Structures of Bombyx m ori and Samia c ynthia r icini Silk Fibroins

Biomacromolecules 2004, 5, 680-688

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Structures of Bombyx mori and Samia cynthia ricini Silk Fibroins Studied with Solid-State NMR Juming Yao, Yasumoto Nakazawa, and Tetsuo Asakura* Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan Received August 7, 2003; Revised Manuscript Received January 21, 2004

There are many kinds of silks spun by silkworms and spiders, which are suitable to study the structureproperty relationship for molecular design of fibers with high strength and high elasticity. In this review, we mainly focus on the structural determination of two well-known silk fibroin proteins that are from the domesticated silkworm, Bombyx mori, and the wild silkworm, Samia cynthia ricini, respectively. The structures of B. mori silk fibroin before and after spinning were determined by using an appropriate model peptide, (AG)15, with several solid-state NMR methods; 13C two-dimensional spin-diffusion solid-state NMR and rotational echo double resonance (REDOR) NMR techniques along with the quantitative use of the conformation-dependent 13C CP/MAS chemical shifts. The structure of S. c. ricini silk fibroin before spinning was also determined by using a model peptide, GGAGGGYGGDGG(A)12GGAGDGYGAG, which is a typical repeated sequence of the silk fibroin, with the solid-state NMR methods. The transition from the structure of B. mori silk fibroin before spinning to the structure after spinning was studied with molecular dynamics calculation by taking into account several external forces applied to the silk fibroin in the silkworm. Introduction Silkworm silks, including Bombyx mori and Samia cynthia ricini silks, which are synthesized in specialized sets of modified salivary glands and extruded from spinnerets located in the head of the larva, have been studied intensively.1 Because of their impressive properties as well as the biocompatibility and relative environmental stability, the silk fibroins have been used as an important set of material options in the fields of controlled release, biomaterials, and scaffolds for tissue engineering for decades.2-4 Recently, much attention has been paid to silk from textile engineers to polymer chemists and biomedical scientists. For example, silk fibroin from silkworms can produce strong and stiff fibers at room temperature and from an aqueous solution, whereas synthetic materials with comparable properties must be processed at higher temperatures and/or from less benign solvents. Thus, it is advantageous to focus on the structural determination of silk fibroins before and after spinning and the factors that contribute to the conformational change, because in this case it can provide some implications for the de novo design of artificial proteins. The primary structure of B. mori silk fibroin contains multiple repeats of AGSGAG which make up 55% of the total fibroin and form the insoluble crystalline Cp fraction after chymotrypsin cleavage. The remaining sequence is rich in Tyr and constitutes the semicrystalline part of the fibroin.1,5 B. mori silk fibroin can assume two distinct structures in the solid state, namely silk I before spinning and silk II after spinning, that is, the silk fiber. The corresponding structures have been investigated by X-ray fiber diffraction,6-10 electron diffraction,9,11,12 conformational energy calculations,13,14 in* To whom correspondence should be addressed. Tel & Fax: +81-42383-7733. E-mail: [email protected].

frared spectroscopy,8,15 and 13C and 15N cross-polarization magic angle spinning (CP/MAS) NMR.8,16-19 Despite a long history of studying silk I, its structure determination was difficult because any attempt to induce a macroscopic orientation of the sample for X-ray diffraction, electron diffraction, or solid state NMR readily causes a conversion of the silk I form to the silk II form.8,16,20-22 The structure of silk II was proposed as a regular array of antiparallel β-sheet model first by Marsh et al. about half century ago, based on a fiber diffraction study of native B. mori silk fibroin fiber.6 Later, Fraser et al. and Lotz et al. pointed out some intrinsic structural disorder in the silk II structure, but they supported the general features of this antiparallel β-sheet model.7,11 Recently, Takahashi et al. reported a more detailed X-ray fiber diffraction analysis of B. mori silk fibroin where two antipolar antiparallel β-sheet structures are statistically stacked with different orientations, occupying the crystal site with a ratio of 1:2.10 On the other hand, the primary structure of S. c. ricini silk fibroin is considerably different from that of B. mori silk fibroin.23 The basic repeat sequence is made of alternating (Ala)12-13 regions and the Gly rich regions. This primary structure is similar to spider (major ampullate) silk but the length of polyalanine is longer, (Ala)5-6 in the case of spider silk.24 The solution-state 13C and 15N NMR studies showed that about 70% of Ala residues in S. c. ricini silk fibroin before spinning form an R helix, whereas the conformation of the other Ala residues is in a random coil sate.25-28 However, in the S. c. ricini silk fibroin fiber, Ala residues were proposed to form the antiparallel β-sheet on the basis of the conformation-dependent 13C chemical shift.22,29 In this article, we reviewed our recent studies on the structural determination of these silkworm silks, mainly the crystalline region of silk fibroins, before and after spinning

