Structural Analysis of Bombyx mori Silk Fibroin Peptides with Formic

In this study we analyzed the structural characteristics of native peptides, derived from B. mori silk fibroin, with formic acid treatment using high-...
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Biomacromolecules 2004, 5, 1763-1769

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Structural Analysis of Bombyx mori Silk Fibroin Peptides with Formic Acid Treatment Using High-Resolution Solid-State 13C NMR Spectroscopy Juming Yao,† Kosuke Ohgo,† Rena Sugino,† Raghuvansh Kishore,‡ and Tetsuo Asakura*,† Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo, 184-8588, Japan, and Institute of Microbial Technology, Sector 39-A, Chandigarh-160 036, India Received March 22, 2004; Revised Manuscript Received April 29, 2004

Bombyx mori silk fibroin fiber is a fibrous protein produced by the silkworm at room temperature and from an aqueous solution whose primary structure is highly repetitive. In this study we analyzed the structural characteristics of native peptides, derived from B. mori silk fibroin, with formic acid treatment using highresolution solid-state 13C NMR. We establish that the Ser residue bearing a short polar side chain has the ability to stabilize the conformation formed in the model peptides due to its ability to form intermolecular hydrogen bonds involving its hydroxyl group as a donor and the carbonyl groups of other residues as acceptors. On the other hand, insertion of Tyr residue in the basic (AG)n and (AGSGAG)n sequence motifs usually exhibited disruptive effects on the preferred conformations. Moreover, the environmental effect was investigated by mixing the native Cp fraction with the model peptides, showing that there is no significant structural difference on the Ser-containing peptides, while structural transformation was observed on the peptides containing the GAAS unit. This may be attributed to the fact that the Cp fraction promotes the formation of an antiparallel β-sheet in the Ala-Ala unit. Such periodically disrupted ordered structures in the semicrystalline region of B. mori silk fibroin may be critical not only for facilitating the conformational transformation from silk I to silk II structural form but also for having some correlation with the unique properties of the silk materials. Introduction Silk, a fibrous protein produced by the domestic silkworm Bombyx mori, has a number of desirable properties, i.e., luster, comfort, high strength, and elasticity for textiles. In addition, recent studies clearly show that silk fibroin may find potential applications in the development and construction of various biotechnological and biomedical devices.1 In fact, such exclusive properties of the silk protein essentially originate from its unique amino acid composition translated into an unusual primary structure and hierarchical structural organization. The amino acid composition (in mol %) of B. mori fibrous protein shows the predominance of four amino acids: Gly (42.9%), Ala (30.0%), Ser (12.2%), and Tyr (4.8%).2 Employing a shotgun cDNA sequencing strategy combined with traditional physical map-directed sequencing of the fibroin gene of the heavy chain, first Mita et al.3 and later Zhou et al.4 predicted the presence of unusual repetitive sequences in the silk fibroin. Their analyses revealed that the primary structure of B. mori silk fibroin includes 12 distinct repetitive regions R connected by 11 amorphous regions A. The amino acid residues in each repetitive region R indicated unique arrangements: (i) the highly repetitive hexameric AGSGAG motif (432 copies) constituting the * To whom correspondence should be addressed. Phone/fax: +81-42383-7733. E-mail: [email protected]. † Tokyo University of Agriculture and Technology. ‡ Institute of Microbial Technology.

crystalline region, (ii) relatively less repetitive GAGAGY and/or GAGAGVGY motifs (120 copies and 11 copies, respectively) comprising semicrystalline regions, and (iii) the motif in i extended by AAS, i.e., AGSGAGAAS (42 copies). The 11 nonrepetitive amorphous regions A usually contain negatively charged, polar, bulky hydrophilic and aromatic residues which have the primary structure TGSSGFGPYVAHGGYSGYEYAWSSESDFGT. It may be presumed that the superior nature of silk fibroin, making it an excellent natural fiber, is likely to originate from the combination of these essential residues in the sequences, which are translated into higher order super secondary structures. The two crystalline forms, silk I and silk II, are reported as dimorphs of silk fibroin on the basis of extensive investigations from X-ray fiber diffraction,5-10 electron diffraction,8,10 conformational energy calculations,11,12 infrared,13 and 13C and 15N solid-state NMR spectroscopy.13-23 While the silk I form of the silk fibroin is the structure in the solid state before spinning, the silk II form is the structure after spinning. Under non-native conditions, it has been feasible to produce both the silk fibroin and the silk fibroinderived peptides in silk I or silk II structural form.12,20-22 For example, if the silk fibroin sample or its derived peptides is dissolved in 9 M LiBr solution and dialyzed against distilled water, the resulting structure will be the silk I form, and if the silk I sample is treated with formic acid and dried, it will give the silk II structural form. Significantly, the unique spectral features of solid-state 13C CP/MAS NMR,

10.1021/bm049831d CCC: $27.50 © 2004 American Chemical Society Published on Web 06/17/2004

