NANO LETTERS
Nanostructure of Natural Fibrous Protein: In Vitro Nanofabric Formation of Samia cynthia ricini Wild Silk Fibroin by Self-Assembling
2003 Vol. 3, No. 10 1329-1332
Shun-ichi Inoue,* Hidetoshi Tsuda, Toshihisa Tanaka, Masatoshi Kobayashi, Yoshiko Magoshi, and Jun Magoshi Japan Science and Technology Corporation, National Institute of Agrobiological Science, 2-1-2 Kannondai Tsukuba, 305-8602 Japan Received January 16, 2003; Revised Manuscript Received August 7, 2003
ABSTRACT Atomic force microscopy was adopted to study the nanoscopic structure formation of natural fibrous protein, fibroin from the Samia cynthia ricini wild silkworm. We have observed highly ordered nanoscale textile-fabric-like structures of fibroin molecules. From AFM and SDS-PAGE experiments, it was revealed that fibroin molecules have a rigid rodlike structure and form aggregates by end-to-end interactions of molecules. By analogy with Bombyx mori fibroin, S. c. ricini fibroin is supposed to assemble by electrostatic interactions of molecules.
Self-assembly is one of the most important ways that biological structures are constructed, and it is also important in engineering applications.1-3 Many kinds of molecules form supramolecular structures by self-assembling, and protein molecules are good examples of such structure formation.2-5 Here we focused on fibroin from the Samia cynthia ricini wild silkworm (Eri silkworm). Fibroin is the water-soluble fibrous protein and the main component of silk fiber. Fibroin is synthesized in the silk glands of a silkworm and stored in a weak gel state. When fibroin is spun into fiber, it changes into a sol state with liquid-crystalline order.6,7 After it is spun into a fiber, the fibroin becomes insoluble in water. Although the spinning process is not yet thoroughly understood, it is anticipated that fibroin molecules selfassemble to form the silk filaments. To confirm the hypothesis that fibroin molecules self-assemble, atomic force microscopy (AFM) was used to observe the higher-order structure of silk fibroin molecules from S. c. ricini. We have also performed SDS-PAGE experiments on fibroin to analyze the molecular weight. From analyzing the size and shape of the fibroin molecule and its aggregates, the structure formation process can be revealed. This provides important information about structural formation within biological systems and will be beneficial to engineering applications concerned with forming nanoscale structures. For the AFM experiments, silk fibroin was obtained from the posterior silk glands (PSG) of wild silkworms S. c. ricini * Corresponding author. E-mail:
[email protected]. 10.1021/nl0340327 CCC: $25.00 Published on Web 08/29/2003
© 2003 American Chemical Society
(Eri silkworm) in their fifth instar. After the tissue surrounding the fibroin gel was taken out, the gel was immersed in distilled water, and the fibroin molecules gradually dissolved in distilled water at 5 °C. The thus-obtained polymer solution was then filtered with a paper filter (mesh size 5 µm) 24 h after removal from the silkworm. Thereafter, the polymer solution was diluted with distilled water to a concentration of 2 × 10-3 wt %; then the polymer solution was divided into two portions (A and B). Portion A was filtered through a 0.45-µm filter 1 h after the paper filtering and was further split into two samples. One portion of polymer solution A was cast over a newly cleaved mica surface immediately after filtering (sample 1), and the other portion was cast 24 h after a second filtering (sample 2). Then both samples were allowed to dry in ambient air. Portion B was stored at 5 °C for 24 h after paper filtering and then also split into two samples. One portion was further filtered by a 0.45-µm filter and cast over a mica surface (sample 3), and the other portion was cast over a mica surface without further filtering (sample 4). The measurements were performed using an SPI-3000S AFM system (Seiko Instruments Inc.) with an SPA-300HV unit both in contact mode and tapping mode. All scanned images were obtained using a 20-µm scanner. For contactmode images, an Olympus SN-AF01 cantilever and tip (Si3N4, tip radius 10 nm, triangular base 100 µm long) with a force constant of 0.09 N/m was used. The scanning line frequencies ranged from 0.3 Hz for large image areas
