Synthesis and Characterization of Chimeric Silkworm Silk

Mar 29, 2003 - A tetramer of the chimeric repeat sequence encoding a ∼29 kDa protein was expressed as a fusion protein in Escherichia coli. In compa...
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Biomacromolecules 2003, 4, 815-820

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Synthesis and Characterization of Chimeric Silkworm Silk Tetsuo Asakura,*,† Koji Nitta,† Mingying Yang,† Juming Yao,† Yasumoto Nakazawa,† and David L. Kaplan‡ Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan, and Department of Chemical & Biological Engineering, Bioengineering Center, Tufts University, Medford, Massachusetts 02155 Received January 17, 2003; Revised Manuscript Received March 4, 2003

A synthetic gene encoding a chimeric silklike protein was constructed that combined a polyalanine encoding region (Ala)18, a sequence slightly longer than the (Ala)12-13 found in the silk fibroin from the wild silkworm Samia cynthia ricini, and a sequence encoding GVGAGYGAGAGYGVGAGYGAGVGYGAGAGY, found in the silk fibroin from the silkworm Bombyx mori. A tetramer of the chimeric repeat sequence encoding a ∼29 kDa protein was expressed as a fusion protein in Escherichia coli. In comparison to S. c. ricini silk, the chimeric protein demonstrated improved solubility because it could be dissolved in 8 M urea. The purified protein assumed an R-helical structure based on solid-state 13C CP/MAS NMR and was less prone to conformational transition to a β-sheet, unlike native silk proteins from S. c. ricini. Model peptides representing the crystalline region of S. c. ricini silk fibroin, (Ala)12 and (Ala)18, formed β-sheet structures. Therefore, the solubility and structural transitions of the chimeric protein were significantly altered through the formation of this chimeric silk. This experimental strategy to the study of silk structure and function can be used to develop an improved understanding of the contributions of protein domains in repetitive silkworm and spider silk sequences to structure development and structural transitions. Introduction Silks are of interest from fundamental and applied directions because of the unique synthesis and processing features of this family of proteins, as well as the unusual structural and functional properties of the fibers.1-3 In addition, because of the unique mechanical properties, as well as biodegradability, thermostability, and biocompatibility, silks are being actively pursued as new biomaterials for enzyme immobilization and scaffolds for tissue engineering.4-6 Bombyx mori and Samia cynthia ricini silkworm silk fibroins have been well studied in terms of primary structure,7 secondary structure,8-16 and mechanical properties.17,18 According to these studies, the primary sequences are composed of repeated motifs. B. mori silk fibroin is mainly composed of repetitions of two motifs, [Gly-Ala-Gly-Ser-Gly-Ala]n and [Gly-Ala-Gly-Xaa-Gly-Ala]n where Xaa ) Tyr or Val.7 The former motif forms crystalline domains in B. mori silk fibroin and the latter contributes to the solubility of the fibroin in water.19 On the other hand, the amino acid composition of silk fibroin from S. c. ricini is considerably different from that of B. mori silk fibroin. The sum of the glycine and alanine residues is 82%, similar to B. mori silk fibroin (73%), but the relative composition of alanine and glycine is reversed with a higher portion of alanine.2 The primary structure of the silk fibroin from S. c. ricini has recently been determined by Yukihiro et al.20 and consists of about 100 repeats of * To whom correspondence should be addressed. Tel & Fax: +81-42383-7733. E-mail: [email protected]. † Tokyo University of Agriculture and Technology. ‡ Tufts University.

alternating polyalanine, (Ala)12-13, and glycine-rich regions. From solution 13C and 15N NMR studies of S. c. ricini silk fibroin in aqueous solution, the polyalanine regions have been shown to form R-helices. A rapid exchange between the helix and coil forms in the NMR time scale has been observed with increasing temperature.14 With time, the conformational transition from R-helix to β-sheet structure occurs and the silk fibroin precipitates.21 When considering fundamental questions of primary sequence and environment on silk structure and function, as well as the use of silks as biomaterials for enzyme immobilization and scaffolds for tissue engineering, it is important to understand solubility to optimize processing options into useful scaffold morphologies, and it is important to control secondary structure because this will influence both processability and cell-specific responses. B. mori silk fibers are soluble in concentrated LiBr or CaCl2 aqueous solutions, and aqueous solutions can be generated after dialysis.1-3 However, S. c. ricini silk fibers are insoluble in these solutions.2 In this paper, a silklike protein chimera that combines motifs from two different species of silkworm was generated to improve solubility in water and to examine the control of structural features. The protein mainly consists of the following two sequences: polyalanine sequences found in S. c. ricini silk fibroin,20 and the sequence GVGAGYGAGAGYGVGAGYGAGVGYGAGAGY found in B. mori silk fibroin.7 Here, the polyalanine sequence is designed as (Ala)18, which is slightly longer than the native sizes found in the crystalline-forming regions, (Ala)12-13 from S. c. ricini