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Table 1. Peptides and Their Amino Acid Sequences Used in This Study peptide

amino acid sequence

1 2 3 4 5 6 7

(AG)7SG(AG)7 (AGSGAG)8 AGSGAGAGSGA[1-13C]G[3-13C]A[2-13C]GSG[2-13C]AG[1-13C]AGSGAGAGSGAG (AG)7SG(AG)4YG(AG)2 (AGSGAG)4AGYGAG (AGSGAG)2(AGYGAG)(YGAGAG)(AGYGAG) AGYGAGAGAGYGAGAGSG[3-13C]A[1-13C]ASGAGAGAGAG

i.e., the characteristic conformation-dependent chemical shifts and the line shape of the carbon resonances, have been primarily used to ascertain, both qualitatively as well as quantitatively, the presence and/or absence of the two distinct structural forms.12-14,16,20-22 Despite a long history of interest in the less stable silk I form of B. mori silk fibroin, its structural determination was difficult because any attempt to induce orientation of the silk I form for X-ray and electron diffraction studies easily causes its conversion into the more stable silk II form.8-13 Nevertheless, we recently presented the conformational characteristics of the silk I form using a 30-residue synthetic peptide (AG)15 as the model for highly repetitive crystalline domains. Employing high-resolution solid-state NMR techniques and analyzing 13C CP/MAS NMR chemical shifts quantitatively, in conjunction with molecular simulations,20 we proposed a repeated β-turn type II structure (φAla ≈ -60 ( 5°, ψAla ≈ 130 ( 5° and φGly ≈ 70 ( 5°, ψGly ≈ 30 ( 5°) stabilized by a classical 4 f 1 intramolecular hydrogen bond for the silk I form. Numerous X-ray diffraction studies established that the structure of silk II is largely an antiparallel β-sheet structure.5-7,10,11 Conversely, using high-resolution solid-state 13 C NMR spectroscopy, we recently proposed a heterogeneous structure of B. mori silk fibroin for the silk II form.21,22 Although the precise disclosure of the amino acid sequence of B. mori silk fibroin has been presented, the structural organizations at the molecular level are not yet understood. Noteworthy in this connection are the structural analyses of the highly repetitive crystalline region, and the derived synthetic peptides of B. mori silk fibroin have been the subject of intensive investigation. In particular, the analysis of simple model peptides (AG)n has attracted considerable attention.10,11,20,21 The deduced primary structure of B. mori silk fibroin revealed the occurrence of polar, bulky aromatic and/or hydrophobic side chains as repetitive AGSGAG and GAGAGY sequence motifs. From the amino acid composition of this fibroin protein, the relative contents of Ser (12.2%) and Tyr (4.8%) residues appear to be reasonably high as they stand third and fourth behind the Gly and Ala residues. Recently, using solid-state 13C, 2H, and 15N NMR spectroscopy, we attempted to establish the relationship between the primary structure of B. mori silk fibroin and the ‘local’ structure of semicrystalline regions of biosynthetically isotope-labeled silk fibroin samples of B. mori, particularly the silk I and silk II forms in the vicinity of Tyr residues.23 A comparison of the observed Tyr CR and Tyr Cβ chemical shifts with those predicted from the contour plots provided evidence in favor of an antiparallel β-sheet structure of the Tyr residues in the silk fibroin fibers, whereas the

residue appears to exhibit random-coil or less ordered conformations in the fibroin film with a silk I form. To investigate the structural role played by Ser and Tyr residues in silk II structural form, we synthesized several model peptides containing Ser and Tyr residues in AG copolypeptides (Table 1), mimicking the primary structure of B. mori silk fibroin.4 The local structure of these peptides with formic acid treatment is studied by employing the highresolution solid-state 13C CP/MAS NMR spectroscopy. The 13 C NMR chemical shifts and the line shape of the predominant Ala Cβ resonance are analyzed quantitatively primarily.21,22 Analysis of experimental results in conjunction with theoretical molecular mechanics calculations provided further insight into the local structure across the Ser and Tyr residues in the highly repetitive sequence motifs of B. mori silk fibroin. These structures permitted further clarification of the short polar hydrophilic and bulky hydrophobic residues on the preferred secondary structure of the B. mori silk fibroin fibers. Materials and Methods Peptide Synthesis. The synthetic model peptides summarized in Table 1 were synthesized using solid-phase Fmoc chemistry (at 0.1 mmol scale) on a fully automated Pioneer Peptide Synthesis System (Applied Biosystems Ltd.). Fmoc amino acids and all reagents used in peptide synthesis were procured from PerSeptive Bio-systems, Warrington, U.K. The resin and N-[(dimethylamino)-1H-1,2,3-triazol[4,5-b]pyridin-1-ylmethylene]-N-methyl methanaminium hexafluorophosphate N-oxide (HATU) were from Applied Biosystems and PE Biosystems, respectively. The solvents of high-purity grade and other chemicals were available locally from Wako Pure Chemical Industries Ltd. Typically the peptide was assembled on poly(ethylene glycol)-polystyrene (Fmoc-GlyPEG-PS) resin (0.19 mmol/g). The coupling of Fmoc amino acids was performed by HATU. After synthesis, the free peptides were released from the resin by treatment with a 40 mL mixture of trifluoroacetic acid (TFA), phenol, triisopropylsilane, and water (88:5:2:5 vol %) for 2 h at room temperature. The crude peptide was precipitated with dry diethyl ether and washed repeatedly with cold ether. The precipitate, collected by centrifugation, was dried in vacuo. The solid sample was dissolved in 9 M LiBr and dialyzed extensively against distilled water for several days. The desired peptide was recovered by filtration and/or lyophilization. All of the samples were accomplished by dissolving the peptides in a minimum amount of formic acid and dried at ambient temperature. Cp Fraction of B. mori Silk Fibroin. The Cp fraction of B. mori silk fibroin was prepared from regenerated silk

