International Journal of Biological Macromolecules 75 (2015) 398–401
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Influence factors analysis on the formation of silk I structure Jinfa Ming a,b , Fukui Pan a , Baoqi Zuo b,∗ a b
The College of Textiles & Fashion, Qingdao University, Qingdao 266071, China National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, China
a r t i c l e
i n f o
Article history: Received 26 September 2014 Received in revised form 2 February 2015 Accepted 3 February 2015 Available online 9 February 2015 Keywords: Silk fibroin Silk I Temperature–concentration–humidity
a b s t r a c t Regenerated silk fibroin aqueous solution was used to study the crystalline structure of Bombyx mori silk fibroin in vitro. By controlling environmental conditions and concentration of silk fibroin solution, it provided a means for the direct preparing silk I structure and understanding the details of silk fibroin molecules interactions in formation process. In this study, silk fibroin molecules were assembled to form random coil at low concentration of solution and then, as the concentration increases, were converted to silk I at 55% relative humidity (RH). At the same time, the structure of silk fibroin forming below 45 ◦ C was mostly in silk I. A partial ternary phase diagram of temperature–humidity–concentration was constructed based on the results. The results showed silk I structure could be controlled by adjusting the external environmental conditions. The enhanced control over silk I structure, as embodied in phase diagram, could potentially be utilized to understand the molecular chain conformation of silk I in further research work. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Many types of silk have been characterized, including silk fibroin (SF) from Bombyx mori, tussah silk, and dragline silk from the spider Nephila clavipes [1–3]. Bombyx mori silk contains two main proteins, fibroin and sericin. Fibroin is the structural protein, whereas sericin is the water-soluble glue like protein surrounding fibroin protein [4,5]. SF consists of two macromolecules, with molecular weights of 390 and 25 kDa for heavy and light chains, respectively [6,7]. The crystal regions of heavy chain are dominated by the hydrophobic sequence Gly-Ala-Gly-Ala-Gly-Ser (GAGAGS) amino acid motif [7,8]. Crystalline structure in SF has been an active topic of study for several decades [9]. The structure of two crystalline forms in SF, silk I and silk II, has been reported based on all kinds of experimental results [10–13]. The silk II form is characterized by an anti-parallel -sheet structure [11,14]. However, the less stable silk I form has remained poorly understand. At present, many investigations of the structure of silk I form have been studied [15–18]. These studies indicate that the conformation of silk I chain is a repeated -turn type II that is capable of forming intra-molecular hydrogen bonds [19]. At the same time, many
models with limited experimental data are proposed to describe the structure of silk I, such as crankshaft model [10], etc. Thus, in this present study, in order to better research the structure of silk I, the important thing is how to prepare the stable silk I structure using regenerated SF aqueous solution at room temperature. At our previous works, SF aqueous solutions were slowly concentrated to form silk I at stable 55% relative humidity (RH) [6]. This study was focused on elucidation of influence factors such as drying time, concentration, and temperature, etc. in controlling the forming process of silk I. At the same time, the role of water in silk I formation process was also discussed in detail. 2. Experimental 2.1. Materials Bombyx mori silks were bought from Zhejiang province, China. All chemicals (sodium carbonate, lithium bromide, ethanol, polyethylene glycol (PEG), etc.) were analytical grade from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China), and also used without further purification. 2.2. Preparation of SF solutions
∗ Corresponding author. Tel.: +86 512 67061157; fax: +86 512 67246786. E-mail addresses: [email protected] (J. Ming), [email protected] (F. Pan), [email protected] (B. Zuo). http://dx.doi.org/10.1016/j.ijbiomac.2015.02.002 0141-8130/© 2015 Elsevier B.V. All rights reserved.
Raw Bombyx mori silks were boiled for 30 min in aqueous solution of 0.02 M Na2 CO3 and then rinsed thoroughly with distilled water to extract the sericin proteins. After drying, degummed
J. Ming et al. / International Journal of Biological Macromolecules 75 (2015) 398–401
silk fibers were dissolved in 9.3 M LiBr solution at 60 ◦ C for 4 h, yielding a 10% (w/v) solution. This solution was dialyzed against distilled water using Slide-A-Lyzer dialysis cassettes (Sigma, USA, molecular weight cut-off 3500) for 3 days to remove the salt. The concentration of aqueous silk solution was ∼3.0 wt%, determined by weighting the remaining solid after drying. And then, SF aqueous solution (3.0 wt%, 50 ml) was dialyzed against 20 wt% PEG (10,000 g/mol) solution at 5 ◦ C by using Slide-A-Lyzer dialysis cassettes (MWCO 3500). The volume ratio of PEG to SF solution was 100:1. By osmotic stress, water molecules in SF solution moved into the PEG solution through the dialysis membrane obtaining higher concentrated solution. After the required time, the concentrated SF solution was collected by syringe to avoid excessive shear and the concentration (about 10.0 wt%) was determined by weighting the remaining solid after drying. Other concentration solution was obtained through diluted 10.0 wt% SF solution using distilled water. 2.3. Atomic force microscopy (AFM) The morphology of SF aqueous solution in silk I formation process was observed by AFM (Veeco, CA) in air. A 225 m long silicon cantilever with a spring constant of 3 N m−1 was used in tapping mode. For clearly observation, different time points of SF solution in silk I formation process were diluted to 1.0 × 106 to disperse SF molecules with deionized water. Once diluted, 1 L diluted SF solution was quickly dropped onto fleshly silica surface and dried under nitrogen gas. 2.4. X-ray diffraction To analyse the crystalline structure of SF films obtained from silk I formation process, X-ray diffraction (XRD) experiments were measured on X Pert-Pro MPD (PANalytical, Netherlands) with Cu K␣ radiation working at 40 kV and 40 mA in the interval range from 5◦ to 45◦ with a scan rate 2◦ min−1 . The incident beam wavelength was 0.154 nm. The intensity was finally corrected for changes in the incident beam intensity, sample absorption, and background. 2.5. Mechanical properties SF films with different crystalline forms (random coil, silk I, and silk II) were cut into 50 mm × 5 mm rectangles with thickness of 70–150 m. The thickness of these films was measured using a micrometer. Before using an automatic tensile tester (model 3365 electronic strength tester, Instron, Boston, USA) to characterize the mechanical properties of these films, these samples were kept for 24 h at standard atmospheric conditions (20 ◦ C, 65%RH). During test process, distances between grips and test speeds were set to 20 and 10 mm min−1 , respectively. At the same time, the pre-tension was 0.2 cN. An average of five measurements was reported as the mean ± standard deviation for each sample.
