Designed biomaterials to mimic the mechanicalproperties of musclesShanshan Lv1, Daniel M. Dudek2{, Yi Cao1, M. M. Balamurali1, John Gosline2 & Hongbin Li1
The passive elasticity of muscle is largely governed by the I-bandpart of the giant muscle protein titin1–4, a complex molecular springcomposed of a series of individually folded immunoglobulin-likedomains as well as largely unstructured unique sequences5. Thesemechanical elements have distinct mechanical properties, and whencombined, they provide the desired passive elastic properties ofmuscle6–11, which are a unique combination of strength, exten-sibility and resilience. Single-molecule atomic force microscopy(AFM) studies demonstrated that the macroscopic behaviour oftitin in intact myofibrils can be reconstituted by combining themechanical properties of these mechanical elements measured atthe single-molecule level8. Here we report artificial elastomericproteins that mimic the molecular architecture of titin throughthe combination of well-characterized protein domains GB112 andresilin13. We show that these artificial elastomeric proteins can bephotochemically crosslinked and cast into solid biomaterials. Thesebiomaterials behave as rubber-like materials showing high resili-ence at low strain and as shock-absorber-like materials at highstrain by effectively dissipating energy. These properties are com-parable to the passive elastic properties of muscles within thephysiological range of sarcomere length14 and so these materialsrepresent a new muscle-mimetic biomaterial. The mechanicalproperties of these biomaterials can be fine-tuned by adjustingthe composition of the elastomeric proteins, providing the oppor-tunity to develop biomaterials that are mimetic of different types ofmuscles. We anticipate that these biomaterials will find applica-tions in tissue engineering15 as scaffold and matrix for artificialmuscles.
The string of folded immunoglobulin domains and unstructuredunique sequences constitute two distinct types of entropic springs intitin7,8. The string of folded immunoglobulin domains has higher per-sistence length than the unstructured sequences and extend first duringstretching. Only under high stretching forces at the high end of thephysiological range of sarcomere length, when the string of immuno-globulin domains are straightened, can a small number of foldedimmunoglobulin domains unfold to extend the length of titin anddissipate energy, effectively preventing damage due to overstretching16.These features are combined to give rise to the passive mechanicalproperties of muscles at the macroscopic level1,2,7,17, which manifestas a Young’s modulus close to 100 kPa, increasing energy dissipation athigher sarcomere length, and stress relaxation at a constant strain4,14,16.To design biomaterials mimicking the fine-tuned passive elastic pro-perties of muscle, it is critical to incorporate these mechanical featuresat both single-molecule and macroscopic biomaterial levels. Towardsthis goal, here we first engineered artificial elastomeric proteins thatmimic the molecular architecture and nanomechanical properties ofindividual titin molecules. Then we used these proteins to constructbiomaterials that mimic the passive elastic properties of muscle.
To engineer titin-mimicking artificial elastomeric proteins (seeMethods and Supplementary Fig. 1), we used the well-characterizedGB1 domains12 to mimic folded titin immunoglobulin domains,because GB1 domains exhibit mechanical properties comparable tothose of titin immunoglobulin domains12, and we used a consensusrepeat of the random-coil-like protein resilin13 to mimic unstruc-tured sequences (such as the N2B sequence in titin), because resilinis a highly elastic and resilient protein13,18,19 and is also largelyunstructured20 (see Supplementary Fig. 2). With these two buildingblocks, we constructed artificial elastomeric proteins (G–R)4 andGRG5RG4R, where G represents individual GB1 domain, and Rrepresents individual resilin repeat.
We first used AFM techniques6,21 to characterize their nanomecha-nical properties at the single-molecule level. Stretching (G–R)4
results in characteristic sawtooth-like force–extension relationships12
(Fig. 1a), where individual force peaks correspond to the mechanicalunfolding of GB1 domains and are characterized by an unfoldingforce of ,180 pN and a contour length increment DLc of ,18 nm.Owing to the dimerization of (G–R)4 via carboxy-terminal cysteineresidues (Supplementary Information), force–extension curves canshow as many as eight GB1 unfolding events. The featureless ‘spacer’,which is of length L0 and occurs before the GB1 unfolding forcepeaks, corresponds to the stretching of random-coil-like resilinsand folded GB1 domains (Fig. 1a), confirming the entropic spring-nature of resilin repeats. Because the persistence length of GB1 ismuch larger than that of unstructured resilin, fitting the Worm-like-chain model of polymer elasticity to the spacer yielded a persist-ence length of 0.49 6 0.09 nm (average 6 s.d., n 5 188) for resilin,comparable to that of the random-coil-like sequence N2B in titin8,9,22
and unfolded polyprotein chains6,21. Stretching polyproteinGRG5RG4R yielded force–extension curves with similar sawtoothpatterns but with shorter spacers owing to the fewer resilin domainsin GRG5RG4R (Fig. 1b and Supplementary Fig. 3). Moreover, themechanical unfolding of GB1 domains is reversible, becauseunfolded GB1 domains can refold to regain mechanical resistanceupon relaxation12. These nanomechanical properties of (G–R)4 andGRG5RG4R largely mimic those of individual titin molecules.