Analysis of B. mori Silk Fibroin Peptides

fibroin solution as described elsewhere.12,22 Chymotrypsin (40 mg), dissolved in a few milliliters of water, was added to an aqueous solution of about 4 g of fibroin buffered with Na2HPO4‚12H2O and NaH2PO4‚2H2O at pH 7.8. The solution (200 mL) was incubated at 40 °C for 24 h, and the precipitate that formed (Cp fraction) was separated by centrifuging at 10 000 rpm followed by washing with 0.03 N HCL to inactivate the enzyme reaction. Then the precipitate was washed several times with distilled water, ethyl alcohol, and ethyl ether. Finally, the precipitate was dried in a vacuum, yielding 55% of original fibroin. NMR Spectroscopy. The 13C CP/MAS NMR measurements were conducted on a Chemagnetics CMX-400 spectrometer operating at 100 MHz for the 13C nucleus. CP was employed for sensitivity enhancement with high-power 1H decoupling during the signal acquisition interval. A 1H 90° pulse width of 5 µs duration was used with a 1 ms contact time and a 3 s repetition time. Approximately 15K FIDs were added to generate the spectra. The chemical shifts were represented in parts per million with respect to the external reference adamantane. To make a direct comparison with previous data,12-14,16 we added 28.8 ppm to the observed chemical shifts to account for TMS as a reference on the silk II forms of B. mori silk fibroins. Molecular Mechanics Calculations. The molecular mechanics (MM) calculations were performed with the Discover 3 module in Insight II (Ver. 4.0.0P+, Accelrys) using the cvff force field on an OCTANE workstation (Silicon Graphics).24,25 The initial conformation of the silk II form with an antiparallel β-sheet backbone conformation (φAla ) -140°, ψAla ) 153° and φGly ) -141°, ψGly ) 152°) including a total of four octapeptide sequences (three peptide chains of (AG)4 and one peptide chain of AGAGSGAG) was accommodated in a cell according to Takahashi et al.7 Before performing the MM calculations, we also considered all possible side-chain conformations accessible to the Ser side chain. In our previous paper the deuterium solid-state NMR data showed that the value of the preferred N-CR-Cβ-O torsion angle (χ1) was -67° by studying the dynamics and molecular structure of the serine side chains in the [3,3-2H2]Ser-labeled B. mori silk fibroin in silk II.26 Thus, considering the three preferred torsion angles χ1 and χ2 [gauche (60°), trans (-180°), and gauche′ (-60°)], three possible combinations for the χ1/χ2 pair [-60°/-60°, -60°/60°, and -60°/180°] were included for MM calculations, and the entire system with four chains in vacuo was minimized by 3000 steps according to the steepest descent method. Variables in the MM calculation are the χ1, χ2 angles of Ser and the φ, ψ backbone torsion angles of Ala, Gly, and Ser in addition to the cell parameters a(Å), b(Å), c(Å), R(°), β(°), and γ(°). Results and Discussion 13 C CP/MAS NMR Study of Sequence Motifs Containing Ser Residue. A typical Cp fraction represents the insoluble crystalline part of B. mori silk fibroin obtained after chymotrypsin cleavage, which comprises about 55% of the total fibroin.27 The amino acid sequence of the Cp fraction has generally been accepted as GAGAGSGAA[SGAGAG-

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Figure 1. 13C CP/MAS NMR spectra of model peptides with formic acid treatment: (a) (AG)15 copolymer; (b) Cp fraction; (c) (AGSGAG)5; (d) peptide 1, (AG)7SG(AG)7, and (e) peptide 2, (AGSGAG)8.