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Fig. 1. Effect of drying times on the conformation formation of silk I; the drying times were as follows: (a) 1 d, (b) 2 d, (c) 3.5 d, and (d) 5 d, respectively.
d, the main diffraction peaks were appeared at 12.4◦ , 20.1◦ , 24.5◦ , 27.9◦ , and 37.1◦ , corresponding to the crystalline spacing of 0.75 (I), 0.46 (I), 0.39 (I), 0.33 (I), and 0.26 (I) nm, respectively, attributing to typical silk I (Fig. 1c and d) [6,15]. 3.2. Effect of SF concentration on silk I formation In order to study the SF concentration affecting the silk I structure formation, this experiment was used equal solution (35 g) to study the SF structure formation at constant environmental condition (20 ◦ C, 55%RH). Fig. 2 depicted XRD results of SF films forming at different concentrations. Fig. 2a and b exhibited the typical random coil structure, characterized by the presence of a broad peak in the diffraction angle range from 5◦ to 45◦ at solution with lower concentrations (0.5 and 1.0 wt%). With the increase of SF concentration, Fig. 2c showed the diffraction peaks at 12.3◦ and 20.1◦ at 3.0 wt% concentrations, attributing to silk I (-turn) and silk II (sheet), respectively. At the same time, the diffraction peaks at 12.3◦ , 24.5◦ , 28.3◦ , 32.7◦ , and 36.9◦ were observed, when SF concentration was more than 7.4 wt% (Fig. 2d and e). These peaks were attributed to typical silk I structure. Therefore, SF molecules were assembled to form random coil at low concentration in solution and then, as the concentration increases, were converted to silk I. 3.3. Effect of temperature on silk I formation As known, SF molecules with different structures were formed in aqueous solution by self-assembly. In self-assembly process, the drying rate of aqueous solution was strongly correlated with the self-assembly rate of SF, which was decided by environmental temperatures. Tsukada studied effect of drying rate on the structure of tussah silk fibroin cast from aqueous solutions at different temperatures [20]. The results showed the drying rate played an important role in determining the molecular conformation taken by silk on drying [21]. At low drying rate, ␣-helix was formed.
3. Results 3.1. Effect of drying times on silk I formation In our previous study, silk I structure was directly formed when the environment humidity was 55% [6]. Therefore, this paper used SF aqueous solution (2.0 wt%) to study drying times affecting the formation of silk I. Fig. 1 showed XRD data of SF films preparing for different drying times at certain conditions (20 ◦ C, 55%RH). After drying 1 d, Fig. 1a depicted the typical random coil of SF, characterized by the presence of a broad peak in the diffraction angle range from 5◦ to 45◦ . When the drying time was 2 d, the main diffraction peaks were also located at 20.4◦ , attributing to random coil structure (Fig. 1b). However, increasing drying times more than 3
Fig. 2. Effect of SF concentration on the formation of silk I; the concentrations of SF aqueous solution were as follows: (a) 0.5 wt%, (b) 1.0 wt%, (c) 3.0 wt%, (d) 7.4 wt%, and (e) 10.0 wt%, respectively.
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4. Discussion
Fig. 3. XRD results of SF films cast at different temperatures: (a) 10 ◦ C, (b) 20 ◦ C, (c) 35 ◦ C, (d) 45 ◦ C, and (e) 60 ◦ C, respectively.
A conformational transition from ␣-helix to -sheet could be induced at low drying rate above 50 ◦ C. However, high drying rates prevented the transition even above 50 ◦ C, probably because the organization of the inter-chain hydrogen bonds typical of -sheet cannot be achieved under these conditions. Bombyx mori silk fibroin had three major conformations in the solid state, which were random coil, silk I (-turn), and silk II (sheet) [22]. Fig. 3 showed XRD results of SF films casting with equal solutions (50 g, 2.0 wt% concentration) at different temperatures with constant humidity (55%RH). At 10 ◦ C, Fig. 3a depicted the main diffraction peaks of SF films at 11.9◦ , 19.8◦ , 24.5◦ , 27.8◦ , and 36.7◦ , which were typical of SF in silk I structure. When the temperature increased to 45 ◦ C, the main diffraction peaks being located at 11.9◦ , 19.8◦ , 24.5◦ , 27.8◦ , and 36.7◦ , were also appeared (Fig. 3b–d), attributing to silk I structure. However, SF films were formed by self-assembly at 60 ◦ C, and its structure was characterized by XRD. Fig. 3e showed the diffraction peaks of SF films at 9.2◦ , 20.2◦ , and 24.5◦ for silk II. These results confirmed that the drying temperature was a key parameter in determining the structure taken by SF. What’s more, the drying temperature was strongly correlated to the drying rates. SF in films forming below 45 ◦ C was mostly in silk I structure. This finding can be explained by slow crystallization for SF from water during casting at lower temperatures.