We then used these miniature-titin-like elastomeric proteins toconstruct biomaterials to mimic the passive mechanical propertiesof muscle. Individual titin molecules are well-aligned and organizedin the filament lattice of muscle3. However, it remains challenging tomimic such ordered structures in synthetic biomaterials. As analternative, we created chemically crosslinked GB1–resilin networksto exploit the nanomechanical properties engineered into individualGB1–resilin molecules. We used the well-developed [Ru(bpy)3]21-mediated photochemical crosslinking strategy23, which allows thecrosslinking of two tyrosine residues in close proximity into dityrosineadducts (Supplementary Fig. 4). This method was used successfully to
1Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada. 2Department of Zoology, University of British Columbia, Vancouver, BritishColumbia V6T 1Z1, Canada. {Present address: Department of Engineering Science and Mechanics, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA.
crosslink recombinant resilins into solid biomaterials13. The use ofresilin repeats, which provide the majority of crosslinking sites inGB1–resilin polyproteins, enables an efficient approach with whichto prepare GB1–resilin-based biomaterials. We found that on illu-mination with white light, GB1–resilin polyproteins can readily becrosslinked into solid and transparent biomaterials at room temper-ature from their concentrated (.150 mg ml21) solutions (Sup-plementary Information). The middle panel of Fig. 2a shows opticalphotographs of moulded rings of both polyproteins. The formation ofdityrosine crosslinks was indicated by their characteristic blue fluor-escence upon ultraviolet irradiation13 (Fig. 2a, top panel).
Protein-based biomaterials, such as those based on elastin24–28,resilin13 and abductin29, are engineered from non-globular elasto-meric proteins that behave like entropic springs. To our knowledge,the GB1–resilin-based biomaterial is the first chemically crosslinkedbiomaterial that incorporates folded, mechanically resistant globulardomains in its constituent elastomeric proteins, enabling us to examinetheir macroscopic mechanical properties and investigate how themicroscopic properties of individual proteins are translated into mac-roscopic ones in biomaterials.
Here we carried out tensile measurements to characterize the mech-anical properties of GB1–resilin-based biomaterials in PBS at roomtemperature. For technical reasons, we used ring-shaped samples fortensile testing30 (see Supplementary Information). Typical stress–strain curves of (G–R)4 and GRG5RG4R-based biomaterials are shownin Fig. 2b-c. It is evident that GB1–resilin-based biomaterials areelastic. GRG5RG4R can be stretched to a strain as high as 135%without breaking. The Young’s modulus is ,70 kPa for (G–R)4 and,50 kPa for GRG5RG4R (at 15% strain), both close to the Young’smodulus measured for myofibrils/myocytes, which is in the range60–100 kPa within the physiological range of sarcomere length4,14.These biomaterials are isotropic (Supplementary Information), sothe measured Young’s modulus reflects the overall isotropic propertyof the biomaterials.
Resilience is a measure of a material’s ability to deform reversiblywithout loss of energy18. Resilin is known for its superb resilience19,and resilin-based biomaterials constructed using the same photoche-mical crosslinking method did not show appreciable hysteresis evenat 250% strain13,31 (Fig. 2d). To examine the influence of folded GB1domains on the resilience of GB1–resilin-based biomaterials, wemeasured the resilience of these biomaterials. The stretching andrelaxation curves of both (G–R)4 and GRG5RG4R at low strain(,15%) were superimposable and no hysteresis was observed(Fig. 2b, c), suggesting high resilience for both materials at lowstrains. However, the stretching and relaxation curves were no longersuperposable at higher strains and hysteresis started to develop, indi-cating that some of the work done during stretching was dissipatedand cannot be recovered upon relaxation. The hysteresis increaseswith the increase of strain (Fig. 2b, c), indicating that the resilience ofGB1–resilin-based materials decreases with the increase of strain(Fig. 2d). This behaviour is similar to that of myofibrils or myo-cytes14,16,32, which showed increasing hysteresis between stretchingand relaxation at increasing sarcomere lengths, indicating thatGB1–resilin-based biomaterials, just like muscles16, behave likeshock-absorbers at higher strains by effectively dissipating energy.
The observed hysteresis, that is, energy dissipation, during cyclicexperiments indicates that stretching GB1–resilin-based biomaterialsto higher strains involved the breakage of weak non-covalent bonds33 inthe crosslinked network. And the breaking of such bonds is reversible,as the hysteresis observed in GB1–resilin-based biomaterials can befully recovered upon relaxation. As shown in the insets of Fig. 2b andc, during subsequent stretching, stress–strain curves superpose on oneanother regardless of the final strain, suggesting a full recovery of thehysteresis. Moreover, the recovery of hysteresis is very fast: during cyclicstretching–relaxation experiments, the stretching–relaxation loopswere identical (Supplementary Fig. 6) even when there was no waitingtime between consecutive cycles, suggesting that the recovery of hys-teresis occurs at a timescale significantly shorter than the dead time ofour Instron, which is estimated to be ,1 s. Again, this reversible hys-teresis behaviour is similar to that for myofibrils and myocytes16,32. It isalso interesting to note that the recovery of hysteresis can occur evenunder residual stress. In partial relaxation experiments (Fig. 2e), whenthe biomaterial was partially relaxed to a strain above 35%, no recoveryof hysteresis was observed. When the biomaterial was relaxed to below35% strain, partial recovery started to occur. The degree of recoverydepends on the residual stress: the lower the residual stress, the higherthe percentage of recovery (Fig. 2e).