]nY, where n is usually 6-10. On the basis of this characteristic primary structure, we synthesized two model peptides, (AG)7SG(AG)7 (1) and (AGSGAG)8 (2), treated the peptides with formic acid, and subsequently dried them. Figure 1d,e shows the fully assigned 13C CP/MAS NMR spectra of the two Ser-containing model peptides 1 and 2. The resonance assignments of the Ala, Gly, and Ser residues in NMR spectra are primarily based on the known chemical shift values reported for model peptides, (AG)15 and (AGSGAG)5, and the Cp fraction in silk II form (Figure 1a-c).22 The conformation-dependent chemical shifts of the three Ala carbons, i.e., CR, Cβ, and carbonyl, along with line-shape analysis of the Ala Cβ peak are established as characteristic markers to identify the existence of silk I and silk II structural forms of B. mori silk fibroin and the model peptides.12-14 As seen in Figure 1, the 13C chemical shifts of the Ala, Gly, and Ser residues in 1 and 2 are in excellent agreement with those of (AG)15, (AGSGAG)5, and the Cp fraction in silk II form. Therefore, it may be inferred that the judicial insertion of Ser residues in the basic sequence of the simple (AG)n copolymer with formic acid treatment does not lead to any dramatic structural differences when compared to the native crystalline Cp fraction extracted from B. mori silk fibroin. Recent focus on the analysis of the broad and asymmetric Ala Cβ peak of the native fibroin sample and synthetic model peptides in the 13C CP/MAS NMR spectrum revealed that the (AG)15 sequence as well as Cp fraction in silk II form is intrinsically heterogeneous, i.e., both backbone conformations and the packings.21,22 In the present study, the Ala Cβ peaks of both 1 and 2 with silk II treatment (i.e., formic acid treatment) are also broad and asymmetric as noted for (AG)15 and Cp fraction under similar conditions. Assuming Gaussian line shape, the peak deconvolution was performed for detailed structural analysis. As shown in Figure 2, the Ala Cβ signals of both peptides consist of three components, yielding three isotropic chemical shifts at 22.3, 19.8, and

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Figure 3. Illustration of the environment surrounding Ser in (AG)n copolymer model calculated by MM from initial silk II conformational parameters. It shows the most favorable Ser side-chain conformations (χ1, χ2) ) (-54°, -87°), which are compatible with a silk II form being maintained. Green line shows the intermolecular hydrogen bond. Figure 2. Expanded Ala Cβ peaks of 13C CP/MAS NMR spectra of (a) peptide 1, (AG)7SG(AG)7, and (b) peptide 2, (AGSGAG)8, with formic acid treatment. Table 2. Side-Chain Torsion Angles χ1/χ2 of Ser Residue before and after Molecular Mechanics Calculations Together with the Resulting Set of Backbone Torsion Angles (φ, ψ) of Ser Residue, Calculated for a Unit Cell Containing Three AGAGAGAG Chains and One AGAGSGAG Chaina

initial (χ1, χ2)

final (χ1, χ2)

Ser5 (φ, ψ)

total potential energy (kcal mol-1)

(-60°, -60°) (-60°, 180°) (-60°, 60°)

(-54°, -87°) (-54°, 81°) (-54°, 81°)

(-152°, 121°) (-141°, 122°) (-152°, 121°)

125.4 126.1 125.4

a

The chain arrangement was described in the text.

16.8 ppm with fractions of 25%, 47%, and 28% for peptide 1 and 22.2, 19.7, and 16.8 ppm with fractions of 27%, 40%, and 32% for peptide 2. The results are quite analogous with the Cp fraction in silk II form, i.e., 22.1, 19.5, and 16.5 ppm with 23%, 45%, and 32%, respectively.22 Consequently, the peak at ∼22.3 ppm is unambiguously assigned to the Ala Cβ groups aligned in parallel, and the peak at ∼19.7 ppm corresponds to those pointing in opposite directions in the antiparallel β-sheet, whereas the peak at ∼16.8 ppm may be safely attributed to a distorted β-turn as established previously.21,22 Molecular Mechanics Calculations of Ser-Containing Peptides in Silk II Form. To gain further insight into the influence of Ser residues on the molecular conformation and intermolecular arrangement of polypeptide chains in the silk II form, MM calculation was performed on a mixture of one AGAGSGAG and three AGAGAGAG chains. While the initial backbone torsion angles of the nonchiral Gly residue were φ ) -141° and ψ ) 152°, the torsion angles of the chiral Ala and Ser residues were φ ) -140° and ψ ) 153°7 and the Ser side-chain orientations were placed in their preferred rotameric combinations: χ1 (-60°) and χ2 (-60°, 60°, -180°). The results of MM calculations and the torsion angles of the Ser backbone and side chain in AGAGSGAG are summarized in Table 2. The data clearly suggests their conformation in the antiparallel β-sheet region, irrespective of initial Ser side-chain orientations. In our previous paper 2 H solid-state NMR was used to study the dynamics and molecular structure of the serine side chains in the [3,3-2H2]Ser-labeled B. mori silk fibroin fibers, and the data suggested that approximately 75% of the Ser residues contribute to the