10.1021/bm034285u CCC: $27.50 © 2004 American Chemical Society Published on Web 02/25/2004

Silk Fibroins Studied with Solid-State NMR

by using solid state NMR techniques and tried to simulate the fiber formation mechanism with molecular dynamics simulation. These studies provide further understanding on the structure of different silk proteins, which is significant not only for the clarification of fiber formation mechanism but also for the further de novo molecular design of silklike materials. Materials and Methods Silk Fibroin Fiber and Film. The cocoons were degummed three times with 0.5 (w/v) % Marseilles soap (for B. mori silk) or 0.1 (w/v) % sodium peroxide (for S. c. ricini silk) solution at 100 °C for 30 min and washed with distilled water in order to remove sericin proteins from the surface of the fibers. The fibroin fibers were dried at 37 °C overnight prior to the next experiments. To prepare the silk fibroin films, the middle silkglands were pulled out from anesthetized 7-day-old fifth instar larva. The silkglands containing the fibroin were then washed twice in ice-cold 1.15 (v/v) % potassium chloride solution. The center of the silkgland was excised and the effluent was immersed in distilled water to remove most of the soluble sericin. The liquid silk was diluted with distilled water and placed in plastic Petri dishes with drying for 2 days to prepare silk fibroin films. Model Peptides. All peptides were synthesized by the F-moc solid-phase method (Pioneer Peptide Synthesizer Co. Ltd). The model peptides for B. mori silk fibroin in silk I form were obtained as powders by dissolving the peptides in 9 M LiBr and then dialyzing against water for 4 days, followed by lyophilization of peptide solutions. The peptides in silk II form were obtained as powders by dissolving the silk I peptides in formic acid and subsequently drying. On the other hand, the model peptides for S. c. ricini silk fibroin were precipitated after 4 days’ dialysis, which takes the β-sheet structure judging from the 13C CP/MAS NMR chemical shifts. To transform the β-sheet structure to the R-helix as a structural model for the silk fibroin before spinning, the precipitated samples were dissolved in TFA followed by the addition of diethyl ether. The dried samples formed an R-helix structure as judged from the 13C CP/MAS NMR chemical shifts. 13 C CP/MAS NMR Spectroscopy. Solid state 13C CP/ MAS NMR spectra were acquired on a Chemagnetics CMX400 spectrometer operating at 100 MHz, with a CP contact time of 1 ms and magic angle spinning at 5 kHz. A 50 kHz radio frequency field strength was used for 1H-13C decoupling. A total of 10 000-25 000 scans were collected over a spectral width of 60 kHz, with a recycle delay of 3 s. Chemical shifts are reported in ppm relative to TMS as reference. 2D Spin Diffusion NMR Spectroscopy. Two-dimensional spin-diffusion NMR spectra were obtained on a Varian Unity INOVA 400 NMR spectrometer, using a 7 mm Jakobsentype double-tuned MAS at off-magic angle condition. Detailed experimental conditions including off-magic angle were reported previously.30-33 The principal axis values of the chemical shift anisotropy tensors for the carbonyl carbons of the isotope labeled Ala and Gly residues were determined from the spinning sidebands, using a Chemagnetics CMX