It is evident that the mechanical behaviours of GB1–resilin-basedbiomaterials differ significantly from those of resilin-based bioma-terials, highlighting the significant roles of folded GB1 domains indetermining the mechanical properties of the resultant biomaterials.Given that photochemically crosslinked resilin-based biomaterialsshow only negligible hysteresis during stretching (Fig. 2d), theobserved hysteresis in GB1–resilin-based biomaterials probablyresulted from folded GB1 domains and the associated structuralchanges of the crosslinked network. The hysteresis observed in tensileexperiments indicates that the stretching of GB1–resilin-based bio-materials involved breaking of weak non-covalent bonds33. It is wellknown from single-molecule AFM experiments that, on stretching,force-induced rupture of non-covalent bonds can lead to the unfold-ing of GB1 domains and dissipation of energy12. Therefore, theunfolding of some GB1 domains during stretching could provide aplausible molecular mechanism to explain the hysteresis observed inGB1–resilin-based biomaterials at high strains. The fast recovery rateof hysteresis and the ability to recover hysteresis under residual stressare consistent with the fast folding kinetics of GB1 domains and theability of GB1 domains to refold under residual force observed insingle-molecule AFM experiments12, providing qualitative evidencethat the hysteresis observed in biomaterials probably originates fromthe unfolding of a small number of GB1 domains.
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Figure 1 | Force–extension curves of two polyproteins. a, (G–R)4.b, GRG5RG4R. The force peaks, characterized by a DLc of ,18 nm and anunfolding force of ,180 pN, result from the mechanical unfolding of GB1domains. Stretching resilins does not result in any unfolding force peaks;instead we see a featureless spacer of length L0. The notable difference betweenthe force–extension curves of (G–R)4 and GRG5RG4R is the shorter featurelessspacer of GRG5RG4R, which is due to fewer resilin repeats in GRG5RG4R. Greylines correspond to the worm-like chain model fits to the experimental data.
To further compare the energy dissipation behaviours of thedesigned biomaterials with those of myofibrils/myocytes, we carriedout stress-relaxation experiments at constant strains. WhenGRG5RG4R was stretched rapidly to a given strain that was heldconstant afterwards, we observed clear stress relaxation (Fig. 3a),again suggestive of the existence of energy dissipation processes.The larger the initial strain, the greater the amplitude of stress relaxa-tion. We found that the stress-relaxation behaviours can be describedreasonably well by double-exponential fits. The relaxation rates (k1
and k2) were observed to increase with the increase of strain, but theincrease of fast-phase rate k1 mainly occurs at higher strain while theincrease of slow-phase rate k2 occurs at lower strain (Fig. 3b).
The stress-relaxation behaviours of GB1–resilin-based biomaterialsare qualitatively similar to those of myofibrils16,17, but they are verydifferent from the behaviour of biomaterials made of resilin, in whichnegligible stress-relaxation was observed in similar experiments31. Theunfolding of a few immunoglobulin domains was proposed as apossible molecular mechanism to explain the stress-relaxation beha-viours of myofibrils31. Similarly, Monte Carlo simulations6 on force-relaxation behaviour of GRG5RG4R at constant extension revealedthat the unfolding of some GB1 domains can lead to force-relaxationbehaviours similar to those seen in our experiments and similardependence of the fast-phase relaxation rate on extension(Supplementary Fig. 7). However, the simulated behaviour of theslow-phase relaxation rate differed from the experimental data. It isclear that the relaxation behaviours simulated at the single-molecule
level cannot be directly compared with the stress-relaxation beha-viours of GB1–resilin-based biomaterials quantitatively, becauseGB1–resilin molecules are not well-aligned in the photochemicallycrosslinked three-dimensional network and the force experienced byindividual molecules cannot be measured directly. A more detailedmodel combining the possibility of GB1 unfolding with a three-dimensional network is required to describe the stress-relaxationbehaviour at the macroscopic level. Moreover, it is important to notethat stress relaxation is considered to be a viscoelastic property macro-scopically. Although domain unfolding can lead to stress relaxation,the direct demonstration of domain unfolding in macroscopicmaterials is yet to be achieved. Therefore, it is possible that othermicroscopic processes or mechanisms, such as friction experiencedby folded domains during stretching, may also contribute to thestress-relaxation behaviours of muscles as well as GB1–resilin-basedbiomaterials.
Our results demonstrate that the incorporation of folded, mech-anically resistant globular domains into elastomeric proteins pro-vides a novel approach with which to construct biomaterials thathave unusual macroscopic mechanical properties. Such a bottom-up approach offers the opportunity to tailor the macroscopic pro-perties of biomaterials by fine-tuning the nanomechanical propertiesof their molecular building blocks at the single-molecule level. Todemonstrate such possibilities, we used chemical denaturant to affectthe nanomechanical properties of individual GB1 domains in orderto modulate the mechanical properties of macroscopic biomaterials.
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Figure 2 | Mechanical properties of (G–R)4 and GRG5RG4R-basedbiomaterials. a, Photographs of moulded rings built from (G–R)4 (left,intact) and GRG5RG4R (right, after being loaded to failure in tensile test)under white light (middle panel) and ultraviolet illumination (top panel).b, c, Representative stress–strain curves of (G–R)4 (b) and GRG5RG4R(c) measured in PBS. For clarity, stress–strain curves are offset relative toone another. Final strains are shown on the curves. Insets show thesuperposition of the stress–strain curves at different strains. d, Resilience ofGB1–resilin-based biomaterials decreases with the increase of strain. Incontrast, biomaterials constructed from resilin do not show any appreciable
hysteresis (data taken from ref. 13). e, GRG5RG4R-based biomaterials canrecover hysteresis under residual stress. During stretching–relaxationexperiments, when the biomaterial is partially relaxed to a strain above 35%,no recovery of hysteresis was observed. When the biomaterial was relaxed tobelow 35% strain, we started to observe partial recovery. The degree ofrecovery increased with the decrease of residual stress. For clarity, the initialstretching trace is coloured blue. The inset shows the experimental protocolof the partial relaxation experiments. The pulling speed used in theexperiments was 25 mm min21. Error bars indicate standard deviation of thedata.
