Rapid Communication Computational Simulation and Analysis of a Candidate for the Design of a Novel Silk-Based Biopolymer Ewa I. Golas, Cezary Czaplewski Department of Chemistry, University of Gda nsk, Ul. Wita Stwosza 63, 80–308 Gda nsk, Poland Received 6 January 2014; revised 26 February 2014; accepted 20 March 2014 Published online 11 April 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22494
ABSTRACT: This work theoretically investigates the mechanical properties of a novel silk-derived biopolymer as polymerized in silico from sericin and elastin-like monomers. Molecular Dynamics simulations and Steered Molecular Dynamics were the principal computational methods used, the latter of which applies an external force onto the system and thereby enables an observation of its response to stress. The models explored herein are single-molecule approximations, and primarily serve as tools in a rational design process for the preliminary assessment of properties C 2014 Wiley Periodicals, in a new material candidate. V
Inc. Biopolymers 101: 915–923, 2014. Keywords:
sericin;
biopolymer;
steered
molecular
dynamics; computational; silk
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected]
INTRODUCTION
T
his study proposes a novel silk-based biopolymer and investigates its properties by purely computational methods. As such, sericin and elastin-like monomers were polymerized in silico to form a model system that was explored by Molecular Dynamics and
Correspondence to: Ewa I. Golas; e-mail: [email protected] C 2014 Wiley Periodicals, Inc. V
Biopolymers Volume 101 / Number 9
Steered Molecular Dynamics (SMD) simulations, the latter of which applies an external force to the system and can, therefore, provide insight into the material’s stress response. The aim of this work is the preliminary assessment of a potential biomaterials candidate by simple theoretical methods. Although it does not attempt to faithfully reproduce a biopolymer environment, it offers a means for introductory physical property estimation, and presents a fast and costeffective tool in the development process. Sericin protein occurs naturally as a waste product in the silk processing industry.1 It is one of the two major components of silk, and envelops the inner silk fibroin protein as a sticky material that enables fiber formation.2,3 Sericin protein does not occur as a single protein, but rather as a family whose molecular mass lies in a range of 20–310 kDa.2 Sericin proteins of various size may be obtained at different stages in silk processing,4 although washing with hot water will dissolve the largest species,4 in a process known as silk degumming.2,5 The protein is generally discarded along with the silk processing waste water,4 which given that sericin accounts for 25–30% of the cocoon’s weight,3 accounts for 50,000 tons annually of waste sericin per 1,000,000 tons of fresh cocoon.4 Sericin may be incorporated into biomaterials via blending, crosslinking, and copolymerization.5 From a physicochemical perspective, sericin is readily hydrolyzed5 and degraded by proteases,1,4 and contains a wealth of functional groups that facilitate crosslinking.3,6 In particular, the sericin chain is characterized by a strongly biased distribution of amino acids: 18 amino acids constitute the protein, with the majority being highly polar (and so containing free hydroxyl, amino, or carboxyl moieties)4; indeed, only 12.2% of all residues are nonpolar.1 Interestingly, 33.4 and 16.7% of those present consist of serine and aspartic acid,4 respectively. The protein is further biodegradable,1 and its addition to resins has produced similarly environmentally friendly materials.4 The recovery of sericin for application in the cosmetic, medical, and
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FIGURE 1 (A) Model of the sericin-only system. S1, S2, and S3 indicate sericin strands 1, 2, and 3, respectively. (B) Model of the sericin and elastomeric-motif system. S1, S2, and S3 indicate sericin strands 1, 2, and 3, respectively. E1 and E2 indicate the elastomeric repeats 1 and 2, respectively. Sericin strands S1, S2, and S3 are copies of the same monomer, which differ only in terms of their orientation and the residues through which individual strands were crosslinked. Analogously, E1 and E2 are monomers of the same elastomeric repeat. Sericin strands are shown in gold, blue, and green. Elastin-like motifs are in red and purple. Serine side chains are indicated in gray.
polymer fields has already advanced many practical biomaterials,1,4,5,7 with the protein exhibiting useful properties as a coagulant, UV protective agent, antioxidant, and chemo-protective species.5 Moreover, the tuning of physical properties for sericin-containing materials is also possible; foams have been optimized for their moisture absorption/desorption qualities,4,5 and coatings for their antifrosting potential.5 Sericin-containing polyurethane has offered desirable mechanical and thermal properties for application in fibers and molded objects.4 Polymerization of sericin with other materials is hence a highly feasible avenue for sericin recycling; although polymers containing sericin and additional synthetic components are common,8 this study suggests a fully silk-based biopolymer, wherein sericin is polymerized with an elastin-like motif derived from silk. Studies of the structure of silk have suggested a module-like nature for the construction of the silk fibroin.9 An amino acid motif associated with silk extendibility has therefore been suggested,9 and its presence in contiguous repeats is confirmed in the most extendible of silk forms, the major ampullate and flagelliform silks.9 This elastomeric repeat bears a structure similar to the fold of elastin,9 and has been successfully synthesized by recombinant DNA techniques,10 which foreshadows an avenue for the production of an eventual sericin material with an added elastomeric component. As pure sericin biopolymers often suffer from a lack of strength and elasticity,8 the addition of such an elastin-like motif could provide for a fully silk-based material that combines the desired properties of sericincontaining materials with enhanced resilience. Current experimental advances in sericin-based materials generate an impetus for and justify theoretical exploration, as
preliminary property prediction and testing can provide orientation to the development process, and render it more efficient. In the midst of a spectrum of sericin-based biomaterials and target properties, a priori in silico insight could provide significant, if not indispensable, advantages in terms of both research time and cost. The prospect of the herein investigated biomaterials candidate is bolstered by its similarity to existing sericinbased biopolymers. In particular, crosslinking of the component sericin and silk-motif units is coherent with the experimentally pursued techniques of crosslinking,3 blending, and copolymerization, which have been used as a response to the brittleness11,12 of pure sericin-only materials. As an example, a hydrogel of sericin blended with polyvinyl alcohol resulted in increased elasticity.11 Similarly, crosslinking in sericin-based membranes and gels was found to augment their strength.1,3,8 The incorporation of an engineered silk motif is also well founded; in fact, fibroin-type biopolymers have been formulated based on the union of fibroin- and elastin-like motifs by recombinant methods.11 Thus, this model offers a credible candidate that may be further investigated and assessed for its capacity as a practical biopolymer. The diversity of sericin-containing materials should encourage the use of theoretical tools in the early stages of materials development, as the exploration of structure-function relationships and the primary assessment of candidates can provide directional insight into their design. The theoretical tools used in this study are not meant to faithfully reproduce the detailed characteristics and physical properties of the models tested, but rather serve to offer preliminary property prediction from a physics-based perspective, which generates an efficient in silico Biopolymers
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FIGURE 2 Select trajectory snap-shot visualizations from the stretching process for each of the stretch types in the sericin-only system. Stretching (A) across the left junction, (B) across the right junction, (C) horizontally, (D) vertically, and (E) diagonally. Large spheres: reference distance (pull) sites. Small yellow spheres: crosslinking cysteine atoms. Sericin strands are shown in gold, blue, and green.