formation of hydrogen bonds in the silk fibroin fibers.26 In particular, the gauche conformer around N-CR-Cβ-O was found to be dominant, suggesting that the hydroxyl groups of Ser interact with carbonyl groups on adjacent chains and thereby contribute to the intermolecular hydrogen-bonding network of the fiber. For example, Figure 3 shows the arrangement of two chains, one of which is AGAGAGAG and another is AGAGSGAG, calculated with the MM method in Table 2, where (φ, ψ) ) (-152°, 121°) and (χ1, χ2) ) (-54°, -87°) for the Ser residue. This calculated structure is consistent with the experimental results mentioned above. We have insisted that the insertion of Ser residue(s) into (AG)n copolymer can readily be accommodated in the β-sheet conformation without any significant deviations in the preferred intermolecular organizations of the adjacent chains. 13 C CP/MAS NMR Spectra of Ser-Containing Peptides Mixed with Native Cp Fraction. Since the preferred conformation adopted by the fibrous fibroin generally depends on intermolecular interactions as well as intramolecular ones, the structure of specific motifs containing Ser residues, mixed with native Cp fraction, were also examined. The 13C CP/MAS NMR spectrum of the 13C isotope-labeled peptide sequence (AGSGAG)5 (3) with formic acid treatment is shown in Figure 4a. The NMR spectrum of peptide 3 and the native Cp fraction mixed with a molar ratio of 1:5 with formic acid treatment is also observed as shown in Figure 4b. The observed 13C chemical shifts, e.g., ∼42.3 ppm for Gly CR and ∼49.3 ppm for Ala CR, and also the carbonyl carbons of these two residues are in excellent agreement with those of Cp fraction in silk II form (Figure 4c) within experimental error, suggesting that the Ser residues in the model peptides studied have the same structural role as those in the native Cp fraction. Further evidence to substantiate this fact was obtained by subtracting the spectrum of the Cp fraction (Figure 4c) from that of the model peptide and Cp fraction mixture (Figure 4b). The difference spectrum (Figure 4d) shows remarkable similarity with that of peptide 3. The deconvolution of broad Ala Cβ peaks yields three components, i.e., the isotropic chemical shifts of 22.3, 19.9, and 16.8 ppm with fractions of 30%, 40%, and 30% for the difference spectrum and 22.5, 19.9, and 16.8 ppm with fractions of 32%, 34%, and 33% for peptide 3, respectively (figures not shown). The results of analysis are basically similar to those observed for the native Cp fraction reported previously.22

Analysis of B. mori Silk Fibroin Peptides

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Figure 5. 13C CP/MAS NMR spectra of model peptides with formic acid treatment: (a) peptide 4, (AG)7SG(AG)4YG(AG)2, (b) peptide 5, (AGSGAG)4AGYGAG, and (c) peptide 6, (AGSGAG)2(AGYGAG)(YGAGAG)(AGYGAG). The peptide (AGYGAG)5 we reported previously was used for comparison (d).23 Figure 4. 13C CP/MAS NMR spectra of model peptides with formic acid treatment: (a) peptide 3, AGSGAGAGSGA[1-13C]G[3-13C]A[213C]GSG[2-13C]AG[1-13C]AGSGA GAGSGAG, (b) peptide 3 and Cp fraction mixed with a molar ratio of 1:5, (c) Cp fraction, and (d) difference spectrum (b - c). ssb represents the spinning sideband. 13

C CP/MAS NMR Study of Sequence Motifs Containing Ser and Tyr Residues. Disclosure of the primary structure of B. mori silk fibroin revealed the complex organization of the repetitive motifs that incorporate Ser and Tyr residues, which in turn connect the crystalline and semicrystalline regions of the fibroin molecule. Our preliminary structural investigations on peptide motifs containing the Tyr residue(s) suggest that the insertion of Tyr at the central region produces local destabilization of both silk I and silk II structural forms in (AG)15 peptide sequence.24 Figure 5a-c shows the 13C CP/MAS NMR spectra and the peak assignments of model peptides containing Ser and Tyr residues with formic acid treatment. The peptide (AGYGAG)5 previously reported was also used for comparison (Figure 5d).23 As expected, the observed 13C NMR chemical shifts of these peptide sequences suggest that the Ala and Gly residues largely accommodate the silk II form, i.e., antiparallel β-sheet structure, as revealed from the unique chemical shift values of the constituent residues: ∼42.6 ppm for Gly CR, ∼49.1 ppm for Ala CR, and ∼64.0 ppm for Ser Cβ. However, 13C NMR peaks for the Tyr-containing peptides 4, 5, and 6 yield different results after deconvolution of broad Ala Cβ peaks. The broad Ala Cβ peaks (Figure 5a and b) indicated three components, i.e., the isotropic chemical shifts of 22.6, 19.9, and 16.5 ppm with fractions of 27%, 39%, and 34% for peptide 4 and 22.2, 19.7, and 16.7 ppm with fractions of 23%, 41%, and 36% for peptide 5, respectively (Figure 6a and b). The overall analysis yields similar results to those observed in Ser-containing model peptides discussed above, although the fraction at ∼16.7 ppm increased insignificantly. Therefore, these three components, from low field to high field, are assigned to the Ala methyl groups aligned in parallel and pointing in opposite directions in