Folded globular domains are mechanically more resistant than theirunfolded conformations, but less extensible. Because chemical dena-turants can affect folded states of globular proteins, we used urea tomodulate the nanomechanical properties of GB1–resilin-basedelastomeric proteins. Figure 4a shows such an example forGRG5RG4R. In the presence of 4 M urea, about half of GB1 domainsare unfolded, resulting in the loss of their mechanical resistance. Sucha change is clearly evident in the force–extension relationships ofGRG5RG4R (Fig. 4a), which are characterized by long featurelessspacers before the unfolding events of the remaining folded GB1domains. Such long featureless spacers correspond to the stretchingof predominantly unfolded GB1 domains. The conversion of foldedGB1 into unfolded sequences leads to a dramatic decrease in Young’smodulus of the biomaterials in a urea-concentration-dependentfashion: the Young’s modulus reduced from ,60 kPa in PBS to,10 kPa in 8 M urea. We note that this change is fully reversible atboth molecular and macroscopic levels. Replacing urea with PBSallowed GB1 domains to refold and thereby regain their mechanicalresistance. Macroscopically, the biomaterial can recover its originalYoung’s modulus when replacing urea with PBS. This macroscopicchange in mechanical properties of biomaterials can readily beexplained with information from the single-molecule level: the con-version of folded GB1 domains into mechanically labile and moreextensible sequences effectively increased the length between cross-linking points, leading to the decrease in Young’s modulus of thematerial. Similarly, it is also possible to modulate the mechanicalproperties of these biomaterials (Supplementary Fig. 8) in otherways, such as adjusting the relative GB1/resilin content, just as thepassive elastic properties of different muscles are mediated by differ-ent isoforms of titin.
To fulfill their biological functions, different biological tissuespossess distinct mechanical properties. For example, mammalian ten-don is highly resilient (resilience .90%) but relatively inextensible(breaking strain of ,13%), whereas elastin is resilient (90%) andextensible (breaking strain of ,150%) but lacks toughness18.Mimicking the biomechanical properties of different tissues has beenan important challenge in biomaterials research. Here we havedesigned a muscle-mimetic biomaterial, which is highly resilient atlow strains, but also extensible and tough at high strain, to mimic thepassive elastic properties of muscles. Titin is largely responsible for thepassive elastic properties of myofibrils. A hallmark of titin-like elasto-meric proteins is their ability to unfold under a stretching force todissipate energy effectively and prevent damage to tissues by over-stretching6,8,10,11,16. The hysteresis and stress-relaxation observed instretching of myofibrils have been explained by force-induced unfold-ing of a small number of immunoglobulin domains16.
All these properties have been well reproduced in biomaterialsconstructed from GB1–resilin-based artificial elastomeric proteins.Therefore, GB1–resilin-based polyproteins mimic the architectureand mechanical properties of titin at the single-molecule level, andbiomaterials based on GB1–resilin polyproteins mimic the titin-mediated passive elastic properties of muscles (Supplementary
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Figure 3 | GB1–resilin-based biomaterials exhibit pronounced stressrelaxation behaviours. a, Representative stress-relaxation curves ofGRG5RG4R at varying strains. b, Relaxation rates of GRG5RG4R-basedbiomaterials depend upon the initial stress. The relaxation rates wereobtained by fitting the stress-relaxation to a double-exponential equation:s(t) 5 s0 1 A1exp(2k1t) 1 A2exp(2k2t), where s(t) is the stress at time t, s0
is the offset, A1 and A2 are decay amplitudes and k1 (filled squares) and k2
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Figure 4 | The macroscopic mechanical properties of GB1–resilin-basedbiomaterials can be fine-tuned by controlling the nanomechanicalproperties of the constituting elastomeric proteins at the single-moleculelevel. a, Force–extension curves of single GRG5RG4R molecules in PBS andin 4 M urea. The long featureless spacers observed in force-extension curvesof GRG5RG4R in 4 M urea largely correspond to the stretching ofmechanically labile, unfolded GB1 domains. The unfolding force of GB1domains that remain folded in 4 M urea is also significantly reduced. Greylines are WLC fits. b, Young’s modulus of GRG5RG4R-based biomaterial canbe modulated by chemical denaturant urea. The conversion of folded GB1domains into unfolded sequence leads to the dramatic decrease in Young’smodulus of the biomaterials in a urea-concentration-dependent manner.Error bars indicate standard deviation of the data.
Information). These designed biomaterials represent a new type ofmuscle-mimic, which is fully hydrated and biodegradable, and weanticipate that they will find applications in material sciences as wellas in tissue engineering by serving as scaffold and matrix for artificialmuscles. Moreover, our results indicate that nanomechanical pro-perties engineered into individual polyproteins can be translated intomacroscopic properties in materials, a new example of obtainingnovel macroscopic mechanical features by designing in such featuresat the single-molecule level. This method represents a new avenuetowards tailoring the macroscopic mechanical properties of bio-materials and can be applied to the design of a wide range of materials.