work-bench from which candidates may be proposed, evaluated, and further pursued.
RESULTS Post equilibration of both systems produced polymerized species of similar structure; Figures 1A and 1B illustrate the sericin-only and dual sericin and elastomeric motif models, respectively. Figures 2 and 3 depict frames from the stretching simulations of select trajectories from each of the stretch experiment types for both the sericin-only and sericin and elastomeric motif systems, respectively. Stretching of Biopolymers
either the sericin-only or sericin-elastomeric dual models displays a parallel initial response with the introduction of tension: the random coil progressively unravels and consequently elongates. The elastomeric repeat remains intact in the dual polymer, despite concurrent sericin monomer extension. Post unraveling, tension in the peptide backbone increases until finally the system reaches either a crosslink in the sericin-only system, or an elastomeric motif in the dual polymer. At this point in the dual system, the spring-like structure of the elastin-like motif begins to unwind.
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FIGURE 3 Select trajectory snap-shot visualizations from the stretching process for each of the stretch types in the sericin and elastin-like motif system. Stretching (A) across the left junction, (B) across the right junction, (C) horizontally, (D) vertically, and (E) diagonally. Large spheres: reference distance (pull) sites. Small yellow spheres: crosslinking cysteine atoms. Sericin strands are in gold, blue, and green. Elastin-like motifs are in red and purple.
Figures 4–7 compare the force curves as a function of the distance stretched for each stretch location. Figure 4A illustrates the force curves generated when the sericin-based sys-
tems are stretched across the left junction. The sericin-only species unravels up to a distance of approximately 36 A˚, after which the tension significantly rises as the simulation attempts Biopolymers
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FIGURE 4 (A) Left junction stretch, sericin-only system. (B) Left junction stretch, sericin and elastomeric repeat system. (C) Right junction stretch, sericin-only system. (D) Right junction stretch, sericin and elastomeric repeat system. All forces scaled by a factor of 100. Independent runs/trajectories are depicted by a different color.
to stretch the crosslinking bond between sericin monomers. Breakage of this bond, although not predicted due to the force field employed, would be expected in the immediate force vicinity; the dual sericin-elastomeric polymer, conversely, releases stress at this point by unwinding the elastomeric sequence, smoothly enabling continued unfolding at low force, as illustrated in Figure 4B. The right junction force curves (Figures 4C and 4D) exhibit a parallel response. Figure 5 portrays force curves for stretches that occur far from points of crosslink. In these stretches, both systems preferentially unwind sericin coil in response to tension, a relatively low-force operation. The dual sericin and elastomeric motif system behaves analogously to the sericin-only model, despite polymerization with the elastomeric repeat, which remains intact. No major peak in force occurs during the unwinding process as the pulling force propagates along the chain. Figure 6 portrays the force curves for a diagonal stretch: the system first uncoils and then reaches either a point of crosslink or the elastomeric motif. At this stage, the sericinonly model begins to climb a force maximum as stress in the Biopolymers
link between sericin monomers increases, analogous to the left and right junction stretches (Figure 4). Such an ascent in force would naturally be associated with eventual bond failure. This rise in force is circumvented in the dual model by the unfolding of the elastin-like motif, as is evident in Figure 6B, which maintains a flat force profile. System elasticity was examined by repeating stretch experiments on species that had already been stretched previously (following a short intervening MD relaxation period). Secondary stretch experiments on the sericin-only polymer (Figures 7A and 7C) indicated a system with a relatively moderate capacity for reproducing the initially obtained force curves; in fact, once stretched along the right junction, the resulting sericin-only system re-entered the final climb of its force curve at an earlier (by 20 A˚) stretch distance, reflecting incomplete elasticity and implicating plastic deformation. The sericin and elastomeric repeat model, however, presented a completely elastic behavior (Figure 7B), generating an analogous force curve to that of the initial stretch (Figure 4D). Figure 7C illustrates the secondary force curve for a repeated diagonal stretch for the
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FIGURE 5 (A) Horizontal junction stretch, sericin-only system. (B) Horizontal junction stretch, sericin and elastomeric repeat system. (C) Vertical junction stretch, sericin-only system. (D) Vertical junction stretch, sericin and elastomeric repeat system. All forces scaled by a factor of 100. Independent runs/trajectories are depicted by a different color.
sericin-only model. As was noted earlier, the secondary force curve peaks sooner on secondary stretching, in this case at a considerably shorter (by 60 A˚) stretch distance. The dual
model (Figure 7D), although eventually tailing-off gradually toward moderate forces at the end of the stretch, reveals a significant improvement in polymer elasticity; increasing the
FIGURE 6 (A) Diagonal junction stretch, sericin-only system. (B) Diagonal junction stretch, sericin and elastomeric repeat system. All forces scaled by a factor of 100. Independent runs/trajectories are depicted by a different color.
