Ala-Bombyx mori Silk Fibroin Stud

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Effect of Water on The Structure and Dynamics of Regenerated [3- C] Ser, [3- C] Tyr and [3- C] Ala-Bombyx mori Silk Fibroin Studied with C Solid-State NMR. 13

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Akio Nishimura, Hironori Matsuda, Yugo Tasei, and Tetsuo Asakura Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01665 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 10, 2018

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Biomacromolecules

Effect of Water on The Structure and Dynamics of Regenerated [3-13C] Ser, [3-13C] Tyr and [3-13C] Ala-Bombyx mori Silk Fibroin Studied with 13C Solid-State NMR. Akio Nishimura, Hironori Matsuda, Yugo Tasei, and Tetsuo Asakura* Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588 Japan Keywords: Bombyx mori Silk Fibroin, 13C Labeling, Silk Sponge, Regenerated Silk Fiber, Hydration, 13C sold-state NMR S : Supporting Information ABSTRACT:

The effects of water on the structure and dynamics of natural and regenerated silk fibroin (SF) samples were studied using 13C solid-state NMR spectroscopy. We prepared different types of SF materials, sponges and fibers, with different preparation methods and compared their NMR spectra in the dry and hydrated states. Three kinds of 13C NMR techniques, r-INEPT, CP/MAS, and DD/MAS, coupled with 13C isotope labeling of Ser, Tyr, and Ala residues were used. In the hydrated sponges, several conformations, that is, Silk I* and two kinds of β-sheets, A and B, random coil and highly mobile hydrated random coil were observed and the fractions were determined. The fractions were different remarkably among three sponges, but only small differences among the regenerated and native fibers. The increase in the fraction of β-sheet B might be one of the structural factors to prepare stronger regenerated SF fiber.

INTRODUCTION Silk fibroin (SF) from Bombyx mori silkworm has been valuable materials in fields ranging from textile industry to biotechnologies.1 This is justified by many inherently superior qualities such as their mechanical properties, environmental stability, biocompatibility, low immunogenicity and biodegradability as well as ease of fabrication into different forms such as fiber, film, gel, powder, sponge and so on.2–6 These superior qualities of SF are especially useful in the application to the medical field. The biomaterials are generally used in a hydrated state, and it is well known that the structure and dynamics of SF changes remarkably by the effect of water.2,6 Thus, it is the key points to understand the structure and dynamics of SF in the hydrated state for the application. Many analytical methods including Raman,7 near-IR,8,9 IR,10,11 DSC,10–13 TGA,12,13 XRD,13 DMTA,14 ESR,15 NMR15–24 and so on have been used for the purpose. They have provided many insights into the molecular structure and dynamics of SF in the hydrated state. In general, water in the silk–water system can be divided into three categories: free water, freezing bound water, and non-freezing bound water.7–12,16–18 Recently, we could identify further distinct components of the freezing bound water in the 2 H solution NMR relaxation measurements for water in SF fiber.22,23 However, a complete picture of the structure and dynamics of SF is still not well understood at molecular level. Because of the heterogeneous dynamic character (distribution from mobile to immobile domains) of SF in the hydrated state, the 13C solid-state NMR observations which emphasized the mobile and immobile components in SF are very effective for the characterization of the hydrated SF. Recently, we used the combinations of 13 C refocused insensitive nuclei enhanced by polarization transfer (13C r-INEPT), 13C cross polarization/ magic angle spinning (13C CP/MAS) NMR and 13C dipolar decoupled-magic angle spinning (13C DD/MAS) NMR for the purpose.23,24 The 13C r-INEPT in which the pulse sequence was developed for solution NMR is sensitive to the component of the hydrated proteins with fast motion.25 In contrast, 13C CP/MAS NMR can observe only the components of the hydrated proteins with very slow motion. Thus, if the presence of water causes an increase in the chain mobility, a loss in cross polarization (CP) signal of the amino acid residues occurs and consequently such a domain cannot be observed in the 13C CP/MAS NMR spectra.26–30 In the dry state, only 13 C CP/MAS NMR was used for the NMR observation of SF. On the other hand, 13C DD /MAS NMR spectra can be used to detect the mobile domains as well as the immobile domains, which make possible to determine the fraction of several structures with different dynamical character in the hydrated state of SF. Thus, the combinations of these three kinds of 13C NMR techniques, 13C r-INEPT, 13C CP/MAS and 13C DD/MAS NMR, provide different perspectives on the dynamical behavior of SF