Crystalline dimorphism in Bombyx mori silk fibroin has been an active topic of study for several decades [9,10,23,24]. There are two known crystalline forms of silk fibroin, silk I and silk II [25]. Silk II has a monoclinic unit cell with the protein chains in a pleated sheet (twofold “zigzag”) conformation [26], and its structure is well understood. The second polymorph, silk I, is not well characterized, because it has not been possible to obtain oriented samples. The exact structure of silk I and its poly(Ala-Gly) analogue may involve a “crankshaft” chain conformation [10]. Therefore, understanding the natural of silk I structure is an essential step to understand the natural silk spinning process. In this study, silk I structure formation was investigated in vitro using regenerated SF aqueous solution. In our previous study, silk I structure was easily obtained using regenerated SF aqueous solution at constant environmental conditions [6]. In the process of silk I formation, SF molecules were assembled from nanospheres to irregular networks through noncovalent bonds (Fig. S1). These bonds included hydrogen bonds, ionic bonds, water-mediated hydrogen bonds, hydrophobic and van der Waals interactions, etc. [6]. For the formation of silk I structure, it was stable at lower temperature such as 5 ◦ C or −20 ◦ C, not transforming silk II (-sheet) (Fig. S2). From Fig. 2, the formation of silk I structure was correlated to the concentration of SF solution. In order to study the existence state of SF molecules in aqueous solution to influence the formation of silk I structure, solution with different SF molecules morphologies was used. Firstly, fleshly SF aqueous solution was prepared with 3.0 wt% concentration. SF nanospheres with 20–50 nm were observed in solution (Fig. 4a). And then, after concentrating, SF solution with 10.0 wt% concentration was obtained. In this concentrated process, SF nanofibrils were formed and composed with many nanospheres by self-assembly (Fig. 4b) [27]. However, through experimental verification, silk I structure can be formed through using SF aqueous solutions with different molecule morphologies (Fig. 5). In addition, the mechanical properties of films with different structures were analysed at dry state (Fig. S3). The mechanical stress of SF films with silk II was 47.51 ± 5.86 MPa. However, the mechanical stress of SF films with silk I had similar to SF films with silk II, reaching 38.68 ± 6.21 MPa. Therefore, the comparison indicated similar mechanical behaviour of SF films with different structures. This phenomenon may be attributed to the self-assembly process of the sequence GAGAGS amino acid motif in structure formation.
Fig. 4. The existence state of SF macromolecules in different concentration solutions: (a) 3.0 wt%, (b) 10.0 wt%.
J. Ming et al. / International Journal of Biological Macromolecules 75 (2015) 398–401
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lower temperature such as 5 ◦ C or −20 ◦ C, not transforming to silk II. Therefore, using environmental factors to control silk I structure may be a useful approach for further understanding the molecular chain conformation of silk I and preparing novel silk-based materials. Acknowledgements We gratefully acknowledge the support of the Second Phase of Jiangsu Universities’ Distinctive Discipline Development Program for Textile Science and Engineering of Soochow University, National Science Foundation of China (No. 81271723), and National Engineering Laboratory for Modern Silk. Appendix A. Supplementary data Fig. 5. Partial ternary phase diagram of silk I structure formation (, random coil; 䊉, silk I; , silk II; , coexistence between random coil and silk I; ♦, coexistence between silk I and silk II).