(10 µm2) to 2 Hz for smaller-scale images. The loading force on the sample is smaller than 10-10 N, which was determined from the force curve. Tapping-mode images utilized an Olympus SI-DF40 cantilever and tip (Si, tip radius 15 nm, rectangular base 125 µm long) with a force constant of 40 N/m. The resonance frequency of the cantilever was 270 kHz, and the applied frequency was set on the lower side of the resonance frequency. All of the measurements were performed in ambient air. For SDS-PAGE experiments, fibroin was obtained from mature S. c. ricini silk glandssthe posterior silk gland (PSG) and the middle silk gland (MSG). Protein from the PSG was squeezed from tissue and then washed twice with water. Thereafter, the protein was dissolved in H2O, 20 mM TrisHCl (pH 8.0) + 5 M urea, H2O + 30 mM DTT, and 20 mM Tris-HCl (pH 8.0) + 5 M urea + 30 mM DTT at 4 °C overnight. Protein from the MSG was obtained in a weak gel state and was cut into small pieces and then immersed in each buffer under the same conditions as those used for PSG samples. The protein concentration of each sample was adjusted by the BCA method to 2 mg/mL. We added an SDS sample buffer (final concentration; 25 mM Tris-HCl (pH 6.5), 5% glycerol, 1% SDS, 0.05% BPB) to each sample without 2-mercaptoethanol, and the samples were loaded on the 10% SDS polyacrylamide gel (SDS-PAGE). After electrophoresis, the gel was stained by 5% CBB and silver stain plus (BioRad). S. c. ricini silk fibroin solutions of various conditions were cast on a mica surface and observed by AFM. In sample 1, many rodlike objects were observed. The size of these objects is about 300 nm in length, 10 nm in width, and 0.4 nm in height. The AFM experiment gives the correct value for the Z direction (height) but not for the X-Y direction because the cantilever tip has a finite radius. The width of the observed objects is nearly equal to the diameter of the cantilever tip, which means that the actual width of them is smaller than the tip radius (10 nm). If these objects do not have a rodlike shape, then a certain fraction of the objects should be observed on the edge. Because the heights of these objects are almost equivalent, this strongly suggests that these objects have a rodlike shape. The size and shape of these rods are similar to each other, and these rods are supposed to be single fibroin molecules. We tried to analyze S. c. ricini protein to confirm that the observed objects by AFM are fibroin molecules. We analyzed protein obtained from S. c. ricini silk gland contents by SDSPAGE. The protein was obtained from the PSG and MSG of mature S. c. ricini larvae. The SDS-PAGE experimental result is shown in Figure 2. There are only two bands observed on PSG lanessone is a distinct band at 330 kDa, and the other is very faint. Because the 330-kDa band was also observed in MSG lanes, this band shows the main protein band of S. c. ricini fibroin. The fact that few bands were observed in Figure 2 means that the purity of the sample solution is very high. From the above result, we conclude 1330
Figure 1. Tapping-mode image of S. c. ricini wild silk fibroin on a mica surface. The sample solution was cast on a mica surface 1 h after filtering. Scale bar: 500 nm.
Figure 2. SDS-PAGE analysis of silk fibroin from posterior and middle silk glands of the Eri silkworm. The contents of each were dissolved in water or a Tris-urea buffer, or each containing 30 mM DTT. The samples were loaded on the 5% stacking gel and 10% separating gel without heat shock. Arrows indicate Eri silkworm fibroin. M1 and M2, molecular weight marker; lanes 1 and 5, PSG contents dissolved in water; lanes 2 and 6, MSG contents dissolved in water; lanes 3 and 7, PSG contents dissolved in a Tris-urea buffer; lanes 4 and 8, MSG contents dissolved in a Tris-urea buffer; lanes 1 to 4, not containing DTT; lanes 5 to 8, containing 30 mM DTT in the solution.
the observed objects in Figure 1 should be assigned to S. c. ricini fibroin. It has been confirmed that the molecular weight of S. c. ricini fibroin is smaller than that of Bombyx mori fibroin, the molecular weight of which is 350 kDa (data not shown). The main protein bands in both MSG and PSG samples are approximately 330 kDa, as shown in Figure 2. However, the DTT digested sample shows a single band at approximately 160 kDa (Figure 2, DTT(+) lanes). This result shows that the S. c. ricini fibroin molecule is a homodimer of a 160-kDa protein linked by single disulfide bond. This is in contrast to B. mori fibroin, which is a heterodimer of a Nano Lett., Vol. 3, No. 10, 2003
Figure 3. Tapping-mode image of S. c. ricini wild silk fibroin on a mica surface. The sample solution was cast over a mica surface 24 h after filtering. Scale bar: 500 nm.