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in the dry and hydrated states, and can be used together to characterize their local structures in view of dynamics. Moreover, conformation-dependent 13C chemical shift31–35 coupled with 13C selective labeling of SF is extremely useful to determine the fraction of several conformations in an amino-acid-specific manner, which has been used frequently in the structural analyses of SF including spider silks.36–38 Especially, [3-13C] Ser, [3-13C] Tyr and [3-13C] Ala labeled SF samples have been prepared by biosynthesis for the 13C NMR analysis of SF.21,22,24 From the reported primary structure of SF, Ser residues are present predominantly in the crystalline domain such as repeated Gly-Ala-Gly-Ser-Gly-Ala sequences, Tyr predominantly in the noncrystalline domain such as Gly-Ala-Gly-Tyr-Gly-Ala sequences and Ala residues in both domains.39,40 Thus, we could obtain information of structure and dynamics of SF fiber from the crystalline and non-crystalline domains independently. In this paper, the effects of water on the structure and dynamics of SF samples were studied using three kinds of 13C solid-state NMR (13C r-INEPT, 13C CP/MAS and 13C DD/MAS) techniques. At first, the effect was compared the natural abundant SF fiber immersed in water directly11,15,21–24 with the SF fiber placed under high humidity atmosphere. 8–10,12–14,17–19 Secondary, the comparison was performed among three kinds of [3-13C] Ser, [3-13C] Tyr and [3-13C] Ala labeled SF sponges. Namely, the watersoluble sponges prepared by freeze-drying treatment and two kinds of water-insoluble sponges prepared by standing them under high humidity atmosphere or by immersing them in methanol.15–17 Thirdly, two kinds of regenerated 13C-labeled SF fibers were prepared from the SF sponges by wet spinning method as “as spun” fiber or the stretched fiber by 3 times relative to the original fiber length. The comparison was extended to three fibers by including natural SF fiber. Fourthly, a chemically modified fiber was prepared by blocking the side chain OH sites of Tyr residues in the regenerated SF fiber with 3 times stretching ratio with hydrophobic group partly.4,41–44 The stress-strain curves of these four kinds of SF fibers were measured and compared. The 13C solid-state NMR spectra of all SF samples were observed in the dry and hydrated states to clarify and compare the effects of water on their structures and dynamics.

EXPERIMENTAL SECTION Preparation of 13C labeled B. mori silk fibroin B. mori silkworms were reared in our laboratory. The 13C labeling of SF was achieved biosynthetically by oral administration of an artificial diet with 13C-enriched amino acids to larvae of the fifth instar, as reported previously.21,22,24 Briefly, the supplementary [3-13C] Tyr and [3-13C] Ser was mixed with 2.0 g of an artificial diet per day. The amount of labelled Tyr and Ser was 10 mg each on the fourth and fifth day of the fifth larval stage. The 13C labeling of Ala Cβ carbon was performed by transamination from [3-13C] Ser in the silkworm.45,46 To prevent amino acid transfer of Ser into Gly, 20 mg non-labeled Gly was also mixed with the artificial diet per day.21,47 Thus, the total amount of [3-13C] Tyr and [3-13C] Ser was 20 mg per silkworm. The 13 C-labeled amino acids, [3-13C] Tyr, and [3-13C] Ser (each 99% enrichment), used for labeling of SF, were purchased from Cambridge Isotope Laboratories, Inc., Andover, MA USA.

Preparation of SF sponges, S1-S3 Cocoons of B. mori were degummed with 0.5 % (w/w) Marseilles soap (Aikuma Senryo, Japan) solution at 100 ºC for 30 min and washed with distilled water in order to remove silk sericin.15,16,31 The degummed SF fibers was dissolved in 9M LiBr solution to a concentration of 10% w/v at 40 ºC for 1 hr and then dialyzed against deionized water for 4 days at 4 ºC. The lyophilized sponge was prepared from the SF aqueous solution by freeze-dry methods which was soluble in water. We named the sponge as S1 here. The insolubilization of the sponges were made by two methods. One is performed while preventing the SF sponge to come into direct contact with water, and allowed to stand in a desiccator filled with water in the bottom ( under high humidity atmosphere).48 We named the sponge as S2 here. Another is that the sponge was immersed in methanol for 1 day and then dried.15,48,49 We named the sponge as S3 here. Thus, three kinds of SF sponges, S1, S2 and S3 were prepared and used for the solid-state NMR observation.