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400 spectrometer under slow MAS conditions. A homemade calculation program was used to simulate the 2D spindiffusion NMR spectra on an OCTANE workstation (Silicon Graphics Inc.). REDOR NMR. REDOR NMR experiments were performed on a Chemagnetics CMX-400 spectrometer equipped with solid-state accessories and a triple-channel magic-angle probe with a 5 mm coil. Detailed experimental conditions have been reported previously.30-32 Values of ∆S/S0 ) 1 S/S0 were computed as the ratios of peak heights in the REDOR spectra. Molecular Dynamics Calculation. The structural change from silk I to silk II was examined with molecular dynamics (MD) simulation. Sixteen (Ala-Gly)6 chains with the repeated β-turn type II structure, where N- and C-terminal were connected to mirror image of themselves, were arranged in the periodic cell of the system reported previously.30 To simulate the infinite bulk system of infinite repeated polymer chains, periodic boundary conditions were used. Using the stress tensor in the Parrinello and Rahman method,34 tensile stress was applied by setting σzz (tensile stress) or σxx and σyy (shear stress) as nonzero value. Temperature of the system was controlled by the Andersen method at 298 K.35 The chains were embedded in 916 water molecules for the initial arrangement of an aqueous solution system, which corresponds to the concentration of silk fibroin in the middle silk gland (about 30 (w/v)%). Both tensile and shear stresses were applied to the system, where the shear stress was changed from 0.3, 0.5, 0.7, to 1.0 GPa with a tensile stress of 0.1 GPa. The process of MD calculation was as follows, initially performed without any external forces till the system reaches to the equilibrium state (equilibrium stage; 5000 steps) and then the calculations continued under tensile stress (simulation stage; 200 000 steps, that is, 200 ps). The 100 structures, which were sampled every 20 steps during the latter steps from 101 to 200 ps in simulation stage, were selected after the MD calculation of the system under both tensile and shear stresses in the presence of water and then the molecular mechanics (MM) calculations were performed after removal of water molecules. All of the MD and MM simulations were carried out by using Discover 3 module in InsightII (4.0.0 P+, Accelrys Inc.) on an OCTANE workstation (Silicon Graphics Inc.). Results and Discussion 13C CP/MAS NMR Spectra. The conformation-dependent chemical shifts of 13C nuclei have been known as characteristic markers, sufficient to identify the structure of silk proteins.8,16,22 Figure 1 shows the 13C CP/MAS NMR spectra and peak assignments of silk fibroin fibers and films, which demonstrates that both B. mori and S. c. ricini silk fibroins occur the structural transition during the fiber spinning. The peaks at 16.7 ppm for Cβ and 51.0 ppm for CR of Ala residues in B.mori silk fibroin film (Figure 1A) suggest that it takes the silk I structure, whereas those at 15.5 ppm for Cβ and 52.5 ppm for CR in S. c. ricini silk film (Figure 1B) take an R-helical structure. The broad and asymmetric peak at 20 ppm for Ala Cβ and 48.8 ppm for CR of Ala residues (Figure 1C for B.mori silk fibroin fibers and Figure 1D for S.c.ricini silk fibroin fibers) indicate an antiparallel β-sheet

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Figure 1. 13C CP/MAS NMR spectra of (A) B. mori and (B) S. c. ricini silk fibroin films prepared from middle silkglands, and the spectra of (C) B. mori and (D) S. c. ricini silk fibroin fibers prepared from the silkworm cocoons. The chemical shifts are represented in ppm downfield from TMS.