METHODS SUMMARYPreparation of GB1–resilin-based polyproteins were performed using previously
published protocols12,21. We used the 15-amino-acid consensus resilin repetitive
sequence (GGRPSDSYGAPGGGN) from the first exon of the Drosophila mela-
nogaster CG15920 gene to construct GB1–resilin-based elastomeric proteins13.
Single-molecule AFM experiments were performed on a custom-designed
atomic force microscope as described12. Hydrogel-like biomaterials of GB1–
resilin was constructed using a photochemical crosslinking strategy as
described13,23. Tensile tests were performed on an Instron-5500R tensometer
with a custom-made force gauge in PBS at constant temperature (22 uC). For
technical reasons, ring-shaped biomaterial specimens were used30 (Supplemen-tary Information). Resilience was calculated from the ratio of the area under the
relaxation curve to the area under the extension curve at a given strain using
custom-written software in Matlab. The local slope at 15% strain on the exten-
sion curve was taken as the modulus at 15% reported in the paper.
Received 22 September 2009; accepted 9 March 2010.
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Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.
Acknowledgements We thank M. Lillie and R. Shadwick for discussions. This workis supported by the Canadian Institutes of Health Research, the Canada ResearchChairs program, the Canada Foundation for Innovation, the Michael SmithFoundation for Health Research, and the Natural Sciences and EngineeringResearch Council of Canada. H.L. is a Michael Smith Foundation for HealthResearch Career Investigator.
Author Contributions H.L. conceived the project. H.L. and J.G. designed the overallexperiments. S.L., D.M.D., Y.C., M.M.B. and J.G. designed, performed individualexperiments and analysed data. H.L. wrote the manuscript and all authors editedthe manuscript.
Author Information Reprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.Correspondence and requests for materials should be addressed to H.L.([email protected]) or J.G. ([email protected]).
Supplementary Information Protein Engineering GB1-resilin-based polyprotein genes were constructed using standard molecular biology techniques following a well-established stepwise construction scheme1. The gene encoding protein GB1 was a generous gift from David Baker of University of Washington. One 15 amino acid consensus resilin repetitive sequence (GGRPSDSYGAPGGGN) from the first exon of the D. melanogaster gene CG15920 (Ref. 2) was used in this study to construct GB1-resilin based polyproteins. The DNA sequence of resilin, flanked with a 5’ BamHI restriction site and 3’ BglII and KpnI restriction sites, was synthesized by PCR (polymerase chain reaction) based oligonucleotide assembly. The expression vector of pQE80L-(GR)4 was constructed by iterative cloning of G and R genes into empty pQE80L vector, on the basis of the identity of the sticky ends generated by BamHI and BglII restriction enzymes. GRG5RG4R, GRG5R, GRG9R and G8 were constructed in the same way. The resulted polyproteins carry two additional cysteines at their C-termini, which can lead to the dimerization of some polyproteins. The expression of polyproteins was carried out in Escherichia coli strain DH5α. Cultures were grown at 37 °C in 2.5% LB containing 100mg/L ampicillin, and induced with 0.8mM isopropyl-1-β-D-thiogalactoside (IPTG) when the optical density reached ~1. Protein expression continued for 5 hours. The cells were harvested by centrifugation at 15,000g for 15min and cell lysis was done using lysozyme from egg white (100 mM, SigmaAldrich). The soluble fraction was purified using Ni2+ affinity chromatography. The yield of the polyproteins is in the range of 40mg to 50mg per liter of culture. Supplementary Fig. 1 shows Coomassie blue stained SDS-PAGE picture for the constructed proteins (G-R)4, GRG5RG4R, GRG5R and GRG9R. The purity of the purified polyproteins is around 90%, as estimated from the SDS-PAGE using AlphaEaseFC software (Version 4.0.0, Alpha Innotech Corporation, San Leandro, CA). The 10% “impurity” is likely to be truncated fragments of polyproteins, which are frequently observed in the expression of polyproteins, such as G8 as well as other polyproteins.
Figure S1. Coomassie blue stained PAGE gel for polyproteins (GR)4, GRG5RG4R, GRG5R, GRG9R and G8 (left to right). The first lane is the broad range molecular weight marker (New England Biolabs).
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The purified proteins were then used in single-molecule atomic force microscopy (AFM) experiments. To prepare GB1-resilin-based biomaterials, the purified proteins were then dialyzed against deionized water for 3 days to remove all the salt from elution buffer. During dialysis, the water was changed every 5 hours. The protein was lyophilized following dialysis.
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Figure S2. Far ultraviolet circular dichroism (CD) spectrum of R12 indicates that R12 is largely unstructured. To confirm that the resilin sequence used in our study is largely unstructured, polyprotein R12, which is composed of 12 identical tandem repeats of the 15 amino acid consensus sequence (GGRPSDSYGAPGGGN), was constructed following the strategy described for the engineering of (G-R)4. There is a two-amino acid linker Arg-Ser between R repeats in the engineered R12, which results from the restriction sites used in the construction of the gene of R12. The CD measurements were carried out on a Jasco-J810 spectropolarimeter. Our CD data showed a minimum in ellipticity at ~200 nm, indicating that the R12 is largely unstructured. Our result contradicts the study by Lyons and et al3. They showed that the CD spectrum of R16, which is composed of 16 identical repeats of the same resilin consensus sequence, is consistent with the existence of β-strand structure3. The origin of the discrepancy between these two studies is unknown. One noted difference between R12 and R16 is that there exists an Arg-Ser linker between R repeats in R12 while there is none in R16. Despite the discrepancy on the secondary structure of this resilin repeat sequence, single-molecule AFM results showed that resilin behaves largely as an entropic spring, and does not show measurable mechanical stability (see Fig. 2 in the main text). This result is consistent with our observation that R is largely unstructured.