Fig. 5 was the partial ternary phase-diagram of silk I structure, based on the above results. Phase boundaries between random coil and silk I and between silk I and silk II were shown by dashed lines. The structure of SF was obviously influenced under the effect of temperature and humidity. The dashed line at 55% RH was the important formation condition for silk I structure, which was confirmed by XRD results (Fig. 3). At the same time, samples indicated in Fig. 5 as displaying phase coexistence occur between random coil and silk I and between silk I and silk II. Coexistence is identified by the superposition of characteristic diffraction patterns. At the same time, Fig. 5 depicted the presence of both silk I and silk II structures in samples at the range of temperature and humidity conditions. This indicated different structures formation of SF was correlated to SF molecules self-assembly process. The self-assembly process of SF molecules was directly affected by the volatilization rate of water molecules in aqueous solution at constant environmental conditions. Sohn [28] studied the phase behaviour and hydration of silk fibroin. The results showed the presence of both silk I and silk II structures in SF samples with ranges of water content. Whether silk I structure was the hydrated crystal structures or mesophases, this question was beyond the scope of this study, as has been suggested for silk I by researchers [29,30]. In addition, silk II with intermolecular hydrogen bonding was identified by diffraction in Fig. 5. What is important is that no such strong statement can be made about the nature of the samples identified as silk I in Fig. 5. 5. Conclusions The crystalline structure of Bombyx mori silk fibroin was investigated in vitro using regenerated SF aqueous solution at different environmental conditions. In our study, silk I structure was directly formed when the environmental humidity was 55%RH. SF molecules were assembled to form random coil at lower solution concentration and then, as the concentration increases, were converted to silk I. At the same time, the drying temperature was a key parameter in determining the structure of SF. What’s more, it was strongly correlated to the drying rates of solution. SF in films forming below 45 ◦ C was mostly in silk I structure. The important point was that a partial ternary phase diagram of temperature–humidity–concentration was drawn based on X-ray data. This diagram suggested silk I structure could be obtained using regenerated SF aqueous solution by adjusting the external environmental conditions. The obtained silk I structure was stable at
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac. 2015.02.002. References [1] [2] [3] [4] [5] [6] [7]
[8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30]
H.J. Jin, D.L. Kaplan, Nature 424 (2003) 1057–1061. J. Magoshi, Y. Magoshi, S. Nakamura, J. Appl. Polym. Sci. 21 (1977) 2405–2407. F. Vollrath, D.P. Knight, Nature 410 (2001) 541–548. M. Roy, S.K. Meena, T.S. Kusurkar, S.K. Singh, N.K. Sethy, K. Bhargava, S. Sarkar, M. Das, Biointerphases 7 (2012) 1–11. H. Zhang, J. Magoshi, M. Becker, J.Y. Chen, R. Matsunaga, J. Appl. Polym. Sci. 86 (2002) 1817–1820. J.F. Ming, B.Q. Zuo, J. Appl. Polym. Sci. 125 (2012) 2148–2154. D.M. Phillips, L.F. Drummy, D.G. Conrady, D.M. Fox, R.R. Naik, M.O. Stone, P.C. Trulove, H.C.D. Long, R.A. Mantz, J. Am. Chem. Soc. 126 (2004) 14350– 14351. T. Asakura, J. Ashida, T. Yamane, T. Kameda, Y. Nakazawa, K. Ohgo, K. Komatsu, J. Mol. Biol. 306 (2001) 291–305. B. Kundu, R. Rajkhowa, S.C. Kundu, X.G. Wang, Adv. Drug Deliv. Rev. 65 (2013) 457–470. K. Okushita, A. Asano, M.P. Williamson, T. Asakura, Macromolecules 47 (2014) 4308–4316. Y. Jin, Y.C. Hang, J. Luo, Y.P. Zhang, H.L. Shao, X.C. Hu, Int. J. Biol. Macromol. 62 (2013) 162–166. M. Demura, M. Minami, T. Asakura, T.A. Cross, J. Am. Chem. Soc. 120 (1998) 1300–1308. T. Asakura, M. Okonogi, Y. Nakazawa, K. Yamauchi, J. Am. Chem. Soc. 128 (2006) 6231–6238. P. Cebe, X. Hu, D.L. David, E. Zhuravlev, A. Wurm, D. Arbeiter, C. Schick, Sci. Rep. 3 (2013) 1130. Q. Lu, X. Hu, X.Q. Wang, J.A. Kluge, S.Z. Lu, P. Cebe, D.L. Kaplan, Acta Biomater. 6 (2010) 1380–1387. T. Asakura, Y. Suzuki, Y. Nakazawa, K. Yazawa, G.P. Holland, J.L. Yarger, Prog. Nucl. Magn. Reson. Spectrosc. 69 (2013) 23–68. X. Chen, D.P. Knight, Z.Z. Shao, Soft Matter 5 (2009) 2777–2781. T. Asakura, R. Sugino, J.M. Yao, H. Takashima, R. Kishore, Biochemistry 41 (2002) 4415–4424. T. Asakura, Y. Suzuki, Y. Nakazawa, Biotechnol. Silk 5 (2014) 49–68. M. Tsukada, J. Polym. Sci. Part B Polym. Phys. 24 (1986) 457–460. P. Monti, G. Freddi, A. Bertoluzza, N. Kasai, M. Tsukada, J. Raman Spectrosc. 29 (1998) 297–304. X. Chen, Z.Z. Shao, N. Marinkovic, L.M. Miller, P. Zhou, M.R. Chance, Biophys. Chem. 89 (2001) 25–34. J.F.C. Mayen, A. Lupan, C. Cosar, A. Kun, R. Silaghi-Dumitrescu, Biophys. Chem. 197 (2015) 10–17. S.W. Ha, H.S. Gracz, A.E. Tonelli, S.M. Hudson, Biomacromolecules 6 (2005) 2563–2569. K. Okuyama, R. Somashekar, K. Noguchi, S. Ichimura, Biopolymers 59 (2001) 310–319. R. Valluzzi, S.P. Gido, W.P. Zhang, W.S. Muller, D.L. Kaplan, Macromolecules 29 (1996) 8606–8614. S. Bai, S. Liu, C. Zhang, W. Xu, Q. Lu, H. Han, D.L. Kaplan, H. Zhu, Acta Biomater. 9 (2013) 7806–7813. S. Sohn, H.H. Strey, S.P. Gido, Biomacromolecules 5 (2004) 751–757. M.T. Krejchi, S.J. Cooper, Y. Deguchi, E.D.T. Atkins, M.J. Fournier, T.L. Mason, D.A. Tirrell, Macromolecules 30 (1997) 5012–5024. S. Sohn, S.P. Gido, Biomacromolecules 10 (2009) 2086–2091.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Influence factors analysis on the formation of silk I structure Jinfa Ming a,b , Fukui Pan a , Baoqi Zuo b,∗ a b
The College of Textiles & Fashion, Qingdao University, Qingdao 266071, China National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, China
a r t i c l e
i n f o
Article history: Received 26 September 2014 Received in revised form 2 February 2015 Accepted 3 February 2015 Available online 9 February 2015 Keywords: Silk fibroin Silk I Temperature–concentration–humidity