Figure 6. Expanded Ala Cβ peaks of 13C CP/MAS NMR spectra of (a) peptide 4, (AG)7SG(AG)4YG(AG)2, (b) peptide 5, (AGSGAG)4AGYGAG, (c) peptide 6, (AGSGAG)2(AGYGAG)(YGAGAG)(AGYGAG), and (d) peptide (AGYGAG)5 with formic acid treatment.

antiparallel β-sheets and the Ala methyl groups in distorted β-turn, as suggested previously.21,22 On the other hand, in the broad Ala Cβ peak in peptides 6 and (AGYGAY)5 (shown in Figure 5c and d), only two components were obtained, i.e., the isotropic chemical shifts of 21.2 and 16.4 ppm with fractions of ∼61% and 39%, for peptide 6 and 21.3 and 16.3 ppm with fractions of ∼45% and 55% for peptide (AGYGAY)5, respectively (Figure 6c and d). Since larger half-height

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Figure 8. Expanded Ala Cβ peaks of 13C CP/MAS NMR spectra of (a) peptide 7, GYGAGAGAGYGAGAGSG[3-13C]A[1-13C]ASGAGAGAGAG, and (b) difference spectrum shown in Figure 7.

of 1:5 (Figure 7b) followed by formic acid treatment. The C chemical shifts of the Ala and Gly carbons in these two cases are basically the same as those of Cp fraction in silk II form (Figure 7c). By subtracting the spectrum of the Cp fraction (Figure 7c) from that of the model peptide and Cp fraction mixture (Figure 7b), we obtained the difference spectrum (Figure 7d). The deconvolutions of broad Ala Cβ peaks of peptide 7 and the difference spectrum are shown in Figure 8. The Ala Cβ peak of peptide 7 yields three components, i.e., the isotropic chemical shifts of 22.4, 20.0, and 16.8 ppm with fractions of 24%, 32%, and 44%, respectively. In the difference spectrum the fraction at lowest field was decreased significantly and replaced by more of the fraction at 20.0 ppm, although it still yields three components, i.e., the isotropic chemical shifts of 22.3, 20.0, and 16.4 ppm with fractions of 13%, 45%, and 42%, respectively. It is noteworthy that the fractions at the highest field are relatively larger, 42-44%, in both cases when compared with the Cp fraction in silk II form, which may due to the high content of Tyr residues in this peptide. However, the effect from the environment around the peptide is clear. The native Cp fraction can easily transform its structure from silk I (repeated β-turn type II) to silk II (antiparallel β-sheet), which may promote the formation of the antiparallel β-sheet in the Ala-Ala unit. Thus, rather than the groups being aligned in parallel, the Ala methyl groups pointing in opposite directions are principally formed due to its stability compared with the former type, although both are in antiparallel β-sheets. We characterized the structure of the 11 nonrepetitive amorphous regions A, TGSSGFGPYVAHGGYSGYEYAWSSESDFGT, showing that these regions predominantly form the random-coil conformation independent of the treatment.28 13

Figure 7. 13C CP/MAS NMR spectra of model peptides with formic acid treatment: (a) peptide 7, GYGAGAGAGYGAGAGSG[3-13C]A[1-13C]ASGAGAGAGAG, (b) peptide 7 and Cp fraction mixed with a molar ratio of 1:5, (c) Cp fraction, and (d) difference spectrum (b c). ssb represents the spinning sideband.

widths were observed when compared with the above two samples, peptides 4 and 5, these two components at 21.2 and 16.4 ppm are assigned to the methyl groups in distorted β-sheet and distorted β-turn structures, respectively. Hence, a single Tyr residue can be readily accommodated by the antiparallel β-sheets formed in both (AG)n and (AGSGAG)n, while the presence of multiple Tyr residues tends to populate disordered structures, like a mixture of distorted β-sheet and distorted β-turn conformations.24 13 C CP/MAS NMR Spectra of Sequence Motifs Containing the GAAS Unit. The characteristic primary structure of the Cp fraction of B. mori silk fibroin shows the occurrence of a GAAS unit in addition to the predominant (AGSGAG)n motif. The periodic appearance of GAAS units in the fibroin molecule stimulated us to analyze the influence of this tetrapeptide segment on the secondary structure of this basic (GA)n sequence. Figure 7a shows the 13C CP/MAS NMR spectrum of model peptide 7 containing GAAS unit, prepared with formic acid treatment as mentioned above. To make the detailed structural analysis of this unit, isotope labeling was performed in this peptide. Expectedly, the specific signals reflecting the local structure of GAAS unit were enriched well at the Ala Cβ and carbonyl regions. From the characteristic chemical shifts of both signals it is clear that both components have the antiparallel β-sheet structure. Previously we introduced a Ala-Ala unit in (AG)15 sequence which dramatically changed the structure from silk I to silk II.17 Consequently, we believe that the presence of Ala-Ala units in B. mori silk fibroin chain may be the regions that may induce/facilitate the structural transition favoring silk fiber formation. Similar processing was performed on this peptide by mixing peptide 7 and native Cp fraction with a molar ratio