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FIGURE 7 (A) Secondary right junction stretch, sericin-only system. (B) Secondary right junction stretch, sericin and elastomeric repeat system. (C) Secondary diagonal junction stretch, sericin-only system. (D) Secondary diagonal junction stretch, sericin and elastomeric repeat system. All forces scaled by a factor of 100. Independent runs/trajectories are depicted by a different color.
interposed MD relaxation period could ameliorate the elastic response achieved. Nonetheless, given the recovery time per size of fragment relaxed, the dual polymer offers both a more rapid recovery rate and higher elasticity than the sericin-only system.
DISCUSSION Undoubtedly, the approximation of a biopolymer with a small polymer fragment in implicit solution, coupled to the lack of a rigorous treatment of the intermolecular and other frictional forces found in the bulk sample, renders a quantitative representation of the systems proposed difficult; nonetheless, locally based physical properties may be efficiently extracted, in addition to qualitative insight with the potential for bulk extrapolation. Stretching beyond typical distance expectations is also a useful exercise, as it enables the observation of inherent mechanical tendencies. In the equilibrated structures of both the sericin-only and dual sericin and elastomeric motif models, the sericin strands adopt an intertwined conformation, which is in accord with experimental work that reported the formaBiopolymers
tion of twisted fibers.2 The rather loose structure of the polymer is further in line with descriptions of sericin as largely dominated by amorphous random coil with occasional inserts of beta-sheet,1 whose manifestation is suggested to be a function of temperature, moisture absorption, and mechanical stretching.1 The structure of the elastomeric motif is concordant with that proposed by literature,10 wherein a spiral-like species similar to elastin is postulated, whose spring-like form is suggested to endow elasticity to silk fibers formed thereof.10 SMD simulations have been notorious for their difficulty in quantitatively reproducing atomic force microscopy (AFM) experiments,13,14 and overshooting forces for comparable in silico stretching experiments by over an order of magnitude is common.13 Responsible for this effect is the force field, which is optimized for equilibrium geometries, and the significantly faster force application rates that are generally used during a simulation.13,14 Nonetheless, SMD and AFM experiments can correlate well, with SMD reproducing the behavior of experimental events13; scaling of the simulated forces can reveal such relationships, and can further potentially provide
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FIGURE 8 (A) Sericin-only system topology. (B) Sericin and elastomeric repeat system topology. (C) Stretch location reference points. The indicated lysine residues in the original Ser1 sequence and elastomeric motif were substituted by cysteine, which enabled monomer crosslinking at these sites through the introduction of a cysteine–cysteine disulfide bond. In the sericin-only topology, strand S1 is linked to S2 through the cysteine-substituted side-chains of residues 89 and 84, respectively. Likewise, strand S2 is linked to S3 at residues 26. In the dual system, these two crosslinks are mediated by an intervening elastomeric monomer. For both models, S1 is crosslinked to S3 through residues 9 and 89, respectively. Stretching was conducted between the illustrated residue pairs on the sericin monomer frame (see Table I for residue numbers); only one stretch distance was pulled at a time. Stretch sites were analogous in both the sericin-only and dual systems.
detailed insight into mechanical events that have been experimentally shrouded by noise.13 Admittedly, mechanical details for structures sensitive to force application speeds cannot be recovered.13 Overestimation of the tensile force was also the case in the current work, although this is as expected, given the significantly faster stretch velocities applied than those typical to experiment.13–15 Similar protein-based experimental systems would anticipate bond breakage,15 although scaling the simulation forces recorded by two orders of magnitude can enable qualitative structure-function relationships to be drawn, and was accordingly done. In the case of new materials groundwork, such functional insight into material behavior can be especially useful. Primary stretch trials of the sericin and elastomeric motif polymer produced force curves with continued extension at low force in situations where the analogous sericin-only control systems had already began their ascent into their bond failure windows. Mechanistically, the elastomeric repeat behaved as a stress release mechanism that was activated only when the uncoiling of sericin chains along the direction of the applied force had already been exhausted. An increase in strength of the sericin polymer could therefore be predicted on the addition of the elastomeric motif to a potential polymerization mixture; indeed, crosslinking essays and blends with synthetic polymers have been already explored for this purpose.3,4,8 In bulk, the elastomeric motif should circumvent bond failure by providing an avenue for stress unloading in conformationally strained situations (especially across crosslink connections).
Moreover, the similar behavior of both models during periods of low stress was found to be primarily the result of random coil unwinding, which suggests that a sericin and elastomeric repeat polymer should exhibit the desired properties of sericin with the concomitant benefit of increased strength. Furthermore, the rapid recovery of the dual model in the intervening relaxation period prior to secondary stretching (when conducted) shows further promise of a resilient biopolymer. The sericin and elastomeric motif system was found to be significantly more elastic than its sericin-only analog. The nearideal elastic recovery of previously stretched systems across the right junction assigns an almost entirely elastic character to the elastomeric repeat, which is concurrent with descriptions of its core motif as having spring-like form and properties.9 Indeed, a spring-like conformation is recovered after initial stretching; this is in accord with the suggestion that the motif’s elasticity is due to its similarity to the b-turn spiral of elastin.9 Secondary stretching diagonally across the copolymer also reiterates a highly elastic species, despite the predominant propagation of the pulling force across not only an elastin-like motif, but also the random coil of component sericin monomers. Extrapolating, this enhanced elasticity should also be anticipated in the case of the prospect bulk copolymer. This work investigated the mechanical properties of a postulated sericin and elastin-like biopolymer by purely in silico methods, with parallel comparison to a sericin-only candidate. Although the methodology used did not aim to reproduce and characterize the biopolymers in their bulk Biopolymers
The Design of a Novel Silk-Based Biopolymer Table I System Stretch Location and Distances Stretch Region
Stretch Residues
Distance Stretched (A˚)
Left junction Right junction Horizontally Vertically Diagonally
S1[97]:S2[87] S2[19]:S3[34] S1[79]:S3[59] S2[55]:S3[104] S1[77]:S2[55]
60 60 240 240 240
E1 and E2 indicate elastomeric repeats 1 and 2, respectively. S1, S2, and S3 indicate sericin strands 1, 2, and 3, respectively.