structure although these two silk fibroins take a completely different structure before spinning. Therefore, in this article, we will try to clarify the structure of both silk fibroins before spinning and also the structure of B. mori silk fiber as an example for the silk fibroin after spinning. Structure of B. mori Silk Fibroin before Spinning (Silk I). The structural features of B. mori silk fibroin are conveniently studied using synthetic peptide, (AG)15, as a model for the crystalline region because the lack of Ser in the model peptide (AG)15 does not make any difference in the 13C CP/MAS NMR chemical shifts of the Ala and Gly residues in the repeated sequence (AGSGAG)n of native silk fibroin.30,33,36 By combining several new solid-state NMR techniques, we have determined the conformation of unoriented samples of a model peptide (AG)15 in the silk I form. The torsional angles of (AG)15 with silk I structure were determined as (-60° ( 5°, 130° ( 5°) and (70° ( 5°, 30° ( 5°) for Ala and Gly residues, respectively.30 The 2D spindiffusion NMR spectrum (only the carbonyl region was expanded) of (AG)6A[1-13C]G14[1-13C]A15G(AG)7 is shown in Figure 2A together with the spectrum calculated with the torsion angles, (φ, ψ) ) (-60°, 130°), of Ala residue (Figure 2B). Figure 2C shows the experimental 2D spin-diffusion NMR spectrum of (AG)6 [1-13C]A13[1-13C]G14(AG)8 together with the spectrum calculated with the torsion angles, (φ, ψ) ) (70°, 30°), of Gly residue (Figure 2D). In both cases, the observed spectra could be reproduced well with the calculated spectra. With these torsion angles of Ala and Gly residues determined here, the structural model of an (AG)15 chain with silk I form was prepared and shown in Figure 3. This can be called as a repeated β-turn type II structure. To confirm this model, REDOR experiments were performed. Namely, the distance between the 13CdO carbon atom of the 14th Gly residue and the 15N nitrogen atom of the 17th Ala residue

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Figure 2. (A) Experimental and (B) simulated 2D spin-diffusion NMR spectra of (AG)6A[1-13C]G14[1-13C]A15G(AG)7 and the (C) experimental and (D) simulated spectra of (AG)6[1-13C]A13[1-13C]G14(AG)8. The torsion angles of Ala15 residue used for the simulation of former spectrum were (φ, ψ) ) (-60°, 130°), whereas the torsion angles of Gly14 residue were (φ, ψ) ) (70°, 30°). Modified and redrawn from ref 30.

Figure 3. Conformation of a repeated β-turn type II-like molecule as a model for silk I. There are intramolecular hydrogen bonds between the carbonyl oxygen atom of the ith Gly residue and the amide hydrogen atom of the (i + 3)th Ala residue. Modified and redrawn from ref 30.

of (AG)15 was determined as shown in Figure 4. The distance was determined to be 4.0 ( 0.1 Å independent of the dilution with unlabeled peptide.30 The observed distance agrees very well with the distance, 4.0 Å, calculated for the distance of the intramolecular hydrogen bond of the repeated β-turn type II-like structure which supports the structural model proposed here.



2.75 Å and 2.70 Å) and angle (NH‚‚‚O; 123.3° and 129.9°). In addition, there are intermolecular hydrogen bonds, in which the geometry of intermolecular hydrogen bond is the length, N‚‚‚O, 2.84 Å and 2.91 Å, and the angle, NH‚‚‚O, 134.5° and 140.0°. Thus, the intra- and intermolecular hydrogen bonds occur alternatively in this structural model. All of the previous models showed only the formation of intermolecular hydrogen bonds and no intramolecular hydrogen bonds.30

Figure 4. Observed plots of ∆S/S0 () 1 - S/S0) values against the corresponding NcTr values for REDOR experiments of (AG)6A[1-13C]GAG[15N]AG(AG)6 for the determination of distance between the 13Cd O carbon of the 14th Gly residue and the 15N nitrogen of the 17th Ala residue. Solid and dotted lines show the theoretical dephasing curves corresponding to the designated distances. The data marked by O are observed for the isotope-labeled compound without dilution of natural abundance (AG)15 and those by 4 are observed for a mixture of equivalent amount of the isotope-labeled compound and natural abundance (AG)15. By comparing the REDOR data and the theoretical dephasing curve, the 13C-15N interatomic distance was determined to be 4.0 ( 0.1 Å, which agrees with the 4.0 Å calculated for intramolecular hydrogen bond for the repeated β-turn type II-like structure. Modified and redrawn from ref 30.

Figure 5. Crystal structure of B. mori silk fibroin in a silk I form, a repeated β-turn type II structure stabilized by a 4 f 1 intramolecular hydrogen bond. The overall planar sheets were held together by a number of intermolecular hydrogen bonding interactions, involving the “central amide-bond” of the β-turn, perpendicular to intramolecular interactions. Redrawn from ref 43.