Figure 4. Tapping-mode image of S. c. ricini wild silk fibroin on a mica surface. The sample solution was cast over a mica surface 24 h after filtering. Scale bar: 500 nm.
heavy chain (350 kDa) and light chain (25 kDa) linked by a disulfide bond.10 S. c. ricini fibroin mainly consists of alanine (45%) and glycine (25%). Seventy-three percent of the total number of amino acid residues are hydrophobic, and the ratio of hydrophobic residues is similar to that of B. mori silk fibroin. Though the primary structure of S. c. ricini fibroin has not yet been completely sequenced, it is known to have polyalanine domains interleaved with glycine-rich domains.8 The polyalanine domains of the fibroin have an R-helix conformation, which has been confirmed by an NMR study.9 Because the molecular weight of S. c. ricini fibroin is about 330 kDa, the fibroin molecules consist of about 3 × 103 amino acid residues. The observed rods in Figure 1 have a height of 0.4 nm, which is equal to the calculated value of the R helix. The length calculated from the molecular weight is about 300 nm, and this is in agreement with the observed object. From the above discussion, the observed rodlike objects in Figure 1 are assigned to the fibroin molecule, and it is also suggested that the major part of the fibroins have an R-helix conformation. Figure 3 shows the tapping-mode image of sample 4, which was prepared 24 h after paper filtering. This sample shows a textile-fabric-like or “nanofabric” structure that seems to consist of warp and weft. The size of this nanofabric is 2 µm in width and some 10 µm in length. The height of both the warp and weft is 0.4 nm, which is the same as that of the single fibroin molecule observed in Figure 1. This means that the molecules assemble in an end-to-end fashion. If the molecules were to assemble side-by-side, then the observed objects would be greater in diameter. The distance between neighboring warps is 50 to 80 nm, and that of neighboring wefts is about the same. Because similar images were obtained by both contact-mode and tapping-mode AFM, it is clear that this structure was not formed artificially (i.e., by scratching the cantilever). Because the purity of the protein solution is proven by the SDS-PAGE experiment as shown in Figure 1, this structure is not caused by impurities. A similar structure was also observed in sample 2 (figures not shown), but in sample 3, we were able to observe neither
rodlike objects nor larger aggregates. This result shows that a fabric-like structure was formed by the self-assembly of fibroin molecules in the solution. Figure 4 shows the tapping-mode image of sample 4, where comblike structures consisting of long threads with many side chains were observed. The distance between neighboring side chains is about 50 to 80 nm, which is the same as that observed in Figure 3. We can further observe that such comblike structure assemble together side-by-side to form a larger structure. In the case of B. mori fibroin, the molecules consist of a rigid rodlike part and flexible parts, which are attached to both ends of the rod. The rigid part is strongly hydrophobic, but the flexible parts are hydrophilic. The molecules form aggregates by end-to-end interactions of the flexible part.11 From the rheological experiments,12,13 the interaction between B. mori fibroin molecules was revealed to be an electrostatic force. The complete amino acid sequence of S. c. ricini fibroin remains unknown. Because the structure-formation mechanism strongly depends on the molecular structure, the detailed mechanism should be discussed after the complete amino acid sequence is revealed. The physical behavior of S. c. ricini fibroin is similar to that of B. mori fibroin, for example, the gel-sol transition or liquid-crystal formation. Because the similarity in the size and shape of the molecules was discussed above, we also expect the interaction mechanism of S. c. ricini fibroin to be similar to that of B. mori fibroin. By the analogy to B. mori fibroin, S. c. ricini wild silk fibroin is considered to form aggregates by an electrostatic interaction. Ten percent of the total number of amino acid residues of S. c. ricini are charged, and this ratio is higher than that of B. mori. Because such charged residues are located in a nonhelical domain, molecules form aggregates by an electrostatic interaction of a nonhelical domain to construct a highly ordered structure. The schematic image of the structure formation of S. c. ricini fibroin was shown in Figure 5. The fibroin
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many similarities between the nuclear lamina and fibroin nanofabric, we expect the structure-formation mechanisms to be similar to each other. To summarize this report, we have observed a highly ordered nanofabric structure formed by S. c. ricini fibroin. Fibroin has an R-helix conformation and assembles end-toend to form rodlike structures. Though the mechanism of higher-order structure formation is not completely revealed, it is supposed to be similar to that of B. mori fibroin. The observed nanofabric might be taken as a model of the higherorder structure formation of biomaterials. Revealing the mechanism of nanofabric formation would provide important information about structure formation in biological systems. References
Figure 5. Schematic image of the structure formation of S. c. ricini fibroin. (a) Single molecule of S. c. ricini fibroin, (b) comblike structure, and (c) fabriclike structure.