Single-Molecule AFM Measurements
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Single molecule AFM experiments were performed on a custom-designed AFM following procedures described previously4. All the force-extension measurements were carried out in PBS, unless noted otherwise. In a typical experiment, ~1 μl polyprotein sample was deposited onto a clean glass cover slip covered by PBS buffer (50 mL) and was allowed to adsorb for approximately 5 min before force-extension measurements. The spring constant of each individual cantilever (Si3N4 cantilevers from Vecco, with a typical spring constant of 40 pN/nm) was calibrated in solution using the Equipartition Theorem before and after each experiment.
Figure S3. Mechanical properties of (G-R)4 and GRG5RG4R at the single-molecule level. A, B) Histogram of spacer length L0 of (G-R)4 and GRG5RG4R. For force-extension curves with similar number of GB1 domains (six or higher) for both polyproteins, the spacer length of (G-R)4 is longer than that for GRG5RG4R, due to the more resilin sequences presented in (G-R)4. C,D) Persistence length of resilins measured in both polyproteins yield similar values of 0.49 nm. E, F) Unfolding force histogram of GB1 domains in both proteins. The unfolding force for GB1 is 191±42 pN in (G-R)4 and 180±41 pN in GRG5RG4R
Preparation of Biomaterials We used a well-developed [Ru(bpy)3]2+-mediated photochemical crosslinking strategy5 to prepare GB1-resilin-based biomaterials. This photochemical strategy allows the crosslinking of two tyrosine residues that are in close proximity into a dityrosine adduct, and leads to rapid and quantitative formation of dityrosine crosslinks between soluble proteins. Supplementary Fig. S4 shows the photocrosslinking scheme and the schematic structure of the resultant biomaterials. It is of note that the majority of crosslinking sites are located in the resilin sequences, and a small fraction of the crosslinking sites originate from the exposed tyrosine residues in GB1. To prepare GB1-resilin-based biomaterials, lyophilized proteins were redissolved in phosphate saline buffer (PBS). In a typical experiment, 18 mg of the protein was weighted using analytical balance and added to a microcentrifuge tube containing 84.4μl
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of PBS (100mM, pH 7.4) and 4.5μl of APS (1M). The trapped air bubbles can be removed by centrifugation at 1000 g for 5 minutes. 0.9μl of [Ru(bpy)3]2+ (20mM) solution was then added to the microcentrifuge tube and quickly mixed with the protein solution by tapping the bottom of the tube. The final solution contains 200mg/ml of GB1-resilin polyprotein, ~200μM [Ru(bpy)3]2+ and 50 mM ammonium persulfate (APS) in PBS buffer. The solution was cast into a custom-made plexiglass mold with inner diameter of 8 mm, outer diameter of 10 mm and height of 3mm. The sample was then irradiated for 30 seconds using a 200 W fiber optical white light source. The irradiation was 10 cm away from the mold. The ring was then taken out from the mold and stored in PBS buffer (10mM with 0.05% (w/v) sodium azide). These chemically crosslinked biomaterials show superior long term stability, and no noticeable erosion was observed in PBS buffer (in the presence of 0.5% azide) over a period of one year.
Figure S4. Photocrosslinking scheme and the schematic structure of hydrogel based on GRG5RG4R. The mini-titin-mimic molecules are mainly crosslinked via tyrosine residues in the R sequences. Some of the solvent exposed tyrosine in the folded GB1 domains may also contribute to the photocrosslinking, as shown in the schematic.
The resultant rings show different degrees of swelling in PBS buffer, resulting in different initial dimensions of the rings (Figure S5, also see Fig. 2a in the main text). The swelling ratio of the rings was found to be consistent within the same batch, but varies across different batches. The Poisson ratio of these biomaterials was measured to be ~0.5. To determine the isotropy of the constructed biomaterials, we used polarized light microscopy to measure the birefringence of the biomaterials. Cubic samples of the biomaterials (5mm×5mm×1mm, width×length×height) were viewed between crossed polarizers on a Leitz Orthoplan polarizing microscope with a 10× achromatic-pol lens, and the retardation was determined at 546 nm using a Leitz Senarmont compensator and a 546 nm interference filter. The rotational angle between the maximum brightness and darkness was determined and the retardation caused by the sample is calculated as 3.03 nm per degree of the analyzer angle. Birefringence was then calculated by dividing the retardation of the cube by its thickness. The smallest birefringence we can resolve is ~3×10-6. Some areas of GB1-resilin-biomaterials showed unmeasurable birefringence,
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while other areas showed maximal birefringence of 5×10-6 to 1×10-5. This birefringence is about 20 to 50 times smaller than that of optically isotropic elastin samples (2×10-4)6, and vanishingly smaller than that of anisotropic major ampullate silks from A.diadematus (birefringence is 2.5×10-2 in the dry state and 6.1×10-3 in the hydrated, supercontracted state)7. This result strongly indicates that GB1-resilin-based biomaterials are optically isotropic.