a b s t r a c t Regenerated silk fibroin aqueous solution was used to study the crystalline structure of Bombyx mori silk fibroin in vitro. By controlling environmental conditions and concentration of silk fibroin solution, it provided a means for the direct preparing silk I structure and understanding the details of silk fibroin molecules interactions in formation process. In this study, silk fibroin molecules were assembled to form random coil at low concentration of solution and then, as the concentration increases, were converted to silk I at 55% relative humidity (RH). At the same time, the structure of silk fibroin forming below 45 ◦ C was mostly in silk I. A partial ternary phase diagram of temperature–humidity–concentration was constructed based on the results. The results showed silk I structure could be controlled by adjusting the external environmental conditions. The enhanced control over silk I structure, as embodied in phase diagram, could potentially be utilized to understand the molecular chain conformation of silk I in further research work. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Many types of silk have been characterized, including silk fibroin (SF) from Bombyx mori, tussah silk, and dragline silk from the spider Nephila clavipes [1–3]. Bombyx mori silk contains two main proteins, fibroin and sericin. Fibroin is the structural protein, whereas sericin is the water-soluble glue like protein surrounding fibroin protein [4,5]. SF consists of two macromolecules, with molecular weights of 390 and 25 kDa for heavy and light chains, respectively [6,7]. The crystal regions of heavy chain are dominated by the hydrophobic sequence Gly-Ala-Gly-Ala-Gly-Ser (GAGAGS) amino acid motif [7,8]. Crystalline structure in SF has been an active topic of study for several decades [9]. The structure of two crystalline forms in SF, silk I and silk II, has been reported based on all kinds of experimental results [10–13]. The silk II form is characterized by an anti-parallel -sheet structure [11,14]. However, the less stable silk I form has remained poorly understand. At present, many investigations of the structure of silk I form have been studied [15–18]. These studies indicate that the conformation of silk I chain is a repeated -turn type II that is capable of forming intra-molecular hydrogen bonds [19]. At the same time, many
models with limited experimental data are proposed to describe the structure of silk I, such as crankshaft model [10], etc. Thus, in this present study, in order to better research the structure of silk I, the important thing is how to prepare the stable silk I structure using regenerated SF aqueous solution at room temperature. At our previous works, SF aqueous solutions were slowly concentrated to form silk I at stable 55% relative humidity (RH) [6]. This study was focused on elucidation of influence factors such as drying time, concentration, and temperature, etc. in controlling the forming process of silk I. At the same time, the role of water in silk I formation process was also discussed in detail. 2. Experimental 2.1. Materials Bombyx mori silks were bought from Zhejiang province, China. All chemicals (sodium carbonate, lithium bromide, ethanol, polyethylene glycol (PEG), etc.) were analytical grade from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China), and also used without further purification. 2.2. Preparation of SF solutions
∗ Corresponding author. Tel.: +86 512 67061157; fax: +86 512 67246786. E-mail addresses: [email protected] (J. Ming), [email protected] (F. Pan), [email protected] (B. Zuo). http://dx.doi.org/10.1016/j.ijbiomac.2015.02.002 0141-8130/© 2015 Elsevier B.V. All rights reserved.
Raw Bombyx mori silks were boiled for 30 min in aqueous solution of 0.02 M Na2 CO3 and then rinsed thoroughly with distilled water to extract the sericin proteins. After drying, degummed
J. Ming et al. / International Journal of Biological Macromolecules 75 (2015) 398–401
silk fibers were dissolved in 9.3 M LiBr solution at 60 ◦ C for 4 h, yielding a 10% (w/v) solution. This solution was dialyzed against distilled water using Slide-A-Lyzer dialysis cassettes (Sigma, USA, molecular weight cut-off 3500) for 3 days to remove the salt. The concentration of aqueous silk solution was ∼3.0 wt%, determined by weighting the remaining solid after drying. And then, SF aqueous solution (3.0 wt%, 50 ml) was dialyzed against 20 wt% PEG (10,000 g/mol) solution at 5 ◦ C by using Slide-A-Lyzer dialysis cassettes (MWCO 3500). The volume ratio of PEG to SF solution was 100:1. By osmotic stress, water molecules in SF solution moved into the PEG solution through the dialysis membrane obtaining higher concentrated solution. After the required time, the concentrated SF solution was collected by syringe to avoid excessive shear and the concentration (about 10.0 wt%) was determined by weighting the remaining solid after drying. Other concentration solution was obtained through diluted 10.0 wt% SF solution using distilled water. 2.3. Atomic force microscopy (AFM) The morphology of SF aqueous solution in silk I formation process was observed by AFM (Veeco, CA) in air. A 225 m long silicon cantilever with a spring constant of 3 N m−1 was used in tapping mode. For clearly observation, different time points of SF solution in silk I formation process were diluted to 1.0 × 106 to disperse SF molecules with deionized water. Once diluted, 1 L diluted SF solution was quickly dropped onto fleshly silica surface and dried under nitrogen gas. 2.4. X-ray diffraction To analyse the crystalline structure of SF films obtained from silk I formation process, X-ray diffraction (XRD) experiments were measured on X Pert-Pro MPD (PANalytical, Netherlands) with Cu K␣ radiation working at 40 kV and 40 mA in the interval range from 5◦ to 45◦ with a scan rate 2◦ min−1 . The incident beam wavelength was 0.154 nm. The intensity was finally corrected for changes in the incident beam intensity, sample absorption, and background. 2.5. Mechanical properties SF films with different crystalline forms (random coil, silk I, and silk II) were cut into 50 mm × 5 mm rectangles with thickness of 70–150 m. The thickness of these films was measured using a micrometer. Before using an automatic tensile tester (model 3365 electronic strength tester, Instron, Boston, USA) to characterize the mechanical properties of these films, these samples were kept for 24 h at standard atmospheric conditions (20 ◦ C, 65%RH). During test process, distances between grips and test speeds were set to 20 and 10 mm min−1 , respectively. At the same time, the pre-tension was 0.2 cN. An average of five measurements was reported as the mean ± standard deviation for each sample.
