer hydrogel arise from the chain conformation and thermodynamics of individual polymer molecules within the gel. In many protein hydrogels, the chain contains folded secondary and tertiary structures, providing more complex force–extension profiles than simple Gaussian coils. Over the last decade, SMFS techniques have been exploited to establish the relationship between tertiary structures and mechanical properties of individual proteins. Based on the folded tertiary structures, stretch ratios and energy dissipation process, currently studied proteins can be divided into three categories: (a) globular proteins, (b) coiled-coils, and (c) α-helical repeat proteins.
When stretched, most globular proteins display similar mechanical properties: large stretch ratios owing to unfolding of the folded structure and relatively strong force hysteresis upon stretching and relaxation, compared to other proteins. For example, when immunoglobulin (Ig) 27 domains (one of the domains in the mechanically active region of gigantic muscle protein titin153) were fully stretched by AFM, force–extension profiles including multiple sawtooth force peaks were obtained [Fig. 5(a)].154–158 Each sawtooth peak corresponds to unfolding of an individual folded Ig 27 domain. Contour length increments (ΔLc = fully stretched length L2 − folded length L1) of 28 nm correspond to the length increase upon unfolding a single Ig 27 domain, whereas the force of 200 pN corresponds to the force required for unfolding. Once unfolded, each Ig 27 domain behaves as a simple worm-like chain (blue curve, Fig. 5(a)),159, 160 resulting in the large force hysteresis between the unfolding and the refolding force curves because of energy dissipation during protein unfolding. Cyclic stretching/relaxation measurements of Ig domains reveal that the unfolding/refolding force profiles are fully reversible.155 Numerous other globular proteins share similar mechanical behaviors, specifically large stretch ratios owing to their well-folded structures and relatively large force hysteresis during stretching and relaxation experiments (e.g., Ig domains, ubiquitin, fibronectin type III domains, Top 7, SNase, and GB1).155, 161–173
In contrast to globular proteins, coiled-coil proteins show little energy dissipation in single-molecule studies. When myosin single molecules, a model coiled-coil, were stretched by AFM,167, 174 the protein initially behaves as worm-like chain. By overstretching the coiled-coil, the force abruptly increased to around 25 pN, and a force plateau appears before they behave as worm-like chain in the AFM force–extension curve [Fig. 5(b)]. As end-to-end distance of the intact coiled-coil structure (L1) is larger than other types of proteins, a relatively small stretch ratio (250%) is observed when compared to the Ig 27 domain (700%). Cyclic stretching/relaxing measurements show that the unfolding and refolding force profiles are reproducible with no energy dissipation, making myosin an ideal elastic biopolymer.167
α-Helical repeat proteins have characteristics of both globular proteins and coiled-coils, exhibiting large stretch ratios and low-energy dissipation.175–178 Individual repeats, composed of two or three α-helixes, are tightly stacked adjacent to each other by a hydrophobic core and form spiral- or solenoidal-shaped α-helical repeat proteins.176 When β-catenin, an α-helical repeat protein, was stretched by AFM [Fig. 5(c)], stepwise unfolding force peaks were captured in the force–extension curve similar to the unfolding force profile of the globular proteins but with lower unfolding forces (<100 pN). Each peak represents unfolding of an individual repeat. Because of closely packed repeats in the solenoidal form, an extremely large 1400% stretch ratio (ΔLc/L0) was generated. When the fully stretched β-catenin was relaxed, fast and stepwise refolding of individual repeats (<2 ms) occurred in a quasi-equilibrium manner.176 Similar to globular proteins and coiled-coils, unfolding/refolding properties of α-helical repeat proteins are reproducible during cyclic stretching/relaxing measurements. Such large stretch ratios with low-energy dissipation distinguish α-helical repeat proteins as a third type of elastic biopolymer with tertiary structure.175–178
Inspired by the design and properties of muscle, tertiary protein structures have been incorporated into the polymer chains of engineered protein gels.179 The giant muscle protein titin (molar mass, ∼3 MDa) has important roles for muscle contraction and passive elasticity.153, 180, 181 The mechanically active regions of titin are I-bands, composed of Ig domains and unstructured PEVK nonmodular sequences. To mimic the structure of I-band regions in smaller engineered proteins amenable to bacterial expression, artificially engineered GB1 (G) and high resilience182 resilin (R) domains were used to replicate the IG domains and PEVK spacers, respectively [Fig. 6(a)]. The artificial globular protein GB1 has a large stretch ratio and force hysteresis, similar to Ig domains at the single-molecule level.168 Two engineered elastomeric protein building blocks, (G-R)4, and GRG5RG4R, were constructed and photochemically crosslinked at tyrosine residues183 to produce elastomeric protein networks [Fig. 6(b)].