Figure S5. Swelling ratio of different types of GB1-resiline-based crosslinked biomaterials samples in PBS (10mM). Error bar indicates standard deviation.
Tensile Testing The tensile tests were performed using an Instron-5500R tensometer with a custom-made force gage as described elsewhere.8 Unless otherwise noted, these tests were done at a constant temperature of 22 oC in PBS buffer (10mM). The fastest strain rate of this tensometer is 20 mm/s.
For technical consideration, the tensile testing did not follow an ASTM (American Society for Testing and Materials) standard through the use of dogbone-shaped specimens. Instead, we used ring-shaped specimens. Tensile testing of rings of materials was conducted to minimize difficulties that arise from gripping soft materials. Because the test strains are large in these experiments, gripped material would thin substantially upon stretching, so the material would need to be clamped so tightly that it would fail at the grips. Self adjusting pneumatic grips that automatically adjust for material thinning are designed for materials much stiffer than our hydrogel and would have the same problem of material failure or slippage. We followed previously published methods for testing arterial elastin rings9 to avoid these problems. An additional benefit of the ring method is that we can likely perform tests at significantly greater strains using rings. Lille found testing had to be limited to 40% strain when testing dogbone shaped purified elastin strips (M. Lillie, personal communication). Non-Gaussian behavior of this material does not begin until at least 60% strain and breaking strain is over 100%. Therefore, approved dogbone shapes could actually limit our ability to adequately describe the mechanical properties of this material.
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Figure S6. Consecutive stretching-relaxation curves of GRG5RG4R at a pulling speed of 200 mm/min. The waiting time between consecutive cycles was zero. These curves are superimposable, indicating that GB1-resilin-based biomaterials can fully recover their hysteresis. This behavior is consistent with the fast folding rate of GB1 domains measured in single-molecule AFM experiments4.
Monte Carlo simulations on the force-relaxation of GRG5RG4R Monte Carlo simulations on the force-relaxation of a single GRG5RG4R protein were carried out according to published protocols10. (GRG5RG4R)6 was used to mimic the polyprotein in a three dimensional network. During the simulations, the polyprotein was quickly stretched to a given extension, and then the stretching force was monitored as a function of time while the extension was kept constant. The unfolding of GB1 domains was described using the Bell-Evans model11, 12, )/exp()( 0 TkxFF BuΔ⋅= αα , where α(F) is the unfolding rate constant at a stretching force F, α0 is the intrinsic unfolding rate constant at zero force, Δxu is the unfolding distance and kBT is the thermal energy. The force-extension relationship of polyproteins was described using the WLC model of polymer elasticity. The persistence length of resilin sequence was taken as 0.5 nm, the same as that of the unfolded polypeptide chain, and the persistence length for the folded GB1 domains were taken as 10 nm. Using an unfolding rate constant of 1x10-4 s-1, an unfolding distance of 0.2 nm and a folding rate constant of 300 s-1, we simulated the force relaxation of GRG5RG4R at constant extensions and measured the extension-dependence of the relaxation rate (Fig. S7). It is clear that the relaxation rate observed in the force-relaxation of GRG5RG4R polyproteins showed similar extension-dependent behaviors and the force-relaxation can be well described using double exponentials. The fast-phase relaxation rate was observed to increase with the increase of strain, suggesting that the fast relaxation process in the stress-relaxation behaviors of GB1-resilin-based biomaterials is qualitatively consistent with the forced-unfolding of a few GB1 domains. However, the extension-dependent behaviors of the slow-phase relaxation rate are different from experimental data. These results suggest that other microscopic processes, which were not accounted for in Monte Carlo simulations, may also contribute to the stress-relaxation behaviors of GB1-resilin-based biomaterials. Moreover, the three
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dimensional networks as well as the random orientation of GB1-resilin-based elastomeric chains in the GB1-resilin-based biomaterials are not considered in Monte Carlo simulations. A more detailed model that combines the unfolding of individual GB1 domains, the three dimensional network architecture and possibly chain friction during stretching is required to adequately describe the stress-relaxation behavior at the macroscopic level.
Figure S7. Monte Carlo simulation on the force-relaxation behaviors of GRG5RG4R under constant extensions. A) Force-relaxation behavior of GRG5RG4R under constant extension. The force-relaxation behavior of GRG5RG4R is resulted from the forced unfolding of GB1 domains in the polyprotein, and the non-single exponential relaxation kinetics is due to the constant change of the stretching force during the experiment at a constant extension. B) Relaxation rate constant increases as a function of the initial force. The initial force is determined by the constant extension applied to the polyprotein during force-relaxation experiments. The bars indicate standard deviation of the fitted rate constants.
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Mimicking the passive elasticity of muscle in the full range of sarcomere length Titin is largely responsible for the passive elasticity of muscles. However, at
longer sarcomere length, which is often beyond the physiological range of sarcomere length, collagen molecules are recruited and contribute to the passive elasticity of muscles. The GB1-resilin-based biomaterials we designed largely mimic the passive elastic properties of muscles within the physiological range of sarcomere length. However, the lack of collagen-like molecules as well as structural organization of titin-mimetic proteins in the designed biomaterials make it impossible to mimic the passive mechanical properties of muscle in the full range of sarcomere length.