Preparation of regenerated SF fibers, F1-F3 The sponge, S1 was dissolved in hexafluoroisopropanol (HFIP) overnight at 40 ºC, yielding a 13.5 % (w/w) solution of SF. The HFIP solutions were extruded with the rate, 25 mm/s through a stainless steel spinneret with 0.8 mm inner diameter using a manual spinning method into the methanol coagulation bath at room temperature through the air gap of 5 mm.50–52 This SF as spun fiber after coagulation was soaked in the methanol bath overnight to remove HFIP from the fiber and then dried overnight at room temperature. We named this regenerated “as spun” fiber as F1 here. The fiber was stretched to 3 times of the original length using a manual stretching method in the air, which was named as F2 here. The F2 fiber was dried overnight at room temperature under the fixed lengths. On the other hand, the stretched fiber, F2 was immersed in 1 M Na2CO3 alkaline aqueous solution for 30 min at 4°C.44 Then, the fiber was picked up and immersed in 0.3 M aqueous solution of 4-(4, 6-dimethoxy-1, 3, 5-triazin-2-yl)-4methylmorpholinium chloride, (DMT-MM) at room temperature again. After 30 min, the fiber treated with DMT-MM aqueous solution was washed with distilled water and then dried overnight at room temperature, which was named as F3 here. The reagents, DMT-MM and HFIP were purchased from Wako Pure Chemical Industries Ltd., Japan. The degummed native SF fiber was named as NF here. 13

C r-INEPT, 13C CP/MAS and 13C DD/MAS NMR measurements


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Biomacromolecules The 13C solid-state NMR spectra of the SF sponges, and regenerated and 13C labeled natural SF fibers were observed using a Bruker Avance 400 NMR spectrometer with a 4-mm double resonance MAS probe and a MAS frequency of 8.5 kHz at ambient temperature. The SF sponge or fiber samples after immersing in water overnight were inserted into a zirconia rotor that was sealed with PTFE insert to prevent dehydration of the hydrated samples.21–24 The natural SF fibers after incubating at 25°C for 2 days under relative humidity of 84% and 97% were obtained with the saturated water vapor of salts, potassium chloride and potassium sulfate, respectively.13 Typical experimental parameters for the 13C CP/MAS NMR experiments included a 3.5-µs 1H 90° pulse, a 1-ms ramped CP pulse with 71.4 kHz rf field strength, TPPM 1H decoupling during acquisition, 2,176 data points, 8 k scans, a 6.2 µs pulse dipolar dephasing time, 310 ppm sweep width, and a 4-s recycle delay. Details of the NMR conditions for the DD/MAS NMR experiments were described in a previous paper.21 Typical experimental parameters for the refocused INEPT NMR experiments included a 3.5-µs 1H and a 3.6-µs 13C 90°pulse, an inter-pulse delay of 1/4 1JCH (1JCH =145 Hz), a refocusing delay of 1/6 1JCH or 1/3 1JCH. TPPM 1H decoupling during acquisition, 1,438 data points, 4 k scans, 200 ppm sweep width, and a 3.5-s recycle delay. The exponential line broadening, 20 Hz was used for these spectra. The 13C chemical shifts were calibrated externally through the methylene peak of adamantane observed at 28.8 ppm relative to TMS at 0 ppm.21–24,31,32

Mechanical property measurements The stress-strain curves of SF fibers, F1-F3 and NF were measured as follows. The both edges of the fibers were mounted on the Scotch tape with a base length of 20 mm and fixed with ethyl cyanoacrylate, respectively.51,52 Before starting the tensile test, the diameter of the fiber was measured with an optical microscope (KEYENCE BIOREVO BZ-9000 Japan). The stress-strain curves of these five kinds of fibers were measured using an EZ-Graph tensile testing machine (EZ-Graph, SHIMADZU Co. Ltd. Japan) at room temperature with a 5 N load cell. The rate of crosshead was 3 mm/min on samples of 20 mm length. Each value was the average of 6 measurements. The breaking strength (MPa) measured as the highest stress value attained during the test was calculated by dividing the cross-sectional area of the fiber. The elongation at break (%) was measured as the change in length divided by the initial length.