environments, the study offered a useful tool for the estimation of mechanical tendencies based on the results of simulated stretching experiments for simplified model systems. Accordingly, the sericin and elastomeric-repeat polymer modeled was shown to exhibit both increased strength and elasticity when compared against its sericin-only analog, two features for which experimentally produced sericin-only biopolymers have generally been deficient.8 A refined potential candidate for a silk-based biopolymer is therefore proposed for further investigation, as arrived upon through the use of computational methods in a rational design approach whose goal is to streamline the biomaterials development process.
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20,000 cycles, using first a steepest decent and then conjugate gradient algorithm at 10,000 cycles. Implicit generalized Born solvent was employed for simulations, with a simulated salt concentration of 0.150 M, and a constant temperature of 300 K and Langevin dynamics. A 2-fs time step was used. Initial heating to 300 K was conducted slowly over 1 million steps, followed by 10 million steps of molecular dynamics simulation at 300 K. The systems velocities were then reset and each system was branched to form three parallel trajectories per system, each having molecular dynamics performed for another 0.75 million steps. Thus, two systems (sericin-only and the dual polymer, respectively), containing three independent trajectories each, were prepared. SMD runs were performed at a constant velocity of 1 mA˚/fs: an external force was thereby applied so as to maintain the above mentioned stretch rate between specified a-carbon atoms (Table I). Stretching was conducted analogously for both systems, at five independent stretch locations (across the left junction, the right junction, diagonally, horizontally, and vertically), as depicted in Figure 8C. The distance stretched was either 60 or 240 A˚ (Figure 8C, Table I), and entailed branching of the original independent trajectory to perform the stretch; accordingly, only one location was stretched at a time. Force data was collected every 25 steps. For both systems, a brief molecular dynamics relaxation run of 0.6 million steps was performed after the diagonal and right junction stretches, after which stretching was repeated on the relaxed structure. A Savitzky–Golay filter was applied to all the force curves generated, with scaling of the filtered forces by two orders of magnitude.
REFERENCES
MATERIALS AND METHODS Two computational biopolymer systems were formed: a control containing only sericin, and another consisting of both sericin and the elastomeric motif. Both were described in terms of an all-atom representation, as per the AMBER12 force field. The sericin-only system consisted of three fragments of the 110-residue fragment sericin 1 (Ser1)16 from Bombix mori (silk moth), which were crosslinked by cysteine SAS bonds between cysteine residues on complementary monomers. The sequence of sericin 1 was point-mutated so as to accommodate a cysteine residue at the indicated crosslink points; these points bore lysine residues in the original Ser1 sequence. Both lysine and cysteine residues occur at a frequency of 1–3% in sericin,5 and both may be readily crosslinked8; in the case of cysteine, heating is a simple and sufficient method.8 Cysteine bonds were selected as the crosslinking method due to existing parameterization in the simulation software used. The topology of the serine-only system is shown in Figure 8A. The sericin and elastomeric motif system contained an intervening elastomeric motif at crosslink points analogous to those of the sericin-only system. The elastomeric motif was based on a double repeat of the elastin-like flagelliform silk motif, and was taken as ((GPGGSGPGGY)2 GPGGC)2, where GPGGC was substituted for the suggested lysine-containing fragment GPGGK.10 The topology of the dual sericin-elastomeric motif system is illustrated in Figure 8B. Minimization and simulations used the AMBER12 force field and package, and were conducted on the Beowulf cluster at the faculty of Chemistry, University of Gdansk. All systems were minimized for
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1. Pawar, A. P.; Padamwar, M. N. J Sci Ind Res 2004, 63, 323–329. 2. Huang, J.; Valluzzi, R.; Bini, E.; Vernaglia, B.; Kaplan, D. J Biol Chem 2003, 278, 46117–46123. 3. Nayak, S.; Talukdar, S.; Kundu, S. C. Cell Tissue Res 2012, 347, 783–794. 4. Zhang, Y. Biotechnol Adv 2002, 20, 91–100. 5. Mondal, M.; Trivedy, K.; Kumar, S. N.; CJES 2007, 5, 63–76. 6. Kundu, S.; Kundu, B. Biomaterials 2012, 33, 7456–7467. 7. Cho, C.; Nahm, J.; Lee, K.; Choi, H.; Ahn, J. J Appl Polym Sci 2000, 80, 274–280. 8. Srichana, T.; Siritientong, T.; Aramwit, P. WM&R 2012, 30, 217–224. 9. Lewis, V.; Shipley, H.; Nichola, H.; Hayashi, C. Int J Biol Macromol 1999, 24, 271–275. 10. Conticello, P.; Wu, S.; Zhou, Y. Biomacromolecules 2001, 2, 111–125. 11. Mondal, M.; Trivedy, K.; Kumar, S. N. CJES 2007, 5, 63–76. 12. Gimenes, M. L.; Lui, L.; Feng, X. J Memb Sci 2007, 295, 71–79. 13. Peplowski, L.; Mikulska, K. J Mol Model 2011, 17, 2313–2323. 14. Ritchie, K.; Evans, E. Biophys J 1999, 76, 2439–2447. 15. Kreuzer, J.; Hanke, F. Biointerphases 2006, 1, 11. 16. K7QTQ2_BOMMO UniProtKB databank. Available at www. uniprot.org. Last accessed on November 1, 2013.