10.1021/bm034020f CCC: $25.00 © 2003 American Chemical Society Published on Web 03/29/2003

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Figure 1. The amino acid and nucleotide sequences of the chimeric silklike protein with contributions from the two silkworms indicated.

silk fibroin, which is expected to enhance the sizes of the domains of the crystalline regions through β-sheet formation. This will allow us to explore the effect of introduction of the glycine-rich sequence into this silklike protein. The gene was constructed, and the silklike protein was expressed, purified, and characterized. Several model peptides for the polyalanine region of S. c. ricini silk fibroin were also prepared to assist in the interpretation of structure and structural transitions. The synthesis and characterization of such silklike chimeric proteins is one approach to explore questions of primary sequence versus the impact of processing environments on silk protein assembly and subsequent material properties. Materials and Methods Materials and Reagents. Luria-Bertani broth (LB), agar, agarose, ampicillin, chloramphenicol, and isopropyl-β-Dthiogalactoside (IPTG) were purchased from Fischer Biotech. Restriction enzymes were purchased from New England Biolabs. Other enzymes and reagents, including polynucleotide kinase, T4 DNA ligase, ATP, and calf intestinal alkaline phosphatase were purchased from Pharmacia Biotech. Wizard Plus Miniprep DNA purification systems were purchased from Promega. DNA Synthesis and Sequence Analysis. The synthesis of the designed oligonucleotide sequences and linkers was carried out at the Tufts University Medical School Protein Facility in Boston, Massachusetts. All DNA sequencing to confirm constructs was also performed at this facility. Gene Construction. Four oligonucleotides encoding the modified S. c. ricini silk fibroin crystalline region (MSc) and B. mori silk fibroin glycine-rich region (Bs) were designed, synthesized, and subsequently cloned, ligated, and subcloned using previously reported strategies involving head-to-tail ligation and orientation for Nhe I and Spe I sites (Figure 1).22-24 Codons were optimized for expression in E. coli. Complementary sequences (MSc I/MSc II and MSc III/MSc IV, Bs I/Bs II and Bs III/Bs IV) were annealed in four separate reactions in equimolar ratios at 95 °C for 5 min and then allowed to cool to room temperature. The final constructs (MSc and Bs) were digested with Bam HI to generate a “monomer” (150 bp). Cloning was conducted in

pUC19 and the subsequent multimer (MScBs, 4-mer) was subcloned into pET30a after digestion with Bam HI and used to transform BL21(DE3) pLysS. Gene sequence and size were confirmed by sequencing and gel electrophoresis. Cloning, Expression Vectors, and Host Strain. pUC19 (Pharmacia Biotech) plasmid with ampicillin resistance and pCR-Script (Stratagene) plasmid with chloramphenicol resistance were used as cloning vectors. pET30a expression vector with kanamycin resistance and competent E. coli strains BL21(DE3)pLysS were purchased from Novagen and used as host cells. Plasmid DNA was isolated from overnight cultures in LB medium containing the appropriate antibiotics. The Promega Wizard Miniprep purification kit was routinely used for plasmid preparations. Purification and concentration of DNA were performed by phenol extraction and ethanol precipitation. Transformations were performed by standard calcium chloride protocols as described by Sambrook et al.25 Media and Growth Conditions. LB medium consisting of 10 g/L of Bacto-tryptone, 5 g/L of Bacto-yeast extract, and 10 g/L of NaCl was used for routine growth experiments at 37 °C. Ampicillin and chloramphenicol were added to a final concentration of 35-50 and 10 µg/mL, respectively. Kanamycin was added to a final concentration of 50 µg/ mL. Protein Expression and Purification. Cultures were grown at 37 °C in 2 × TY medium containing 25 µg/mL kanamycin and chloramphenicol to an OD600 ) 0.5-1.0. Expression of the silklike protein was induced by the addition of 1 mM IPTG. After 4 h, 1 mL of cell culture was harvested, and the cells were lysed in 0.1 mL of loading buffer. Lysates were resolved by SDS-PAGE using 12.5% polyacrylamide. For larger-scale expression, cultures were grown in 1.2 L of TB medium to OD600 ) 0.5-0.7, and then IPTG was added to 1 mM. After 4 h, the cells were harvested by centrifugation. Recombinant proteins were purified by affinity chromatography on a Ni-NTA column in accordance with manufacturer’s protocols. The recombinant protein containing the hexa-His fusion was monitored by Western blot using a hexa-His antibody and horseradish peroxidase (HRP)conjugated rabbit IgG. Purified protein was exhaustively dialyzed against deionized water and then lyophilized for subsequent biophysical characterization.