The intermolecular arrangement of this silk I chain was calculated with X-ray diffraction data9 complementary and shown in Figure 5. The geometry of the intramolecular hydrogen bond is normal, both in terms of length (N‚‚‚O;

Structure of S. c. ricini Silk Fibroin before Spinning. The peptide, GGAGGGYGGDGG(A)12GGAGDGYGAG, was synthesized as a model of the repeated typical sequence of S. c. ricini silk fibroin. The observed and calculated 2D spin-diffusion NMR spectra of several 13C labeled model peptides are summarized in Figure 6. Figure 6B shows the 2D spin-diffusion NMR spectrum of the model peptide, GGAGGGYGGDGG(A)5[1-13C]A18[1-13C]A19(A)5GGAGDGYGAG, after TFA treatment together with the calculated spectrum of the Ala residue with the torsion angles (φ, ψ) ) (-59°, -48°) (Figure 6E).31,32 This is a typical R-helix structure, and the torsion angles of the Ala residue are essentially the same with those reported for the S. c. ricini silk fibroin film using the 2D DOQSY NMR measurements.37 Thus, the central Ala residue of the poyalanine region is concluded to take a typical R-helix structure. Moreover, the torsion angles of the N-terminal and C-terminal Gly residues adjacent to the polyalanine region were determined with the 2D spin-diffusion NMR method for two model peptides, GGAGGGYGGD[1-13C]G11[1-13C]G12(A)12GGAGDGYGAG (Figure 6A) and GGAGGGYGGDGG(A)11[1-13C]A24[113 C]G25GAGDGYGAG (Figure 6C), respectively. The spectral patterns are slightly different from that of the Ala19 residue in R-helix form (Figure 6B), indicating that the torsion angles of Gly12 and Gly25 residues are slightly different from those of a typical R-helix. From the error analysis, the torsion angles of the Gly12 and Gly25 residues were determined to be (φ, ψ) ) (-70°, -30°) and (φ, ψ) ) (-66°, -22°), respectively, as shown in Figure 6, parts D and F.32 To obtain further structural information on the C-terminal region, REDOR experiments were performed on GGAGGGYGGDGG(A) 8 [1- 13 C]A 21 AAA[ 15 N]G 25 GAGDGYGAG. Figure 7 shows the experimental 13C REDOR difference curve ∆S/S0 vs NcTr (ms). By comparing the REDOR data with the theoretical dephasing curve, the 13C15N interatomic distance was determined to be 4.8Å.32 The broken lines show that the experimental error is (0.1Å. When the torsion angles of the Ala22 residue are (φ, ψ) ) (-59°, -48°) and those of other two Ala residues, Ala23 and Ala24, are (φ, ψ) ) (-66°, -22°), the distance between [1-13C]A21 and [15N]G25 atoms was calculated to be 4.8 Å. The similar REDOR method was applied to determine the local structure at the N-terminal region of the polyalanine chain. The distance between [1-13C]Gly12 and [15N]Ala16 atoms in the peptide, GGAGGGYGGDG[1-13C]G12AAA[15N]A16(A)8GGAGDGYGAG, was determined to be 5.0 ( 0.2 Å. Only the combination of Ala14;(φ, ψ) ) (-70°, -30°) and Ala15; (φ, ψ) ) (-59°, -48°) satisfies the observed distance, 5.0 Å.

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Figure 6. 2D spin-diffusion NMR spectra of model peptides, (A) GGAGGGYGGD[1-13C]G11[1-13C]G12(A)12GGAGDGYGAG, (B) GGAGGGYGGDGG(A)5[1-13C]A18[1-13C]A19(A)5GGAGDGYGAG, and (C) GGAGGGYGGDGG(A)11[1-13C]A24 [1-13C]G25GAGDGYGAG, after TFA treatment, and their corresponding simulated spectra. The torsion angles used for the simulation were (D) (φ, ψ) ) (-70°, -30°) for Gly12 residue, (E) (φ, ψ) ) (-59°, -48°) for Ala19 residue, and (F) (φ, ψ) ) (-66°, -22°) for Gly25 residue. Parts are redrawn from ref 32.