Summary In this study we analyzed the structural characteristics of native peptides derived from B. mori silk fibroin with formic acid treatment using high-resolution solid-state 13C NMR. The roles of Ser and Tyr amino acid residues in determining the local conformation of silk fibroin and the related conformational transitions were investigated. The role of Ser

Analysis of B. mori Silk Fibroin Peptides

residues is suggested to stabilize the extended structure in the solid-state silk fibroins, while the insertion of more Tyr residues in the basic (AG)n and (AGSGAG)n sequence motifs usually exhibited disruptive effects on the preferred conformations. Further, the present conclusion is somewhat consistent with the proposal of Kaplan and co-workers, who indicated that amorphous Tyr-containing blocks of the native sequence of B. mori silk fibroin do not play a crucial role in selection of the silk I, II, and III polymorphs.29 Such disrupted ordered structures in the semicrystalline region of B. mori silk fibroin may be critical for facilitating the conformational transformation from silk I to silk II structural form and are presumed to have some correlation with the unique properties of the silk materials. Acknowledgment. T.A. acknowledges research grant support from the Insect Technology Project and Ashahi Glass Foundation, Japan. R.K. was supported by the Japan Society for the Promotion of Science Invitation Fellowship.

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References and Notes (1) Altman, G. H.; Diaz, F.; Jakuba, C.; Calabro, T.; Horan, R. L.; Chen, J.; Lu, H.; Richmond, J.; Kaplan, D. L. Silk-based biomaterials. Biomaterials 2003, 24, 401-416. (2) Kaplan, D. L.; Adams, W. W.; Farmer, B.; Viney, C. Silk: Biology, structure, properties and genetics. In Silk Polymers: Materials Science and Biotechnology; Kaplan, D. L., Adams, W. W., Farmer, B., Viney, C., Eds.; ACS Symposium Series 544; American Chemical Society: Washington, D.C., 1994; pp 2-16. (3) Mita, K.; Ichimura, S.; James, T. C. Highly repetitive structure and its organization of the silk fibroin gene. J. Mol. EVol. 1994, 38, 583592. (4) Zhou, C.-Z.; Confalonieri, F.; Medina, N.; Zivanovic, Y.; Esnault, C.; Yang, T.; Jacquet, M.; Janin, J.; Duguet, M.; Perasso, R.; Li, Z.-G. Fine organization of Bombyx mori fibroin heavy chain gene. Nucleic Acids Res. 2000, 28, 2413-2419. (5) Marsh, R. E.; Corey, R. B.; Pauling, L. An investigation of the structure of silk fibroin. Biochim. Biophys. Acta. 1955, 16, 1-34. (6) Fraser, R. D. B.; MacRae, T. P.; Stewart, F. H. C. Poly-l-alanylglycyll-alanylglycyl-l-serylglycine: a model for the crystalline regions of silk fibroin. J. Mol. Biol. 1966, 19, 580-582. (7) Takahashi, Y.; Gehoh, M.; Yuzuriha, K. Structure refinement and diffuse streak scattering of silk (Bombyx mori). Int. J. Biol. Macromol. 1999, 24, 127-138. (8) Okuyama, K.; Takanashi, K.; Nakajima, Y.; Hasegawa, Y.; Hirabayashi, K.; Nishi, N. Analysis of silk I structure by X-ray and electron diffraction methods. J. Seric. Sci. Jpn. 1988, 57, 23-30. (9) Anderson, J. P. Morphology and crystal structure of a recombinant silk-like molecule, SLP4. Biopolymers 1998, 45, 307-321. (10) Lotz, B.; Keith, H. D. Crystal structure of poly(L-Ala-Gly) II: A model for silk I. J. Mol. Biol. 1971, 61, 201-215. (11) Fossey, S. A.; Nemethy, G.; Gibson, K. D.; Scheraga, H. A. Conformational energy studies of β-sheets of model silk fibroin peptides. I. Sheets of poly(AG) chains. Biopolymers 1991, 31, 15291541. (12) Asakura, T.; Kuzuhara, A.; Tabeta, R.; Saito, H. Conformational characterization of B. mori silk fibroin in the solid state by highfrequency 13C cross polarization-magic angle spinning NMR, X-ray diffraction and infrared spectroscopy. Macromolecules 1985, 18, 1841-1845. (13) Saito, H.; Tabeta, R.; Asakura, T.; Iwanaga, Y.; Shoji, A.; Ozaki, T.; Ando, I. High-resolution 13C NMR study of silk fibroin in the solid state by the cross-polarization-magic angle spinning method.