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Chimeric Silkworm Silk

Preparation of Model Peptides. Three model peptides for the polyalanine region of S. c. ricini silk fibroin,14 GGAGGGYGGDGG(A)12GGAGDGYGAG, GDGG(A)12GGAG, GDGG(A)18GGAG, and the model peptide for the glycine-rich region of B. mori silk fibroin,10 (AGVGAGYG)3(AG)3, were synthesized by solid-phase synthesis. The synthesis was performed in a stepwise fashion on FmocGly-PEG-PS resin (PE Biosystems) by a Pioneer peptide synthesizer using Fmoc chemistry. Fmoc amino acids (PE Biosystems) were used with the following side-chain protection: Tyr(tBu), Asp(tBu). The Fmoc group was deblocked with 20% piperidine in N,N-dimethylformamide (DMF) for 5 min (flow rate ) 8.8 mL/min). During the deprotection of each Fmoc group, the amount of Fmoc released was monitored by UV spectroscopy. The coupling reaction was carried out using each Fmoc amino acid (0.8 mmol), N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-Nmethylmethanaminium hexafluorophosphate N-oxide (HATU) (0.8 mmol), and diisopropylethylamine (DIPEA) (0.95 mmol) in DMF for 30 min (flow rate ) 30 mL/min). Each amino acid (0.8 mmol) and HATU (0.8 mmol) were dissolved in 3.8 mL of the base solution (0.25 M DIEPA in DMF). The mixture was purged with a N2 stream and added within 2 min to a column containing the resin with a flow rate of 30 mL/min. After each condensation step, the unreacted amino terminus was blocked with 5% acetic anhydride-pyridine in DMF for 5 min. DMF was used for the flow washes throughout the synthesis. After synthesis, the peptides were cleaved from the resin by treatment with a 40 mL mixture of TFA, phenol, triisopropylsilane, and water (88:5:2:5 vol %) for 2 h at room temperature, and then the crude peptides were precipitate and washed repeatedly with cold diethyl ether. The samples were dissolved in 9 M LiBr and then dialyzed against water for 4 days. The precipitated samples were collected and dried. Preparation of S. c. ricini Silk Fiber and Film. S. c. ricini silkworms were reared in our laboratory. The cocoons of S. c. ricini were degummed three times with 0.1% sodium peroxide (Na2O2) solution at 100 °C for 30 min and washed with distilled water to remove sericin proteins from the surface of the fibers. The fibroin fibers were dried at 37 °C overnight prior to the next experiment. The middle silk gland of the silkworm was pulled out from anesthetized 7-day-old fifth instar larva. The silk glands containing the fibroins were then washed twice in ice-cold 1.15% potassium chloride solution. The center of the silk gland 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 to dry for 2 days to prepare silk fibroin films. 13C CP/MAS NMR Measurement. The 13C CP/MAS NMR experiments were performed at 25 °C with CMX Infinity 400 NMR spectrometer (Chemagnetics) the operating frequency of which was 100.04 MHz for 13C nucleus. Samples were contained in a cylindrical rotor and spun at 10 kHz. The number of acquisitions was 8K, and the pulse delays were 5 s. A 50 kHz radio frequency field strength was used for decoupling with a decoupling period of 12.8 ms. A 90° pulse width of 5 µs with 1 ms CP contact time

Figure 2. Production of MScBs-4-mer in E. coli: (1) 1.5 h after induction; (2) 2 h; (3) 2.5 h; (4) 3 h; (5) 3.5 h; (6) 4 h; (P) positive control (silklike protein, SLP4, (GGAGSGYGGGYGHGYGSDGG(GAGAGS)3)4, MW ) 29 kDa). Samples at 0.5 and 1 h after induction did not show detectable expression.

was employed. Phase cycling was used to minimize artifacts. chemical shifts were calibrated indirectly through the adamantane methyl peak observed at 28.8 ppm relative to tetramethylsilane at 0 ppm.