Figure 7. Observed plots of ∆S/S0 () 1 - S/S0) values against the corresponding NcTr values for REDOR experiments of GGAGGGYGGDGG(A)8[1-13C]A21AAA[15N]G25GAGDGYGAG (b) and GGAGGGYGGDG[1-13C]G12AAA[15N]A16(A)8GGAGDGYGAG (2). Solid and dotted lines show the theoretical dephasing curves corresponding to the designated distances. Redrawn from ref 32.

According to the torsion angles determined here, the structure of the model peptide, GGAGGGYGGDGG(A)12GGAGDGYGAG, was proposed as a structural model of the repeated typical sequence of S. c. ricini silk fibroin in Figure 8. As shown in the left side, the local structure of N-terminal and C-terminal residues besides the R-helical polyalanine chain is more strongly wound than those found

Figure 8. Structure of polyalanine region of the model peptide, GGAGGGYGGDGG(A)12GGAGDGYGAG of polyalanine region of S. c. ricini silk fibroin before spinning. The left side presentation shows that R-helix structure of polyalanine region tends to be wound strongly at the both terminal ends. The right side presentation shows the corresponding intramolecular hydrogen-bonding pattern by broken lines. Redrawn from ref 32.

in a typical R helix. Namely, at the terminals of the helical region, five residues, Gly12, Ala21, Ala22, Ala23, and Ala24 contribute to the formation of i f i+3 hydrogen bonding (see right side in Figure 8), suggesting that there are mechanism to stabilize the R-helix structure of polyalanine region of the silk fibroin in S. c. ricini silkworm. Structure of B. mori Silk Fibroin after Spinning (Silk II). As mentioned in the Introduction, although Lotz, Brack



Figure 10. 2D spin-diffusion NMR spectrum of model peptide (AG)6A[1-13C]G[1-13C]AG(AG)7. By simulation, the torsion angles of the 15th Ala residue were determined to be (φ, ψ) ) (-150° ( 10°, 150° ( 10°). Redrawn from ref 33. Figure 9. Expanded Ala Cβ peak of (AG)15 in silk II form, model peptide of the crystalline fraction of B. mori silk fibroin fibers. Shown as dotted lines underneath are the spectral deconvolutions with Gaussion peaks.

and Spach,11 and Fraser et al.7 generally supported the antiparallel β-sheet model proposed by Marsh et al.,6 the former researchers also pointed out the presence of an irregular structure in the silk fibers. More recently, Takahashi et al.10 proposed that each crystal site of B.mori silk fiber is statistically occupied by two antiparallel β-sheet chains with different relative orientations. Actually, we recently found out that the Ala Cβ peak in the 13C CP/MAS NMR spectrum of B.mori silk fibroin is broad and asymmetric in the silk II form, and we demonstrated that this reflects the heterogeneous structure of the fiber in terms of backbone conformation as well as the packing.33,36 With a deconvolution assuming Gaussian line shapes, the Ala Cβ peak of the model peptide (AG)15 in silk II form yields three isotropic chemical shifts of 22.2, 19.6, and 16.7 ppm, with fractions of 27%, 46%, and 27%, respectively (Figure 9). This observation suggests that silk II possesses an intrinsically heterogeneous structure. The broad component at the highest field has essentially the same chemical shift as the sharp Ala Cβ peak at 16.7 ppm of silk I (data not shown).33 Therefore, we assigned the broad component at 16.7 ppm in Figure 9 to distorted β-turn.8 The other two components with the chemical shifts of 19.6 and 22.2 ppm in Figure 9 can be assigned to antiparallel β-sheet.8,22,33 Actually, the 2D spin-diffusion NMR spectrum indicates that the conformation of (AG)15 in silk II form is mainly an antiparallel β-sheet, and the torsion angles of the Ala residue are determined to be (φ, ψ) ) (-150°, 150°) (Figure 10). Since the Ala Cβ methyl groups are located outside of the protein backbone, the occurrence of two peaks suggests that there are difference in the mode of side-chain packing although the same backbone torsion angles are maintained. To confirm this interpretation, we used an ab initio molecular orbital method to calculate the 13C shielding constants of the Ala Cβ carbons. Two different kinds of stacks were generated from (AG)n by packing the protein chains according to the two models proposed by Takahashi et al. as shown in Figure 9.10 Both of these models possess