molecule is a homodimer of a rodlike polypeptide (Figure 5a) and forms long comblike structures by an electrostatic interaction of hydrophilic residues located on both ends of the rodlike molecule (Figure 5b). The comblike structures assemble by their side-by-side interactions (i.e., end-to-end interactions of side chains), and fabriclike structures are formed (Figure 5c). These textile-fabric-like structures are very similar to that of nuclear lamina, from which the inner nuclear membrane is constructed.14,15 A nuclear lamina consists of an intermediate-type filament protein, lamin, and it has an R-helix domain and nonhelical domains, which are called the head and tail and are attached to both ends of the helix. The lamin molecules form dimers by an interaction between the R-helix domains. Then the dimers form aggregates in a head-to-tail manner to form long threadlike structures. These threadlike structures stick together side-by-side to form a larger macrostructure.14-16 The structure-formation mechanism of the nuclear lamina is also not revealed. Because there are
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(1) Chen, L.; Jenekhe, S. Macromolecules 2000, 33, 4610. (2) Pralle, M. U.; Whitaker, C. M.; Braun, P. V.; Stupp, S. I. Macromolecules 2000, 33, 3550. (3) Klok, H.-A.; Langenwalter, J. F.; Lecommandoux, S. Macromolecules 2000, 33, 7819. (4) Seto, C. T.; Mathias, J. P.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 1321. (5) Aggeli, A.; Bell, M.; Boden, N.; Keen, J. N.; Knowles, P. F.; McLeish, T. C. B.; Pitkeathly, M.; Radford, S. E. Nature 1997, 386, 259. (6) Magoshi, J.; Magoshi, Y.; Nakamura, S. Polym. Commun. 1985, 26, 60. (7) Willcox, P. J.; Gido, S. P.; Muller, W.; Kaplan, D. L. Macromolecules 1996, 29, 5106. (8) Ishida, M.; Asakura, T.; Yokoi, M.; Saito, H. Macromolecules 1996, 29, 2920. (9) Beek, J. D.; Beaulieu, L.; Scha¨fer, H.; Demura, M.; Asakura, T.; Meier, B. H. Nature 2000, 405, 1077. (10) Yamaguchi, K.; Kikuchi, Y.; Takagi, T.; Kikuchi, A.; Oyama, F.; Shimura, K.; Mizuno, S. J. Mol. Biol. 1989, 210, 127. (11) Inoue, S.; Magoshi, J.; Tanaka, T.; Magoshi, Y.; Becker, M. J. Polym. Sci., Part B: Polym. Phys. 2000, 38, 1436. (12) Kobayashi, M.; Tanaka, T.; Inoue, S.; Tsuda, H.; Magoshi, J.; Magoshi, Y. ACS DiV. Polym. Mater.: Sci. Eng., Proc. 2001, 84, 620. (13) Kobayashi, M.; Tanaka, T.; Inoue, S.; Tsuda, H.; Magoshi, J.; Magoshi, Y. Trans. MRS-J 2001, 26, 577. (14) Stuurman, N.; Heins, S.; Aebi, U. J. Struct. Biol. 1998, 122, 42. (15) Aebi, U.; Cohn, J.; Buhle, L.; Gerace, L. Nature 1986, 323, 560. (16) Heitlinger, E.; Peter, M.; Haner, M.; Lustig, A.; Aebi, U.; Nigg, E. J. Cell Biol. 1991, 113, 485.
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