13C

Results and Discussion Construction and Expression of Silklike Protein. The crude extract of the successful construction and cloning of MScBs-4-mer was analyzed by western-blotting analysis using a [His]6 antibody as shown in Figure 2. Another silklike protein, (GGAGSGYGGGYGHGYGSDGG(GAGAGS)3)4 expressed in our lab was used as the marker with a MW 29 kDa.26 After sonication of the cell pellet followed by centrifugation, western-blotting analysis was performed to assess the solubility of the supernatant and precipitated silk samples (Figure 3). MScBs-4-mer was present in the precipitate. The recombinant protein was subsequently dissolved in 8 M urea buffer and purified by Ni-NTA affinity chromatography under denaturing conditions, dialyzed against deionized water, and then lyophilized. The yield of MScBs4mer was 34.2 mg/L of culture. 13C CP/MAS NMR Spectra of Silklike Protein and S. c. ricini Silk Fibroin. Figure 4 shows the solid-state 13C CP/MAS NMR spectra of (a) a S. c. ricini silk fibroin film with an R-helix structure, (b) a S. c. ricini silk fiber with an antiparallel β-sheet structure,27,28 and (c) the lyophilized MScBs-4-mer. When the recombinant protein sample was dissolved in 8 M urea and then precipitated in acetonitrile, the spectrum in Figure 4d was obtained. The peak assignments of the S. c. ricini silk fibroin in both R-helix and β-sheet forms have been reported.27,28 Sharp peaks at 15.5 ppm for Cβ and 52.5 ppm for CR of alanine residues in Figure 4a suggest that the (Ala)12-13 region in S. c. ricini

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Figure 3. Western-blotting analysis of MScBs-4-mer after sonication in lysis buffer (10 mM imidazole, 50 mM NaH2PO4, pH 8.0, 300 mM NaCl): (1) supernatant; (2) precipitate sample. SLP4 was used as a control (see legend for Figure 2).

Figure 5. 13C CP/MAS NMR spectra of (a) the silklike protein, MScBs-4-mer, in R-helix form, the model peptides (b) GGAGGGYGGDGG(A)12GGAGDGYGAG, (c) GDGG(A)12GGAG, and (d) GDGG(A)18GGAG of the crystalline domain of S. c. ricini silk fibroin, and (e) the model peptide (AGVGAGYG)3(AG)3 of B. mori silk fibroin.

Figure 4. 13C CP/MAS NMR spectra of S. c. ricini silk fibroin in (a) film and (b) fiber forms. The spectra of silklike protein, MScBs-4mer, is also shown (c) after dialysis of the 9 M LiBr solution and (d) after acetonitrile exposure.

silk fibroin film assumes an R-helical structure. The broad and asymmetric peak at 20 ppm for Cβ and 48.8 ppm for CR of the alanine residues (Figure 4b for the silk fiber) indicate an antiparallel β-sheet structure. From the comparison of these spectra with the silklike recombinant chimeric protein, both (c) R-helix and (d) antiparallel β-sheet structures are observed. However, there are also valine and alanine residues in the glycine-rich sequence of B. mori silk fibroin, and therefore, the valine Cγ and alanine Cβ peaks from this

sequence overlap with the alanine Cβ peaks of the (Ala)18 regions. 13C CP/MAS NMR Spectra of Model Peptides of Crystalline Region of S. c. ricini Silk Fibroin. The model peptides GGAGGGYGGDGG(A)12GGAGDGYGAG, GDGG(A)12GGAG, and GDGG(A)18GGAG representing the crystalline domain of S.c. ricini silk fibroin and the model peptide (AGVGAGYG)3(AG)3 selected from B. mori silk fibroin were synthesized to compare with MScBs-4-mer to assist in structural interpretations. 13C CP/MAS NMR chemical shifts and 13C 2D spin-diffusion NMR have been used to determine the detailed structure of the peptide GGAGGGYGGDGG(A)12GGAGDGYGAG and the shorter sequence G(A)12G considered to be essential components of the crystalline domain, which can form R-helices in S. c. ricini silk fibroin.29 Therefore, the shorter peptide GDGG(A)12GGAG was also used to help in the study. The peptide GDGG(A)18GGAG was also synthesized to account for the sequence of the MScBs-4-mer. (AGVGAGYG)3(AG)3 consists of the sequence involving tyrosine or valine residues instead of alanine in the repeat AG sequences.10 The four peptides were soluble in 9 M LiBr. After dialysis against water, the peptides were freeze-dried and 13C CP/MAS NMR spectra were observed (Figure 5b-e), as well as for the MScBs-4-mer (Figure 5a). The alanine CR, CO, and Cβ peaks in the spectra b, c, and d show typical β-sheet patterns. The asymmetric line shapes of the alanine Cβ carbon indicate a heterogeneous structure for the polyalanine region in the model peptides.