an antiparallel β-sheet structure; however, the methyl groups are oriented differently between the adjacent sheets. In the first case, all Ala methyl groups point in the same direction. In the second case, they alternately point in opposite directions. The initial coordinates were energy-minimized at the B3LYP/6-31G* levels. Then the 13C chemical shielding of the Ala Cβ carbon was calculated by the GIAO method using the RHF/6-31G* levels.38 The calculated shielding values of the Ala Cβ carbons in the former case (parallel Ala in Figure 9) predict a shift of 2.5 ppm toward lower field compared to the latter case (alternating Ala in Figure 9). This is consistent with the observed chemical shift difference of 2.6 ppm between the two deconvoluted components (Figure 9). The peak at 22.2 ppm is therefore assigned to the Ala methyl groups aligned in parallel, whereas the peak at 19.6 ppm corresponds to those pointing in opposite directions. The relative peak intensities at 22.2 and 19.6 ppm are approximately 1:2, which is in good agreement with the ratio of different packing modes suggested from X-ray diffraction.10 Structural Change of B. mori Silk Fibroin from Silk I to Silk II. Since the Ala Cβ peak in the 13C CP/MAS NMR spectrum of model peptide such as (AG)3YG(AG)2VGYG (AG)3Y(AG)3, for semicrystalline domain does not change significantly after the silk I and silk II treatments,17 it was concluded that the structural change from silk I to silk II occurs exclusively in the crystalline region (around 55% of total silk fibroin) of B. mori silk fibroin. Thus, on the basis of determination of the detailed structures of the peptides for the crystalline domain in both silk I and silk II forms, it is possible to discuss the mechanism of the structural transition during fiber formation. It may be noted that the concentration of the silk fibroin stored in the middle silk gland is about 30% in water.39 Magoshi et al.15,40 and Kataoka et al.41,42 described that the silk fibroin in the spinneret of the B. mori silkworm undergoes a structural change under tensile and shear stresses and loss of water. So that, the MD simulation was performed by taking into account these factors to clarify the structural change from silk I to silk II.43 Sixteen (Ala-Gly)6 chains with repeated β-turn type II structure were arranged in the periodic cell of the system.30 The MD calculation was tried by changing the shear stress when the tensile stress is fixed to be 0.1 GPa because the

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Figure 11. Snapshot of the structure of sixteen (Ala-Gly)6 under 0.1 GPa of the tensile stress and 0.7 GPa of the shear stress with time. The chains were embedded in 916 water molecules for the initial arrangement of an aqueous solution system, which corresponds to the concentration of silk fibroin in the middle silkgland (about 30 (w/v)%). Detailed experimental conditions were described in the text.

presence of strong shear stress applied to the silk fibroin at the press part of the spinneret has been emphasized. Figure 11 shows the structural snapshots of sixteen (Ala-Gly)6 under 0.1 GPa of the tensile stress and 0.7 GPa of the shear stress at different calculation steps. Further structural information was shown in Figures 12 and 13. Figure 12 shows the time dependences of conformational probability distributions for Ala and Gly residues in the aqueous solution system where the shear stress is 0.7 GPa and the tensile stress is 0.1 GPa, respectively. The conformational maps at the equilibrium stages were described in detail previously.43 At the time of 80-82 ps, the preferred conformation was changed to mainly C5 conformation for both residues. However, there is also one small minimum region at around ((φ, ψ) ) (90°,150°)) in the map of Ala residue. Figure 13A shows the distribution of the torsion angles of Ala residues after MD simulation at the time of 80-82 ps under 0.7 GPa of shear stress and 0.1 GPa of weak tensile stress, where water molecules are present. Then the water molecules are removed and MM calculation was performed by taking into account the fact that the water molecules are rapidly evaporated just after spinning of silkworm.43 After the removal of water molecules, the change in the Ramachandran map of Ala residues is remarkable as shown in Figure 13B, where the scattering of the conformations in the Ramachandran map becomes larger, and the C5 and PII regions tend to appear separately. In addition, one small minimum region at around (φ, ψ) ) (90°, 150°) in Figure 13A tends to disappear. As we reported above,33,36 the crystalline region of B. mori silk fiber takes a heterogeneous structure as shown in Figure 9. By considering the distribution of the torsion angles of Ala residues, the fractions of β-sheet (φ, ψ) ) (-150° ( 30°, 150° ( 30°)33 and β-turn (φ, ψ) ) (-60° ( 40°, 130° ( 40°)30 are calculated by changing the shear stress. The experimental data, 73% for β-sheet and 27% for β-turn, are the same results calculated at a shear stress of 0.7 Gpa, suggesting that the heterogeneous structure in the crystalline region of