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(21)

(22) (23)

(24)

(25)

(26)

(27) (28)

(29)

Conformational characterization of silk I and silk II type forms of B. mori fibroin by the conformation-dependent 13C chemical shifts. Macromolecules 1984, 17, 1405-1412. Ishida, M.; Asakura, T.; Yokoi, M.; Saito, H. Solvent- and mechanical-treatment-induced conformational transition of silk fibroin studied by high-resolution solid-state 13C NMR spectroscopy. Macromolecules 1990, 23, 88-94. Nicholson, L. K.; Asakura, T.; Demura, M.; Cross, T. A. A method for studying the structure of uniaxially aligned biopolymers using solid state 15N-nmr: application to Bombyx mori silk fibroin fibers. Biopolymers 1993, 33, 847-861. Asakura, T.; Demura, M.; Miyashita, N.; Ogawa, K.; Williamson, M. P. NMR study of silk I structure of B. mori silk fibroin with 15Nand 13C-NMR chemical shifts contour plots. Biopolymers 1997, 41, 193-203. Asakura, T.; Sugino, R.; Okumura, T.; Nakazawa, Y. The role of irregular unit, GAAS, on the secondary structure of Bombyx mori silk fibroin studied with 13C CP/MAS NMR and wide-angle X-ray scattering. Protein Sci. 2002, 11, 1873-1877. Asakura, T.; Minami, M.; Shimada, R.; Demura, M.; Osanai, M.; Fujito, T.; Imanari, M.; Ulrich, A. S. 2H-labeling of silk fibroin fibers and their structural characterization by solid-state 2H NMR. Macromolecules 1997, 30, 2429-2435. Demura, M.; Minami, M.; Asakura, T.; Cross, T. A. Structure of B. mori silk fibroin based on solid-state NMR orientational constraints and fiber diffraction unit cell parameter. J. Am. Chem. Soc. 1998, 120, 1300-1308. Asakura, T.; Ashida, J.; Yamane, T.; Kameda, T.; Nakazawa, Y.; Ohgo, K.; Komatsu, K. A repeated β-turn structure in poly(Ala-Gly) as a model for silk I of Bombyx mori silk fibroin studied with twodimensional spin-diffusion NMR under off magic angle spinning and rotational echo double resonance. J. Mol. Biol. 2001, 306, 291305. Asakura, T.; Yao, J.; Yamane, T.; Umemura, K.; Ulrich, A. S. Heterogeneous structure of silk fibers from Bombyx mori resolved by 13C solid-state NMR spectroscopy. J. Am. Chem. Soc. 2002, 124, 8794-8795. Asakura, T.; Yao, J. 13C CP/MAS NMR study on structural heterogeneity in Bombyx mori silk fiber and their generation by stretching. Protein Sci. 2002, 11, 2706-2713. Asakura, T.; Sugino, R.; Yao, J.; Takashima, H.; Kishore, R. Structural analysis of semi-crystalline Bombyx mori silk fibroin chain with Silk I and Silk II forms by 13C, 15N and 2H stable isotope labeling, conformation-dependent chemical shifts and solid-state NMR spectroscopy. Biochemistry 2002, 41, 4415-4424. Asakura, T.; Suita, K.; Kameda, T.; Afonin, S.; Ulrich, A. S. Structural role of tyrosine in Bombyx mori silk fibroin, studied by solid-state NMR and molecular mechanics on a model peptide prepared as silk I and II. Magn. Reson. Chem. 2004, 42, 258-266 Yamane, T.; Umemura, K.; Nakazawa, Y.; Asakura, T. Molecular dynamics simulation of conformational change of poly(Ala-Gly) from repeated β-turn type II to β-sheet in relation to fiber formation mechanism of Bombyx mori silk fibroin. Macromolecules 2003, 36, 6766-6772. Kameda, T.; Ohkawa, Y.; Yoshizawa, K.; Naito, J.; Ulrich, A. S.; Asakura, T. Hydrogen-bonding structure of serine side chains in Bombyx mori and Samia cynthia ricini silk fibroin determined by solid-state 2H NMR. Macromolecules 1999, 32, 7166-7171. Strydom, D. J.; Haylett, T.; Stead, R. H. The amino-terminal sequence of silk fibroin peptide CPsa reinvestigation. Biochem. Biophys. Res. Commun. 1977, 3, 932-938. Asakura, T.; Ashida, J.; Yamane, T. Structure of Bombyx mori silk fibroin before spinning in silkworm. In NMR Spectroscopy of Polymers in Solution and in the Solid State; Cheng, H. N., English, A. D., Eds.; ACS Symposium Series 834; American Chemical Society: Washington, D.C., 2002; pp 71-82. Wilson, D.; Valluzzi, R.; Kaplan, D. Conformational transitions in model silk peptides. Biophys. J. 2000, 78, 2690-2701.

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