Reviewing Editor: Alan Mackie
ABSTRACT: This work theoretically investigates the mechanical properties of a novel silk-derived biopolymer as polymerized in silico from sericin and elastin-like monomers. Molecular Dynamics simulations and Steered Molecular Dynamics were the principal computational methods used, the latter of which applies an external force onto the system and thereby enables an observation of its response to stress. The models explored herein are single-molecule approximations, and primarily serve as tools in a rational design process for the preliminary assessment of properties C 2014 Wiley Periodicals, in a new material candidate. V
Inc. Biopolymers 101: 915–923, 2014. Keywords:
sericin;
biopolymer;
steered
molecular
dynamics; computational; silk
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected]
INTRODUCTION
T
his study proposes a novel silk-based biopolymer and investigates its properties by purely computational methods. As such, sericin and elastin-like monomers were polymerized in silico to form a model system that was explored by Molecular Dynamics and
Correspondence to: Ewa I. Golas; e-mail: [email protected] C 2014 Wiley Periodicals, Inc. V
Biopolymers Volume 101 / Number 9
Steered Molecular Dynamics (SMD) simulations, the latter of which applies an external force to the system and can, therefore, provide insight into the material’s stress response. The aim of this work is the preliminary assessment of a potential biomaterials candidate by simple theoretical methods. Although it does not attempt to faithfully reproduce a biopolymer environment, it offers a means for introductory physical property estimation, and presents a fast and costeffective tool in the development process. Sericin protein occurs naturally as a waste product in the silk processing industry.1 It is one of the two major components of silk, and envelops the inner silk fibroin protein as a sticky material that enables fiber formation.2,3 Sericin protein does not occur as a single protein, but rather as a family whose molecular mass lies in a range of 20–310 kDa.2 Sericin proteins of various size may be obtained at different stages in silk processing,4 although washing with hot water will dissolve the largest species,4 in a process known as silk degumming.2,5 The protein is generally discarded along with the silk processing waste water,4 which given that sericin accounts for 25–30% of the cocoon’s weight,3 accounts for 50,000 tons annually of waste sericin per 1,000,000 tons of fresh cocoon.4 Sericin may be incorporated into biomaterials via blending, crosslinking, and copolymerization.5 From a physicochemical perspective, sericin is readily hydrolyzed5 and degraded by proteases,1,4 and contains a wealth of functional groups that facilitate crosslinking.3,6 In particular, the sericin chain is characterized by a strongly biased distribution of amino acids: 18 amino acids constitute the protein, with the majority being highly polar (and so containing free hydroxyl, amino, or carboxyl moieties)4; indeed, only 12.2% of all residues are nonpolar.1 Interestingly, 33.4 and 16.7% of those present consist of serine and aspartic acid,4 respectively. The protein is further biodegradable,1 and its addition to resins has produced similarly environmentally friendly materials.4 The recovery of sericin for application in the cosmetic, medical, and
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FIGURE 1 (A) Model of the sericin-only system. S1, S2, and S3 indicate sericin strands 1, 2, and 3, respectively. (B) Model of the sericin and elastomeric-motif system. S1, S2, and S3 indicate sericin strands 1, 2, and 3, respectively. E1 and E2 indicate the elastomeric repeats 1 and 2, respectively. Sericin strands S1, S2, and S3 are copies of the same monomer, which differ only in terms of their orientation and the residues through which individual strands were crosslinked. Analogously, E1 and E2 are monomers of the same elastomeric repeat. Sericin strands are shown in gold, blue, and green. Elastin-like motifs are in red and purple. Serine side chains are indicated in gray.
polymer fields has already advanced many practical biomaterials,1,4,5,7 with the protein exhibiting useful properties as a coagulant, UV protective agent, antioxidant, and chemo-protective species.5 Moreover, the tuning of physical properties for sericin-containing materials is also possible; foams have been optimized for their moisture absorption/desorption qualities,4,5 and coatings for their antifrosting potential.5 Sericin-containing polyurethane has offered desirable mechanical and thermal properties for application in fibers and molded objects.4 Polymerization of sericin with other materials is hence a highly feasible avenue for sericin recycling; although polymers containing sericin and additional synthetic components are common,8 this study suggests a fully silk-based biopolymer, wherein sericin is polymerized with an elastin-like motif derived from silk. Studies of the structure of silk have suggested a module-like nature for the construction of the silk fibroin.9 An amino acid motif associated with silk extendibility has therefore been suggested,9 and its presence in contiguous repeats is confirmed in the most extendible of silk forms, the major ampullate and flagelliform silks.9 This elastomeric repeat bears a structure similar to the fold of elastin,9 and has been successfully synthesized by recombinant DNA techniques,10 which foreshadows an avenue for the production of an eventual sericin material with an added elastomeric component. As pure sericin biopolymers often suffer from a lack of strength and elasticity,8 the addition of such an elastin-like motif could provide for a fully silk-based material that combines the desired properties of sericincontaining materials with enhanced resilience. Current experimental advances in sericin-based materials generate an impetus for and justify theoretical exploration, as
preliminary property prediction and testing can provide orientation to the development process, and render it more efficient. In the midst of a spectrum of sericin-based biomaterials and target properties, a priori in silico insight could provide significant, if not indispensable, advantages in terms of both research time and cost. The prospect of the herein investigated biomaterials candidate is bolstered by its similarity to existing sericinbased biopolymers. In particular, crosslinking of the component sericin and silk-motif units is coherent with the experimentally pursued techniques of crosslinking,3 blending, and copolymerization, which have been used as a response to the brittleness11,12 of pure sericin-only materials. As an example, a hydrogel of sericin blended with polyvinyl alcohol resulted in increased elasticity.11 Similarly, crosslinking in sericin-based membranes and gels was found to augment their strength.1,3,8 The incorporation of an engineered silk motif is also well founded; in fact, fibroin-type biopolymers have been formulated based on the union of fibroin- and elastin-like motifs by recombinant methods.11 Thus, this model offers a credible candidate that may be further investigated and assessed for its capacity as a practical biopolymer. The diversity of sericin-containing materials should encourage the use of theoretical tools in the early stages of materials development, as the exploration of structure-function relationships and the primary assessment of candidates can provide directional insight into their design. The theoretical tools used in this study are not meant to faithfully reproduce the detailed characteristics and physical properties of the models tested, but rather serve to offer preliminary property prediction from a physics-based perspective, which generates an efficient in silico Biopolymers
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FIGURE 2 Select trajectory snap-shot visualizations from the stretching process for each of the stretch types in the sericin-only system. Stretching (A) across the left junction, (B) across the right junction, (C) horizontally, (D) vertically, and (E) diagonally. Large spheres: reference distance (pull) sites. Small yellow spheres: crosslinking cysteine atoms. Sericin strands are shown in gold, blue, and green.