B. mori silk fibroin fiber could be reproduced in this research. Moreover, the distance between two Cβ carbons of the Ala residues in the β-sheet (φ, ψ) ) (-150° ( 30°, 150° ( 30°) region was calculated except the Ala Cβ carbons within the same chain. The number of the pairs with the distance less than 10 Å was calculated under the shear stress of 0.7 Gpa. There are two distributions centered at around 4 and 5 Å. Actually, these data, around 4 and 5 Å coincide with the distances between two Ala Cβ carbons cycled in “alternating Ala” and “parallel Ala” as shown in Figure 9, respectively. The fraction of the distribution centered at 4 Å is larger than that centered at 5 Å, which is in agreement with our experimental NMR results, that is, 27% β-sheet (parallel Ala residues) and 46% β-sheet (alternating Ala residues) in model peptide for the crystalline region. Interestingly, this MD calculation result agrees well with our previous experimental data, in which the structural change from silk I to silk II was monitored by carefully stretching the gellike liquid silk stored in the middle silkgland of the fifth larval stage B. mori silkworm as well as native silk fibroin fibers.36 With increasing the stretching ratio of B. mori silk fibroin, the relative intensity of the peaks at around 22.1 and 19.9 ppm increases and that at around 16.7 ppm decreases simultaneously. The fraction of the peaks at 22.1 and 19.9 ppm increases gradually with keeping the ratio of relative intensities, 2:1. This means the appearance of heterogeneous silk II form. Summary In this paper, we reviewed our recent studies on the structural determination of silk fibroins before and after spinning using a combination of advanced solid-state NMR methods. The mechanism of fiber formation corresponding to B.mori silk fibroin was also reviewed. Silks are a wide class of fibrous proteins, which are composed of repeated sequences of amino acids and suitable to study the structureproperty relationship. Artificial polymeric proteins are of increasing interest to biotechnology. In the past decade, the



Figure 13. Conformational distributions of Ala residues in sixteen (Ala-Gly)6 chains in aqueous solution under 0.1 GPa of the tensile stress and 0.7 GPa of the shear stress (A) and then the distributions calculated with molecular mechanics method after removal of water molecules (B). Redrawn from ref 43.

Acknowledgment. T.A. acknowledges the support from the Asahi Glass Foundation and the Insect Technology Project, Japan. References and Notes Figure 12. Conformational probability maps of Ala (A) and Gly (B) residues in sixteen (Ala-Gly)6 chains in aqueous solution under 0.1 GPa of the tensile stress and 0.7 GPa of the shear stress with time. Contour lines of its probability are drawn every 0.0005 from 0.0005 to 0.002. Redrawn from ref 43.

design and synthesis of silk-like artificial proteins is an emerging area of research with important implications for structural biology, materials science, and biomedical engineering.44-50 Significant progress has been reported in the design of fibrous proteins that adopt predictable secondary structures and have higher order protein folding. With virtually absolute control of amino acid sequence, chain length, and stereochemical purity, the artificial proteins can be designed to represent a new class of macromolecular materials, with properties potentially quite different from those of the synthetic polymers currently available and in widespread use. Lazaris et al.46 described the production of a number of silk proteins in mammalian cells and showed, for the first time, that harvested recombinant proteins could be spun into strong, lightweight fibers. Although the fibers are not as good as natural silk, some improvements might be gained from making larger proteins by spinning the constituent proteins of natural dragline silk.

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Structures of Bombyx m ori and Samia c ynthia r icini Silk Fibroins

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