Tensile tests of (G-R)4- and GRG5RG4R-based hydrogels reveal that the single-molecule mechanical properties of resilin and GB1 blocks translate into hydrogels. At strains <15% where GB1 domains do not unfold, ideal elastic behavior was observed in the gels. However, at strains more than 15%, increasing energy dissipation was observed with increasing strains [Fig. 6(c)]. After deformation, the gels recover their original properties in <1 s. As resilin-based materials possess high resilience up to 250% strain,182 the unfolding/refolding of GB1 domains168 was suggested to explain the fast recoverable hysteresis. Stress–relaxation tests of GRG5RG4R-based hydrogels present similar mechanical behaviors to myofibrils in muscle at the macroscopic level.184 When a step strain is applied to the hydrogel, stress–relaxation occurs by GB1 unfolding to dissipate energy [Fig. 6(d)]. This process can reduce damage to the material owing to overstretching, similar to elastomeric proteins in muscle.155, 157, 163, 179, 184–186
The effect of crosslinking density on the stiffness, strength, and viscoelastic properties of globular protein hydrogels has been demonstrated by crosslinking polyprotein (GB1)8 building blocks and its derivatives.187 Tensile tests of (GB1)8-based gels show that the Young's modulus and failure stress increase with increasing crosslink density on GB1 building blocks, whereas failure strains stayed similar between 60 and 80% for each hydrogel. At constant strain, stress and the amount of fully recoverable hysteresis were increased with increasing crosslinking density. Increased stress and hysteresis of (GB1)8-derivative hydrogels at the macroscopic level was explained by more unfolded GB1 domains at the nanoscale level owing to increased number of crosslinks between GB1 domains. Tandem modular (GB1)8 has also been incorporated as a midblock into physically associating telechelic protein gels with tetrameric coiled-coil endblocks (A) to form the protein A(GB1)8A.188 The relatively rigid tandem modular GB1 midblock is speculated to result in decreased loop formation in the gel, resulting in lower erosion rates by about one order of magnitude than gels formed with traditional flexible protein midblocks.47, 73, 188
Although energy dissipation in GB1 hydrogels reflects many of the properties seen in SMFS experiments, the full extensibility promised by stored length in folded protein structures has yet to be realized. At the single-molecule level, individual GB1 domains can be unfolded more than 600% (=ΔLc/L1).168, 179 However, chemically crosslinked GB1-based hydrogels can be maximally extended to 135%.179, 187 One hypothesis is that random 3D networks formed by chemical crosslinks can limit full unfolding of elastomeric proteins, resulting in relatively low maximum extension protein-based hydrogels. Physical crosslinkers, such as coiled-coils, potentially allow site-specific connections that could maintain the full unfolding potential of the proteins. However, the rupture strength of such physical crosslinkers is weaker than unfolding of other types of elastomeric protein domains in most known cases;161–163, 166, 167, 174, 189 therefore, elastomeric protein-based hydrogels fail to demonstrate the high extensibility of single molecules when connected by weak physical crosslinkers. If strong physical crosslinkers were developed, passive elasticity of well-ordered three-dimensional structures of muscle could be more accurately mimicked.