399
Fig. 1. Effect of drying times on the conformation formation of silk I; the drying times were as follows: (a) 1 d, (b) 2 d, (c) 3.5 d, and (d) 5 d, respectively.
d, the main diffraction peaks were appeared at 12.4◦ , 20.1◦ , 24.5◦ , 27.9◦ , and 37.1◦ , corresponding to the crystalline spacing of 0.75 (I), 0.46 (I), 0.39 (I), 0.33 (I), and 0.26 (I) nm, respectively, attributing to typical silk I (Fig. 1c and d) [6,15]. 3.2. Effect of SF concentration on silk I formation In order to study the SF concentration affecting the silk I structure formation, this experiment was used equal solution (35 g) to study the SF structure formation at constant environmental condition (20 ◦ C, 55%RH). Fig. 2 depicted XRD results of SF films forming at different concentrations. Fig. 2a and b exhibited the typical random coil structure, characterized by the presence of a broad peak in the diffraction angle range from 5◦ to 45◦ at solution with lower concentrations (0.5 and 1.0 wt%). With the increase of SF concentration, Fig. 2c showed the diffraction peaks at 12.3◦ and 20.1◦ at 3.0 wt% concentrations, attributing to silk I (-turn) and silk II (sheet), respectively. At the same time, the diffraction peaks at 12.3◦ , 24.5◦ , 28.3◦ , 32.7◦ , and 36.9◦ were observed, when SF concentration was more than 7.4 wt% (Fig. 2d and e). These peaks were attributed to typical silk I structure. Therefore, SF molecules were assembled to form random coil at low concentration in solution and then, as the concentration increases, were converted to silk I. 3.3. Effect of temperature on silk I formation As known, SF molecules with different structures were formed in aqueous solution by self-assembly. In self-assembly process, the drying rate of aqueous solution was strongly correlated with the self-assembly rate of SF, which was decided by environmental temperatures. Tsukada studied effect of drying rate on the structure of tussah silk fibroin cast from aqueous solutions at different temperatures [20]. The results showed the drying rate played an important role in determining the molecular conformation taken by silk on drying [21]. At low drying rate, ␣-helix was formed.
3. Results 3.1. Effect of drying times on silk I formation In our previous study, silk I structure was directly formed when the environment humidity was 55% [6]. Therefore, this paper used SF aqueous solution (2.0 wt%) to study drying times affecting the formation of silk I. Fig. 1 showed XRD data of SF films preparing for different drying times at certain conditions (20 ◦ C, 55%RH). After drying 1 d, Fig. 1a depicted the typical random coil of SF, characterized by the presence of a broad peak in the diffraction angle range from 5◦ to 45◦ . When the drying time was 2 d, the main diffraction peaks were also located at 20.4◦ , attributing to random coil structure (Fig. 1b). However, increasing drying times more than 3
Fig. 2. Effect of SF concentration on the formation of silk I; the concentrations of SF aqueous solution were as follows: (a) 0.5 wt%, (b) 1.0 wt%, (c) 3.0 wt%, (d) 7.4 wt%, and (e) 10.0 wt%, respectively.
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4. Discussion
Fig. 3. XRD results of SF films cast at different temperatures: (a) 10 ◦ C, (b) 20 ◦ C, (c) 35 ◦ C, (d) 45 ◦ C, and (e) 60 ◦ C, respectively.
A conformational transition from ␣-helix to -sheet could be induced at low drying rate above 50 ◦ C. However, high drying rates prevented the transition even above 50 ◦ C, probably because the organization of the inter-chain hydrogen bonds typical of -sheet cannot be achieved under these conditions. Bombyx mori silk fibroin had three major conformations in the solid state, which were random coil, silk I (-turn), and silk II (sheet) [22]. Fig. 3 showed XRD results of SF films casting with equal solutions (50 g, 2.0 wt% concentration) at different temperatures with constant humidity (55%RH). At 10 ◦ C, Fig. 3a depicted the main diffraction peaks of SF films at 11.9◦ , 19.8◦ , 24.5◦ , 27.8◦ , and 36.7◦ , which were typical of SF in silk I structure. When the temperature increased to 45 ◦ C, the main diffraction peaks being located at 11.9◦ , 19.8◦ , 24.5◦ , 27.8◦ , and 36.7◦ , were also appeared (Fig. 3b–d), attributing to silk I structure. However, SF films were formed by self-assembly at 60 ◦ C, and its structure was characterized by XRD. Fig. 3e showed the diffraction peaks of SF films at 9.2◦ , 20.2◦ , and 24.5◦ for silk II. These results confirmed that the drying temperature was a key parameter in determining the structure taken by SF. What’s more, the drying temperature was strongly correlated to the drying rates. SF in films forming below 45 ◦ C was mostly in silk I structure. This finding can be explained by slow crystallization for SF from water during casting at lower temperatures.