Chimeric Silkworm Silk

Figure 6. Peak decomposition of 13C CP/MAS NMR spectra of the silklike protein MScBs-4-mer (a) after dialysis of the 9 M LiBr solution and (b) after acetonitrile exposure. There are 18 alanine residues in the polyalanine region and eight isolated alanine residues and three isolated valine residues in the sequence MScBs, ASAAAAAAAAAAAAAAAAAATSGVGAGYGAGYGVGAGYGAGVGYGAGAGYTS. The fraction (5) determined by the peak decomposition is included.

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process is relatively large because of severe peak overlap. However, the content is close to the expected values, 19% and 25%, respectively. The content of β-sheet is 31%, which is the same content as an R-helix. Thus, the structure of half of the polyalanine chain in the MScMBs-4mer transitioned from an R-helix to β-sheet because of exposure to the solvent. Although polyalanine regions easily form β-sheets, as shown in Figure 5, the presence of the noncrystalline region in the chimeric protein prevented the conformational transition from R-helix to β-sheet. These types of transitions and the relationship with primary sequence are important in considering sequence-function relationships among the various silk proteins in silkworms and spiders. Studies with native silk proteins and genetically engineered variants have provided a solid base of insight into issues of sequence and structure; however, many subtle interactions remain to be understood to fully exploit this novel family of proteins for materials science and engineering applications. The chimeric approach described here suggests a useful path forward to add to this understanding. Acknowledgment. We thank the NSF-DMR (D.L.K.) for grant support. References and Notes

On the other hand, the alanine Cβ chemical shift and the broad line shape in the 13C CP/MAS NMR spectrum of (AGVGAGYG)3(AG)3 indicate that the structure is a random coil. Thus, the chemical shift of the alanine Cβ carbon of MScBs-4-mer is clearly different from these peaks and supports an R-helical structure for this protein. The alternative sequence of the modified S. c. ricini silk fibroin crystalline region and the region of B. mori silk fibroin prevented the conformational change of the polyalanine region from R-helix to β-sheet. Detailed Analysis of Alanine Cβ Peak in the 13C CP/ MAS NMR Spectra of the Silklike Protein. Although the alanine Cβ peak can be used as a indication of silk structure, the valine Cγ and alanine Cβ peaks from B. mori silk fibroin region overlap with the alanine Cβ peaks of (Ala)18 chain as mentioned above. Therefore, peak decomposition was performed and is shown in Figure 6. There are 18 alanine residues in the polyalanine region and eight isolated alanine residues and three isolated valine residues in the sequence, MScBs; ASAAAAAAAAAAAAAAAAAATSGVGAGYGAGAGYGVGA GYGAGVGYGAGAGYTS. Therefore, in the region 10-25 ppm, 56% alanine Cβ carbons of the polyalanine region, 25% alanine Cβ carbons of isolated alanine residues, and 19% valine Cγ carbons of the isolated valine residues are expected. In the spectrum a, the peak at 21 ppm (21%) is assigned to the valine residue. Usually, the 13C chemical shift of valine Cγ peak is conformationindependent. The alanine Cβ peak at 18 ppm (24%) is assigned to the isolated alanine residues in a random coil. The main peak at 15 ppm (55%) is assigned to an R-helix. Thus, only the polyalanine region forms an R-helix in the MScBs-4mer after 9 M LiBr/dialysis treatment. On the other hand, after treatment with acetonitrile, the content of valine Cγ and isolated alanine Cβ carbons are 18% and 20%, respectively, although the error in the peak decomposition