HYDROGELS WITH BIOLOGICAL RECOGNITION PROPERTIES
Hydrogels that possess specific biological recognition capabilities enable dynamic changes in gel properties in response to a wide variety of biomolecular cues.190–192 The design of these systems relies on the specific tertiary structure of proteins that selectively recognize ligands and bind them with a high affinity. Actuation of gel properties originates from one of two mechanisms: changes in the chain length between crosslinks based on the changes in protein tertiary structure within the backbone of the polymer chain, or changes in the physical crosslink density within the gel based on binding/unbinding of pendant protein crosslinking functionalities.
Calmodulin (CaM) has been studied as a model protein that actuates gel properties based on the conformational change [Fig. 7(a,b)]. The decrease in length of an engineered CaM from 5 to 1.5 nm upon binding of the ligand trifluoperazine (TFP) modulates the chain length between crosslinks in the gel. CaM has been introduced into the main polymer chains of hydrogels both using tetra-PEG gels crosslinked by CaM193 and photocrosslinkable PEG-CaM-PEG/PEG diacrylate gels.194 The key determinant of deswelling upon ligand binding is the change in the effective length of bridging chains. A larger extent of gel deswelling is observed for shorter PEG segments as the change in CaM length represents a larger percentage decrease in bridge length.195 Gels are tunable to exhibit 20–80% decrease in volume upon ligand binding. Optical transparency decreases with deswelling, likely owing to the formation of inhomogeneous regions within the material as solvent is rejected from the hydrogel during contraction. Most of this change in volume was reversible upon addition of Ca2+ to displace the TFP ligand.196 For PEG segments with a degree of polymerization of ∼15,194 each PEG arm contributes a length of approximately 3 nm to the overall polymer chain.197 Therefore, a change in chain length from ∼11 to ∼7.5 nm upon ligand binding is anticipated. This 32% decrease in chain length would result in a 69% reduction in gel volume for isotropic deswelling, consistent with the volume decrease observed for the largest swelling levels. This suggests that simple chain length arguments can estimate the magnitude of deswelling, but quantitative theoretical predictions for these responsive transitions have not been developed.
Investigations performed on a variety of proteins demonstrate both the general utility of conformational change to modulate gel swelling and the importance of controlling the magnitude of the change in chain length to modulate the extent of swelling. The ability of glucose-binding protein to undergo a conformational change that triggers a change in gel size and porosity has been demonstrated,198 where the change in gel linear dimension is 2–3%. Gels crosslinked with metallothioneins199 show decrease in volume down to 20% of the original (change in linear dimension of 58% of the original) owing to a very large change from an unfolded to a folded state of the protein in response to heavy metal ions. However, this change is not fully reversible on repeated cycling. A further approach to volumetric change is to use the binding of ATP by adenylate kinase to trigger a change in the gel properties200; this method achieved an approximately 15% reduction in gel volume. Protein denaturation is not an effective mechanism for chain extension in these type of gels; it is hypothesized that aggregation in the denatured state causes an irreversible loss in swelling owing to the formation of physical crosslinks.201
The dynamics of hydrogel actuation in conformationally responsive protein gels are complex as ligand binding modifies hydrogel porosity during diffusion, modulating transport of further ligand into the hydrogel. The swelling time in CaM hydrogels depends strongly on the initial molecular weight between crosslinks, with a lower network porosity, resulting in longer actuation times owing to slower transport through the polymer network.193, 194 This is qualitatively consistent with the well-established theories of transport through polymeric hydrogels202; however, the detailed transport behavior presents interesting and complex physics that merit further study.