Crystalline dimorphism in Bombyx mori silk fibroin has been an active topic of study for several decades [9,10,23,24]. There are two known crystalline forms of silk fibroin, silk I and silk II [25]. Silk II has a monoclinic unit cell with the protein chains in a pleated sheet (twofold “zigzag”) conformation [26], and its structure is well understood. The second polymorph, silk I, is not well characterized, because it has not been possible to obtain oriented samples. The exact structure of silk I and its poly(Ala-Gly) analogue may involve a “crankshaft” chain conformation [10]. Therefore, understanding the natural of silk I structure is an essential step to understand the natural silk spinning process. In this study, silk I structure formation was investigated in vitro using regenerated SF aqueous solution. In our previous study, silk I structure was easily obtained using regenerated SF aqueous solution at constant environmental conditions [6]. In the process of silk I formation, SF molecules were assembled from nanospheres to irregular networks through noncovalent bonds (Fig. S1). These bonds included hydrogen bonds, ionic bonds, water-mediated hydrogen bonds, hydrophobic and van der Waals interactions, etc. [6]. For the formation of silk I structure, it was stable at lower temperature such as 5 ◦ C or −20 ◦ C, not transforming silk II (-sheet) (Fig. S2). From Fig. 2, the formation of silk I structure was correlated to the concentration of SF solution. In order to study the existence state of SF molecules in aqueous solution to influence the formation of silk I structure, solution with different SF molecules morphologies was used. Firstly, fleshly SF aqueous solution was prepared with 3.0 wt% concentration. SF nanospheres with 20–50 nm were observed in solution (Fig. 4a). And then, after concentrating, SF solution with 10.0 wt% concentration was obtained. In this concentrated process, SF nanofibrils were formed and composed with many nanospheres by self-assembly (Fig. 4b) [27]. However, through experimental verification, silk I structure can be formed through using SF aqueous solutions with different molecule morphologies (Fig. 5). In addition, the mechanical properties of films with different structures were analysed at dry state (Fig. S3). The mechanical stress of SF films with silk II was 47.51 ± 5.86 MPa. However, the mechanical stress of SF films with silk I had similar to SF films with silk II, reaching 38.68 ± 6.21 MPa. Therefore, the comparison indicated similar mechanical behaviour of SF films with different structures. This phenomenon may be attributed to the self-assembly process of the sequence GAGAGS amino acid motif in structure formation.
Fig. 4. The existence state of SF macromolecules in different concentration solutions: (a) 3.0 wt%, (b) 10.0 wt%.
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lower temperature such as 5 ◦ C or −20 ◦ C, not transforming to silk II. Therefore, using environmental factors to control silk I structure may be a useful approach for further understanding the molecular chain conformation of silk I and preparing novel silk-based materials. Acknowledgements We gratefully acknowledge the support of the Second Phase of Jiangsu Universities’ Distinctive Discipline Development Program for Textile Science and Engineering of Soochow University, National Science Foundation of China (No. 81271723), and National Engineering Laboratory for Modern Silk. Appendix A. Supplementary data Fig. 5. Partial ternary phase diagram of silk I structure formation (, random coil; 䊉, silk I; , silk II; , coexistence between random coil and silk I; ♦, coexistence between silk I and silk II).