Figure S8. Mechanical properties of GB1-resilin-based biomaterials can be modulated by adjusting the content of GB1 and resilin. Experimental data is presented as average±standard deviation. To demonstrate such
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feasibility, we constructed elastomeric proteins GRG5R, GRG9R and G8, and their corresponding biomaterials. Preliminary results indicate that the modulus declines significantly (P ≤0.05) as GB1 domains increase when rings are tested to a strain of ~0.15. The significant trend continues when calculating the modulus from breaking tests. Rings with blocks of G-repeats break at significantly higher strains (P = 0.0001). However, there is no clear trend for breaking stress and ring type, although GRG5RG4R breaks at significantly higher stress than the other ring types. These results indicated that it is feasible to modulate the macroscopic properties of these biomaterials in a wide range by controlling the relative GB1/resilin content as well as crosslinking density. However, systematic work is needed to fully explore such approaches, including varying the modular sequences with the same GB1/resilin content, to modulate the mechanical properties of these novel biomaterials, just like how the passive elastic properties of different muscles are mediated by different isoforms of titin.
References 1. Carrion-Vazquez, M., Oberhauser, A. F., Fowler, S. B., Marszalek, P. E., Broedel,
S. E., Clarke, J. & Fernandez, J. M. Mechanical and chemical unfolding of a single protein: a comparison. Proc Natl Acad Sci U S A 96, 3694-3699 (1999).
2. 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 437, 999-1002 (2005).
3. 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 10, 3009-3014 (2009).
4. Cao, Y. & Li, H. Polyprotein of GB1 is an ideal artificial elastomeric protein. NatMater 6, 109-114 (2007).
5. Fancy, D. A. & Kodadek, T. Chemistry for the analysis of protein-protein interactions: rapid and efficient cross-linking triggered by long wavelength light. Proc Natl Acad Sci U S A 96, 6020-6024 (1999).
6. Aaron, B. B. & Gosline, J. M. Optical properties of single elastin fibres indicate random protein conformation. Nature 287, 865-867 (1980).
7. Savage, K. N. & Gosline, J. M. The role of proline in the elastic mechanism of hydrated spider silks. J Exp Biol 211, 1948-1957 (2008).
8. Bell, E. & Gosline, J. Mechanical design of mussel byssus: material yield enhances attachment strength. J Exp Biol 199, 1005-1017 (1996).
9. Lillie, M. A., Chalmers, G. W. & Gosline, J. M. The effects of heating on the mechanical properties of arterial elastin. Connect Tissue Res 31, 23-35 (1994).
10. Rief, M., Fernandez, J. M. & Gaub, H. E. Elastically coupled two-level systems as a model for biopolymer extensibility. Physical Review Letters 81, 4764-4767 (1998).
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11. Bell, G. I. Models for the specific adhesion of cells to cells. Science 200, 618-627 (1978).
12. Evans, E. Probing the relation between force--lifetime--and chemistry in single molecular bonds. Annu Rev Biophys Biomol Struct 30, 105-128 (2001).
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feasibility, we constructed elastomeric proteins GRG5R, GRG9R and G8, and their corresponding biomaterials. Preliminary results indicate that the modulus declines significantly (P ≤0.05) as GB1 domains increase when rings are tested to a strain of ~0.15. The significant trend continues when calculating the modulus from breaking tests. Rings with blocks of G-repeats break at significantly higher strains (P = 0.0001). However, there is no clear trend for breaking stress and ring type, although GRG5RG4R breaks at significantly higher stress than the other ring types. These results indicated that it is feasible to modulate the macroscopic properties of these biomaterials in a wide range by controlling the relative GB1/resilin content as well as crosslinking density. However, systematic work is needed to fully explore such approaches, including varying the modular sequences with the same GB1/resilin content, to modulate the mechanical properties of these novel biomaterials, just like how the passive elastic properties of different muscles are mediated by different isoforms of titin.
References 1. Carrion-Vazquez, M., Oberhauser, A. F., Fowler, S. B., Marszalek, P. E., Broedel,
S. E., Clarke, J. & Fernandez, J. M. Mechanical and chemical unfolding of a single protein: a comparison. Proc Natl Acad Sci U S A 96, 3694-3699 (1999).
2. 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 437, 999-1002 (2005).
3. 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 10, 3009-3014 (2009).
4. Cao, Y. & Li, H. Polyprotein of GB1 is an ideal artificial elastomeric protein. NatMater 6, 109-114 (2007).
5. Fancy, D. A. & Kodadek, T. Chemistry for the analysis of protein-protein interactions: rapid and efficient cross-linking triggered by long wavelength light. Proc Natl Acad Sci U S A 96, 6020-6024 (1999).
6. Aaron, B. B. & Gosline, J. M. Optical properties of single elastin fibres indicate random protein conformation. Nature 287, 865-867 (1980).
7. Savage, K. N. & Gosline, J. M. The role of proline in the elastic mechanism of hydrated spider silks. J Exp Biol 211, 1948-1957 (2008).
8. Bell, E. & Gosline, J. Mechanical design of mussel byssus: material yield enhances attachment strength. J Exp Biol 199, 1005-1017 (1996).
9. Lillie, M. A., Chalmers, G. W. & Gosline, J. M. The effects of heating on the mechanical properties of arterial elastin. Connect Tissue Res 31, 23-35 (1994).
10. Rief, M., Fernandez, J. M. & Gaub, H. E. Elastically coupled two-level systems as a model for biopolymer extensibility. Physical Review Letters 81, 4764-4767 (1998).