(1) 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, DC, 1994; pp 2-16. (2) Asakura, T.; Kaplan, D. L. Silk production and processing. In Encyclopedia of Agricultural Science; Arutzen, C. J., Ed.; Academic Press: New York, 1994; Vol. 4, pp 1-11. (3) Kaplan, D. L.; Mello, C. M.; Arcidiacono, S.; Fossey, S.; Senecal, K.; Muller, W. Silk. In Protein based materials; McGrath, K., Kaplan D. L., Eds.; Birkhauser: Boston, 1998; pp 103-131. (4) Demura, M.; Asakura T. Immobilization of glucose oxidase with Bombyx mori silk fibroin by only stretching treatment and its application to glucose sensor. Biotechnol. Bioeng. 1989, 33, 598603. (5) Altman, G.; Horan, R.; Lu, H.; Moreau, J.; Martin, I.; Richmond, J.; Kaplan, D. L. Silk matrix for tissue engineered anterior cruciate ligaments. Biomaterials 2002, 23, 4131-4141. (6) 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. (7) 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 and references therein. (8) 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. (9) Zhao, C.; Asakura, T. Structure of silk studied with NMR. Prog. Nucl. Magn. Reson. Spectrosc. 2001, 39, 301-352. (10) Asakura, T.; Sugino, R.; Yao, J.; Takashima, H.; Kishore, R. Comparative structure analysis of tyrosine and valine residues in unprocessed silk fibroin (silk I) and in the processed silk fiber (silk II) from Bombyx mori using solid-state 13C, 15N, and 2H NMR. Biochemistry 2002, 41, 4415-4424. (11) 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. (12) 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.

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(13) Ashida, J.; Ohgo, K.; Asakura, T. Determination of the torsion angles of alanine and glycine residues of Bombyx mori silk fibroin and the model peptides in the silk II forms using 2D spin diffusion solidsate NMR under off magic angle spinning. J. Phys. Chem. B. 2002, 106, 6434-9439. (14) Nakazawa, Y.; Asakura, T. Heterogeneous exchange behavior of Samia cynthia ricini silk fibroin during helix-coil transition studied with 13C NMR. FEBS Lett. 2002, 529, 188-192. (15) 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. (16) Yamane, T.; Umemura, K.; Asakura, T. The structural characteristics of Bombyx mori silk fibroin before spinning as studied with molecular dynamics simulation. Macromolecules 2002, 35, 8831-8838. (17) Iizuka, E.; Teramoto, A.; Lu, Q.; Min, S.-J.; Shimizu, O. Comparative studies on the physical properties of extrathick and extrafine silk thread produced by different varieties of Bombyx mori. J. Seric. Sci. Jpn. 1996, 65, 134-136. (18) Iizuka, E.; Sekiguchi, S.; Okachi, Y.; Ohbayashi, S. Studies on the physical properties of Antheraea yamamai silks. Int. J. Wild Silkmoth Silk 1994, 1, 143-146. (19) 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, DC, 2003; pp 71-81. (20) Yukuhiro, K. Personal communication. (21) Nakazawa, Y.; Nakai, T.; Kameda, T.; Asakura, T. A 13C NMR study on the structural change of silk fibroin from Samia cynthia ricini. Chem. Phys. Lett. 1999, 311, 362-366.

Asakura et al. (22) Prince, J. T.; McGrath, K. P.; DiGirolamo, C. M.; Kaplan, D. L. Construction, cloning, and expression of synthetic genes encoding spider dragline silk. Biochemistry 1995, 34, 10879-10885. (23) Winkler, S.; Wilson, D.; Kaplan, D. L. Controlling β-Sheet Assembly in Genetically Engineered Silk by Enzymatic Phosphorylation/ Dephosphorylation. Biochemistry 2000, 39, 12739-12746. (24) Yao, J.; Asakura, T. Synthesis and structural characterization of silklike materials incorporated with an elastic motif. J. Biochem. 2003, 133, 147-154. (25) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning. A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989. (26) Asakura, T.; Kato, H.; Yao, J.; Kishore, R.; Shirai, M. Design, expression and structural characterization of hybrid proteins of Samia cynthia richini and Bombyx mori silk fiborins. Polym. J. 2002, 34, 936-943. (27) Ishida, M.; Asakura, T.; Yokoi, M.; Saito, H. Solvent- and mechanical-treatment-induced conformational transition of silk fibroins studied by high-resolution solid-state 13C NMR. Macromolecules 1990, 23, 88-94. (28) Nakazawa, Y.; Asakura, T. High-resolution 13C CP/MAS NMR study on structure and structural transition of Antheraea pernyi silk fibroin containing Poly(L-alanine) and Gly-rich regions. Macromolecules 2002, 35, 2393-2400. (29) Nakazawa, Y.; Bamba, M.; Nishio, S.; Asakura, T. Tightly winding structure of sequential model peptide for repeated helical region in Samia cynthia ricini silk fibroin studied with solid-state NMR. Protein Sci. 2003, 12, 666-671.

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