Fig. 5 was the partial ternary phase-diagram of silk I structure, based on the above results. Phase boundaries between random coil and silk I and between silk I and silk II were shown by dashed lines. The structure of SF was obviously influenced under the effect of temperature and humidity. The dashed line at 55% RH was the important formation condition for silk I structure, which was confirmed by XRD results (Fig. 3). At the same time, samples indicated in Fig. 5 as displaying phase coexistence occur between random coil and silk I and between silk I and silk II. Coexistence is identified by the superposition of characteristic diffraction patterns. At the same time, Fig. 5 depicted the presence of both silk I and silk II structures in samples at the range of temperature and humidity conditions. This indicated different structures formation of SF was correlated to SF molecules self-assembly process. The self-assembly process of SF molecules was directly affected by the volatilization rate of water molecules in aqueous solution at constant environmental conditions. Sohn [28] studied the phase behaviour and hydration of silk fibroin. The results showed the presence of both silk I and silk II structures in SF samples with ranges of water content. Whether silk I structure was the hydrated crystal structures or mesophases, this question was beyond the scope of this study, as has been suggested for silk I by researchers [29,30]. In addition, silk II with intermolecular hydrogen bonding was identified by diffraction in Fig. 5. What is important is that no such strong statement can be made about the nature of the samples identified as silk I in Fig. 5. 5. Conclusions The crystalline structure of Bombyx mori silk fibroin was investigated in vitro using regenerated SF aqueous solution at different environmental conditions. In our study, silk I structure was directly formed when the environmental humidity was 55%RH. SF molecules were assembled to form random coil at lower solution concentration and then, as the concentration increases, were converted to silk I. At the same time, the drying temperature was a key parameter in determining the structure of SF. What’s more, it was strongly correlated to the drying rates of solution. SF in films forming below 45 ◦ C was mostly in silk I structure. The important point was that a partial ternary phase diagram of temperature–humidity–concentration was drawn based on X-ray data. This diagram suggested silk I structure could be obtained using regenerated SF aqueous solution by adjusting the external environmental conditions. The obtained silk I structure was stable at
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac. 2015.02.002. References [1] [2] [3] [4] [5] [6] [7]
[8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30]
H.J. Jin, D.L. Kaplan, Nature 424 (2003) 1057–1061. J. Magoshi, Y. Magoshi, S. Nakamura, J. Appl. Polym. Sci. 21 (1977) 2405–2407. F. Vollrath, D.P. Knight, Nature 410 (2001) 541–548. M. Roy, S.K. Meena, T.S. Kusurkar, S.K. Singh, N.K. Sethy, K. Bhargava, S. Sarkar, M. Das, Biointerphases 7 (2012) 1–11. H. Zhang, J. Magoshi, M. Becker, J.Y. Chen, R. Matsunaga, J. Appl. Polym. Sci. 86 (2002) 1817–1820. J.F. Ming, B.Q. Zuo, J. Appl. Polym. Sci. 125 (2012) 2148–2154. D.M. Phillips, L.F. Drummy, D.G. Conrady, D.M. Fox, R.R. Naik, M.O. Stone, P.C. Trulove, H.C.D. Long, R.A. Mantz, J. Am. Chem. Soc. 126 (2004) 14350– 14351. T. Asakura, J. Ashida, T. Yamane, T. Kameda, Y. Nakazawa, K. Ohgo, K. Komatsu, J. Mol. Biol. 306 (2001) 291–305. B. Kundu, R. Rajkhowa, S.C. Kundu, X.G. Wang, Adv. Drug Deliv. Rev. 65 (2013) 457–470. K. Okushita, A. Asano, M.P. Williamson, T. Asakura, Macromolecules 47 (2014) 4308–4316. Y. Jin, Y.C. Hang, J. Luo, Y.P. Zhang, H.L. Shao, X.C. Hu, Int. J. Biol. Macromol. 62 (2013) 162–166. M. Demura, M. Minami, T. Asakura, T.A. Cross, J. Am. Chem. Soc. 120 (1998) 1300–1308. T. Asakura, M. Okonogi, Y. Nakazawa, K. Yamauchi, J. Am. Chem. Soc. 128 (2006) 6231–6238. P. Cebe, X. Hu, D.L. David, E. Zhuravlev, A. Wurm, D. Arbeiter, C. Schick, Sci. Rep. 3 (2013) 1130. Q. Lu, X. Hu, X.Q. Wang, J.A. Kluge, S.Z. Lu, P. Cebe, D.L. Kaplan, Acta Biomater. 6 (2010) 1380–1387. T. Asakura, Y. Suzuki, Y. Nakazawa, K. Yazawa, G.P. Holland, J.L. Yarger, Prog. Nucl. Magn. Reson. Spectrosc. 69 (2013) 23–68. X. Chen, D.P. Knight, Z.Z. Shao, Soft Matter 5 (2009) 2777–2781. T. Asakura, R. Sugino, J.M. Yao, H. Takashima, R. Kishore, Biochemistry 41 (2002) 4415–4424. T. Asakura, Y. Suzuki, Y. Nakazawa, Biotechnol. Silk 5 (2014) 49–68. M. Tsukada, J. Polym. Sci. Part B Polym. Phys. 24 (1986) 457–460. P. Monti, G. Freddi, A. Bertoluzza, N. Kasai, M. Tsukada, J. Raman Spectrosc. 29 (1998) 297–304. X. Chen, Z.Z. Shao, N. Marinkovic, L.M. Miller, P. Zhou, M.R. Chance, Biophys. Chem. 89 (2001) 25–34. J.F.C. Mayen, A. Lupan, C. Cosar, A. Kun, R. Silaghi-Dumitrescu, Biophys. Chem. 197 (2015) 10–17. S.W. Ha, H.S. Gracz, A.E. Tonelli, S.M. Hudson, Biomacromolecules 6 (2005) 2563–2569. K. Okuyama, R. Somashekar, K. Noguchi, S. Ichimura, Biopolymers 59 (2001) 310–319. R. Valluzzi, S.P. Gido, W.P. Zhang, W.S. Muller, D.L. Kaplan, Macromolecules 29 (1996) 8606–8614. S. Bai, S. Liu, C. Zhang, W. Xu, Q. Lu, H. Han, D.L. Kaplan, H. Zhu, Acta Biomater. 9 (2013) 7806–7813. S. Sohn, H.H. Strey, S.P. Gido, Biomacromolecules 5 (2004) 751–757. M.T. Krejchi, S.J. Cooper, Y. Deguchi, E.D.T. Atkins, M.J. Fournier, T.L. Mason, D.A. Tirrell, Macromolecules 30 (1997) 5012–5024. S. Sohn, S.P. Gido, Biomacromolecules 10 (2009) 2086–2091.