work-bench from which candidates may be proposed, evaluated, and further pursued.
RESULTS Post equilibration of both systems produced polymerized species of similar structure; Figures 1A and 1B illustrate the sericin-only and dual sericin and elastomeric motif models, respectively. Figures 2 and 3 depict frames from the stretching simulations of select trajectories from each of the stretch experiment types for both the sericin-only and sericin and elastomeric motif systems, respectively. Stretching of Biopolymers
either the sericin-only or sericin-elastomeric dual models displays a parallel initial response with the introduction of tension: the random coil progressively unravels and consequently elongates. The elastomeric repeat remains intact in the dual polymer, despite concurrent sericin monomer extension. Post unraveling, tension in the peptide backbone increases until finally the system reaches either a crosslink in the sericin-only system, or an elastomeric motif in the dual polymer. At this point in the dual system, the spring-like structure of the elastin-like motif begins to unwind.
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FIGURE 3 Select trajectory snap-shot visualizations from the stretching process for each of the stretch types in the sericin and elastin-like motif system. Stretching (A) across the left junction, (B) across the right junction, (C) horizontally, (D) vertically, and (E) diagonally. Large spheres: reference distance (pull) sites. Small yellow spheres: crosslinking cysteine atoms. Sericin strands are in gold, blue, and green. Elastin-like motifs are in red and purple.
Figures 4–7 compare the force curves as a function of the distance stretched for each stretch location. Figure 4A illustrates the force curves generated when the sericin-based sys-
tems are stretched across the left junction. The sericin-only species unravels up to a distance of approximately 36 A˚, after which the tension significantly rises as the simulation attempts Biopolymers
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FIGURE 4 (A) Left junction stretch, sericin-only system. (B) Left junction stretch, sericin and elastomeric repeat system. (C) Right junction stretch, sericin-only system. (D) Right junction stretch, sericin and elastomeric repeat system. All forces scaled by a factor of 100. Independent runs/trajectories are depicted by a different color.
to stretch the crosslinking bond between sericin monomers. Breakage of this bond, although not predicted due to the force field employed, would be expected in the immediate force vicinity; the dual sericin-elastomeric polymer, conversely, releases stress at this point by unwinding the elastomeric sequence, smoothly enabling continued unfolding at low force, as illustrated in Figure 4B. The right junction force curves (Figures 4C and 4D) exhibit a parallel response. Figure 5 portrays force curves for stretches that occur far from points of crosslink. In these stretches, both systems preferentially unwind sericin coil in response to tension, a relatively low-force operation. The dual sericin and elastomeric motif system behaves analogously to the sericin-only model, despite polymerization with the elastomeric repeat, which remains intact. No major peak in force occurs during the unwinding process as the pulling force propagates along the chain. Figure 6 portrays the force curves for a diagonal stretch: the system first uncoils and then reaches either a point of crosslink or the elastomeric motif. At this stage, the sericinonly model begins to climb a force maximum as stress in the Biopolymers
link between sericin monomers increases, analogous to the left and right junction stretches (Figure 4). Such an ascent in force would naturally be associated with eventual bond failure. This rise in force is circumvented in the dual model by the unfolding of the elastin-like motif, as is evident in Figure 6B, which maintains a flat force profile. System elasticity was examined by repeating stretch experiments on species that had already been stretched previously (following a short intervening MD relaxation period). Secondary stretch experiments on the sericin-only polymer (Figures 7A and 7C) indicated a system with a relatively moderate capacity for reproducing the initially obtained force curves; in fact, once stretched along the right junction, the resulting sericin-only system re-entered the final climb of its force curve at an earlier (by 20 A˚) stretch distance, reflecting incomplete elasticity and implicating plastic deformation. The sericin and elastomeric repeat model, however, presented a completely elastic behavior (Figure 7B), generating an analogous force curve to that of the initial stretch (Figure 4D). Figure 7C illustrates the secondary force curve for a repeated diagonal stretch for the
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FIGURE 5 (A) Horizontal junction stretch, sericin-only system. (B) Horizontal junction stretch, sericin and elastomeric repeat system. (C) Vertical junction stretch, sericin-only system. (D) Vertical junction stretch, sericin and elastomeric repeat system. All forces scaled by a factor of 100. Independent runs/trajectories are depicted by a different color.
sericin-only model. As was noted earlier, the secondary force curve peaks sooner on secondary stretching, in this case at a considerably shorter (by 60 A˚) stretch distance. The dual
model (Figure 7D), although eventually tailing-off gradually toward moderate forces at the end of the stretch, reveals a significant improvement in polymer elasticity; increasing the
FIGURE 6 (A) Diagonal junction stretch, sericin-only system. (B) Diagonal junction stretch, sericin and elastomeric repeat system. All forces scaled by a factor of 100. Independent runs/trajectories are depicted by a different color.