An alternative method of biologically specific actuation is to use the protein as a physical crosslinker, where the introduction of soluble ligands causes competitive binding and therefore release of the physical crosslinks [Fig. 7(c,d)]. Glucose-sensitive hydrogels have been prepared by functionalizing a synthetic polymer backbone with glucose and using concanavalin A tetramers which possess four glucose-binding sites as tetravalent crosslinkers.203, 204 In the absence of glucose, each concanavalin A binds the polymer in four places, forming a physically crosslinked network. Free glucose in solution competes with the polymer-tethered glucose for binding, inducing a sol–gel transition at high glucose concentration. Using both chemical and physical crosslinks enables gel swelling to be modulated, with a volumetric increase of approximately 10% observed at the highest physical crosslink densities.205 Heparin-functionalized tetra-PEG may be similarly crosslinked by association with VEGF, where the association of VEGF with cell receptors causes erosion of the gel in the presence of cells.206 The binding action of CaM can also be used to modulate physical crosslink density in gels by tethering the CaM as a side group rather than incorporating it into the polymer main chain enabling responsive swelling or deswelling of the gels to create microfluidic actuators207 and switchable lenses.18 Finally, the dimerization of gyrase subunit B in the presence of coumermycin has been demonstrated as a method for responsive crosslinking and drug release.208
Antigen–antibody interactions provide one of the most attractive methods of implementing responsive crosslinking in materials owing to the wide range of antibody–antigen pairs and their extremely high specificity. Semi-interpenetrating networks with goat anti rabbit IgG antibodies attached to the linear polymer and rabbit immunoglobulin G (IgG) attached to the chemically crosslinked network were prepared to demonstrate this concept.209–211 In the presence of free antigen that breaks the physical crosslinks, an approximately 10% increase in the gel volume could be achieved. Hydrogels were designed that contained pendant antifluorescein antibody fragments, and upon binding of fluorescein a reduction to 60% of the original gel volume was observed.212 This effect is ascribed to changes in hydrophobicity as no change in crosslink density can be achieved with this design.
Artificially engineered proteins can be designed with numerous intriguing physical properties: self-association into well-defined structures, sequence-dependent LCST behavior and structural transitions, fully reversible unfolding/refolding under tension, and highly specific biological recognition. The incorporation of these functional proteins into hydrogels offers the opportunity to produce materials with new physical properties, such as precisely defined network topologies and ligand-specific responsive swelling transitions. Although a great deal has been learned about the polymer physics of these biofunctional hydrogels, a great deal still remains to be discovered to exploit the full potential of proteins in gels. Within this broad field, we have highlighted four areas with new challenges in polymer physics: understanding the effect of network junction structure and valency on the properties of associating gels, mimicking the thermodynamics and mechanics of structural proteins through the use of minimal protein repeats, translating the complex force–extension curves of structured proteins into polymer networks, and developing new responsive systems with biological specificity. These and other questions will provide a rich area for exploration in polymer science for years to come, defining an exciting new direction in the field that will continue to lead to the discovery of new polymeric materials.
This work was supported by the U.S. Army Research Office through the Institute for Soldier Nanotechnologies under contract W911NF-07-D-0004.
Bradley Olsen is an Assistant Professor in the Department of Chemical Engineering at MIT. He earned his S.B. in Chemical Engineering at MIT in 2003, his Ph.D. in Chemical Engineering at the University of California, Berkeley in 2007 and was a postdoctoral scholar at the California Institute of Technology from 2008–2009. Olsen's research in polymer physics focuses on molecular self-assembly, block copolymers, polymer networks and gels, and protein materials.
Minkyu Kim is currently a postdoctoral researcher in the Department of Chemical Engineering at MIT. He received his B.S. (2004) in Mechanical Engineering from Kyung Hee University (Korea) and M.S. (2006) in Biomedical Engineering and Ph.D. (2011) in Mechanical Engineering and Materials Science from Duke University. Current research interests include nanomechanics and molecular self-assembly of biopolymers and their applications for bioinspired materials.
Shengchang Tang is currently a Ph.D. candidate in Chemical Engineering Department at MIT, under the supervision of Prof. Bradley Olsen. He received his B.S. degree in Polymer Materials and Engineering in Tsinghua University (China) in 2010. His thesis project focuses on mimicking the mechanical properties of tissues using hierarchically structured protein-polymer hydrogels.