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FIGURE 7 (A) Secondary right junction stretch, sericin-only system. (B) Secondary right junction stretch, sericin and elastomeric repeat system. (C) Secondary diagonal junction stretch, sericin-only system. (D) Secondary diagonal junction stretch, sericin and elastomeric repeat system. All forces scaled by a factor of 100. Independent runs/trajectories are depicted by a different color.
interposed MD relaxation period could ameliorate the elastic response achieved. Nonetheless, given the recovery time per size of fragment relaxed, the dual polymer offers both a more rapid recovery rate and higher elasticity than the sericin-only system.
DISCUSSION Undoubtedly, the approximation of a biopolymer with a small polymer fragment in implicit solution, coupled to the lack of a rigorous treatment of the intermolecular and other frictional forces found in the bulk sample, renders a quantitative representation of the systems proposed difficult; nonetheless, locally based physical properties may be efficiently extracted, in addition to qualitative insight with the potential for bulk extrapolation. Stretching beyond typical distance expectations is also a useful exercise, as it enables the observation of inherent mechanical tendencies. In the equilibrated structures of both the sericin-only and dual sericin and elastomeric motif models, the sericin strands adopt an intertwined conformation, which is in accord with experimental work that reported the formaBiopolymers
tion of twisted fibers.2 The rather loose structure of the polymer is further in line with descriptions of sericin as largely dominated by amorphous random coil with occasional inserts of beta-sheet,1 whose manifestation is suggested to be a function of temperature, moisture absorption, and mechanical stretching.1 The structure of the elastomeric motif is concordant with that proposed by literature,10 wherein a spiral-like species similar to elastin is postulated, whose spring-like form is suggested to endow elasticity to silk fibers formed thereof.10 SMD simulations have been notorious for their difficulty in quantitatively reproducing atomic force microscopy (AFM) experiments,13,14 and overshooting forces for comparable in silico stretching experiments by over an order of magnitude is common.13 Responsible for this effect is the force field, which is optimized for equilibrium geometries, and the significantly faster force application rates that are generally used during a simulation.13,14 Nonetheless, SMD and AFM experiments can correlate well, with SMD reproducing the behavior of experimental events13; scaling of the simulated forces can reveal such relationships, and can further potentially provide
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FIGURE 8 (A) Sericin-only system topology. (B) Sericin and elastomeric repeat system topology. (C) Stretch location reference points. The indicated lysine residues in the original Ser1 sequence and elastomeric motif were substituted by cysteine, which enabled monomer crosslinking at these sites through the introduction of a cysteine–cysteine disulfide bond. In the sericin-only topology, strand S1 is linked to S2 through the cysteine-substituted side-chains of residues 89 and 84, respectively. Likewise, strand S2 is linked to S3 at residues 26. In the dual system, these two crosslinks are mediated by an intervening elastomeric monomer. For both models, S1 is crosslinked to S3 through residues 9 and 89, respectively. Stretching was conducted between the illustrated residue pairs on the sericin monomer frame (see Table I for residue numbers); only one stretch distance was pulled at a time. Stretch sites were analogous in both the sericin-only and dual systems.
detailed insight into mechanical events that have been experimentally shrouded by noise.13 Admittedly, mechanical details for structures sensitive to force application speeds cannot be recovered.13 Overestimation of the tensile force was also the case in the current work, although this is as expected, given the significantly faster stretch velocities applied than those typical to experiment.13–15 Similar protein-based experimental systems would anticipate bond breakage,15 although scaling the simulation forces recorded by two orders of magnitude can enable qualitative structure-function relationships to be drawn, and was accordingly done. In the case of new materials groundwork, such functional insight into material behavior can be especially useful. Primary stretch trials of the sericin and elastomeric motif polymer produced force curves with continued extension at low force in situations where the analogous sericin-only control systems had already began their ascent into their bond failure windows. Mechanistically, the elastomeric repeat behaved as a stress release mechanism that was activated only when the uncoiling of sericin chains along the direction of the applied force had already been exhausted. An increase in strength of the sericin polymer could therefore be predicted on the addition of the elastomeric motif to a potential polymerization mixture; indeed, crosslinking essays and blends with synthetic polymers have been already explored for this purpose.3,4,8 In bulk, the elastomeric motif should circumvent bond failure by providing an avenue for stress unloading in conformationally strained situations (especially across crosslink connections).
Moreover, the similar behavior of both models during periods of low stress was found to be primarily the result of random coil unwinding, which suggests that a sericin and elastomeric repeat polymer should exhibit the desired properties of sericin with the concomitant benefit of increased strength. Furthermore, the rapid recovery of the dual model in the intervening relaxation period prior to secondary stretching (when conducted) shows further promise of a resilient biopolymer. The sericin and elastomeric motif system was found to be significantly more elastic than its sericin-only analog. The nearideal elastic recovery of previously stretched systems across the right junction assigns an almost entirely elastic character to the elastomeric repeat, which is concurrent with descriptions of its core motif as having spring-like form and properties.9 Indeed, a spring-like conformation is recovered after initial stretching; this is in accord with the suggestion that the motif’s elasticity is due to its similarity to the b-turn spiral of elastin.9 Secondary stretching diagonally across the copolymer also reiterates a highly elastic species, despite the predominant propagation of the pulling force across not only an elastin-like motif, but also the random coil of component sericin monomers. Extrapolating, this enhanced elasticity should also be anticipated in the case of the prospect bulk copolymer. This work investigated the mechanical properties of a postulated sericin and elastin-like biopolymer by purely in silico methods, with parallel comparison to a sericin-only candidate. Although the methodology used did not aim to reproduce and characterize the biopolymers in their bulk Biopolymers
The Design of a Novel Silk-Based Biopolymer Table I System Stretch Location and Distances Stretch Region
Stretch Residues
Distance Stretched (A˚)
Left junction Right junction Horizontally Vertically Diagonally
S1[97]:S2[87] S2[19]:S3[34] S1[79]:S3[59] S2[55]:S3[104] S1[77]:S2[55]
60 60 240 240 240
E1 and E2 indicate elastomeric repeats 1 and 2, respectively. S1, S2, and S3 indicate sericin strands 1, 2, and 3, respectively.
environments, the study offered a useful tool for the estimation of mechanical tendencies based on the results of simulated stretching experiments for simplified model systems. Accordingly, the sericin and elastomeric-repeat polymer modeled was shown to exhibit both increased strength and elasticity when compared against its sericin-only analog, two features for which experimentally produced sericin-only biopolymers have generally been deficient.8 A refined potential candidate for a silk-based biopolymer is therefore proposed for further investigation, as arrived upon through the use of computational methods in a rational design approach whose goal is to streamline the biomaterials development process.
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20,000 cycles, using first a steepest decent and then conjugate gradient algorithm at 10,000 cycles. Implicit generalized Born solvent was employed for simulations, with a simulated salt concentration of 0.150 M, and a constant temperature of 300 K and Langevin dynamics. A 2-fs time step was used. Initial heating to 300 K was conducted slowly over 1 million steps, followed by 10 million steps of molecular dynamics simulation at 300 K. The systems velocities were then reset and each system was branched to form three parallel trajectories per system, each having molecular dynamics performed for another 0.75 million steps. Thus, two systems (sericin-only and the dual polymer, respectively), containing three independent trajectories each, were prepared. SMD runs were performed at a constant velocity of 1 mA˚/fs: an external force was thereby applied so as to maintain the above mentioned stretch rate between specified a-carbon atoms (Table I). Stretching was conducted analogously for both systems, at five independent stretch locations (across the left junction, the right junction, diagonally, horizontally, and vertically), as depicted in Figure 8C. The distance stretched was either 60 or 240 A˚ (Figure 8C, Table I), and entailed branching of the original independent trajectory to perform the stretch; accordingly, only one location was stretched at a time. Force data was collected every 25 steps. For both systems, a brief molecular dynamics relaxation run of 0.6 million steps was performed after the diagonal and right junction stretches, after which stretching was repeated on the relaxed structure. A Savitzky–Golay filter was applied to all the force curves generated, with scaling of the filtered forces by two orders of magnitude.
REFERENCES
MATERIALS AND METHODS Two computational biopolymer systems were formed: a control containing only sericin, and another consisting of both sericin and the elastomeric motif. Both were described in terms of an all-atom representation, as per the AMBER12 force field. The sericin-only system consisted of three fragments of the 110-residue fragment sericin 1 (Ser1)16 from Bombix mori (silk moth), which were crosslinked by cysteine SAS bonds between cysteine residues on complementary monomers. The sequence of sericin 1 was point-mutated so as to accommodate a cysteine residue at the indicated crosslink points; these points bore lysine residues in the original Ser1 sequence. Both lysine and cysteine residues occur at a frequency of 1–3% in sericin,5 and both may be readily crosslinked8; in the case of cysteine, heating is a simple and sufficient method.8 Cysteine bonds were selected as the crosslinking method due to existing parameterization in the simulation software used. The topology of the serine-only system is shown in Figure 8A. The sericin and elastomeric motif system contained an intervening elastomeric motif at crosslink points analogous to those of the sericin-only system. The elastomeric motif was based on a double repeat of the elastin-like flagelliform silk motif, and was taken as ((GPGGSGPGGY)2 GPGGC)2, where GPGGC was substituted for the suggested lysine-containing fragment GPGGK.10 The topology of the dual sericin-elastomeric motif system is illustrated in Figure 8B. Minimization and simulations used the AMBER12 force field and package, and were conducted on the Beowulf cluster at the faculty of Chemistry, University of Gdansk. All systems were minimized for
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1. Pawar, A. P.; Padamwar, M. N. J Sci Ind Res 2004, 63, 323–329. 2. Huang, J.; Valluzzi, R.; Bini, E.; Vernaglia, B.; Kaplan, D. J Biol Chem 2003, 278, 46117–46123. 3. Nayak, S.; Talukdar, S.; Kundu, S. C. Cell Tissue Res 2012, 347, 783–794. 4. Zhang, Y. Biotechnol Adv 2002, 20, 91–100. 5. Mondal, M.; Trivedy, K.; Kumar, S. N.; CJES 2007, 5, 63–76. 6. Kundu, S.; Kundu, B. Biomaterials 2012, 33, 7456–7467. 7. Cho, C.; Nahm, J.; Lee, K.; Choi, H.; Ahn, J. J Appl Polym Sci 2000, 80, 274–280. 8. Srichana, T.; Siritientong, T.; Aramwit, P. WM&R 2012, 30, 217–224. 9. Lewis, V.; Shipley, H.; Nichola, H.; Hayashi, C. Int J Biol Macromol 1999, 24, 271–275. 10. Conticello, P.; Wu, S.; Zhou, Y. Biomacromolecules 2001, 2, 111–125. 11. Mondal, M.; Trivedy, K.; Kumar, S. N. CJES 2007, 5, 63–76. 12. Gimenes, M. L.; Lui, L.; Feng, X. J Memb Sci 2007, 295, 71–79. 13. Peplowski, L.; Mikulska, K. J Mol Model 2011, 17, 2313–2323. 14. Ritchie, K.; Evans, E. Biophys J 1999, 76, 2439–2447. 15. Kreuzer, J.; Hanke, F. Biointerphases 2006, 1, 11. 16. K7QTQ2_BOMMO UniProtKB databank. Available at www. uniprot.org. Last accessed on November 1, 2013.
Reviewing Editor: Alan Mackie