RESULTS 1. 13C solid-state NMR spectra of natural SF fibers immersed in water and incubated under high relative humidity In this experiment, we used natural abundant NF without 13C labeling. At first, the effects of water on the structure and dynamics of NF were compared when the fibers were set under high humidity atmosphere or immersed in water directly. The 13C solidstate NMR spectra, that is, 13C CP/MAS NMR spectrum in the dry state, 13C CP/MAS ,13C DD/MAS and 13C r-INEPT NMR spectra in the hydrated state were observed. Figure 1A shows the 13C CP/MAS NMR spectrum of NF in the dry state. As reported previously,31,37,38 the spectrum was assigned to Ala Cβ, Gly Cα, Ala Cα, Ser Cα, Ser Cβ and C=O peaks to lower field and the chemical shifts indicate to form mainly anti-parallel β-sheet structure. The 13C CP/MAS NMR spectra did not change significantly before and after hydration independent of the way of hydration as shown in the expanded spectra (10-75 ppm) of Figures 1B-a and 1C-a although only Ala Cβ peak became slightly sharper in the spectrum, Figure 1C-a. On the other hand, the 13 C DD/MAS NMR spectra of NF were changed from the 13C CP/MAS NMR spectra in the hydrated state depending on the way of the hydration. Namely, Figure 1B-b shows that the relative intensities of Ala Cβ and Ser Cβ peaks increase considerably compared with those of Ala Cα, Gly Cα and Ser Cα peaks. On the other hand, Figure 1C-b shows appearance of new sharp peaks at the higher field sides of main Ala Cβ and Ser Cβ peaks which were assigned to hydrated random coil peaks with high mobility.21–24,53The Ala Cα and Ser Cα backbone carbons also show asymmetric peaks, indicating the appearance of sharp peaks with random coil chemical shifts. The differences in the spectral patterns were observed more clearly in Figures 1B-c and 1C-c: Namely, there are no peaks in the 13C r-INEPT spectrum of NF setting under 84% relative humidity, which is the same even for the higher humidity atmosphere, 97% relative humidity (Supporting information Figure S2). On the other hand, sharp peaks with random coil chemical shifts of both backbone and side chain carbons were observed in the hydrated 13C r-INEPT spectra of NF. Thus, it is clear that the effects of water on the structure and dynamics of NF are quite different between two methods of the hydration. The water vapor is adsorbed mainly on the surface of the SF fibers. Therefore, the mobilities of the side chains which are considered to be faced to the outside of the fibers increase, but the backbone chains located inside of the fibers are not affected by the water vapor. On the other hand, when the fibers were immersed in water directly, water molecules can partly penetrate into random coil region of the fibers and the mobilities of the carbons in both side and main chains increase partly. In this paper, we concentrate only on NMR hydration experiments when SF samples were immersed in water because the combination of 13C solid-state NMR spectra of SF in the dry(CP/MAS) and hydrated states (CP/MAS, DD/MAS and r-INEPT) changes significantly, which provides unique and useful information on the change in the structure and dynamics of SF by hydration.



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Figure 1. A.13C CP/MAS NMR spectrum of natural SF fiber in the dry state together with the assignment. B. and C. are the expanded a. 13 C CP/MAS NMR b.13C DD/MAS, and c. 13C r-INEPT spectra of the hydrated natural SF fibers under high humidity atmosphere (84% relative humidity) and immersed in water, respectively. SSB stands for spinning side band.

2.13C solid-state NMR spectra of three kinds of SF sponges in the dry and hydrated states


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Biomacromolecules We prepared three kinds of [3-13C] Ser-, [3-13C] Tyr- and [3-13C] Ala-labeled sponges, S1, S2 and S3 as described in EXPERIMENTAL SECTION. The expanded four 13C solid-state NMR spectra for each sponge were shown in Figures 2A, 2B and 2C. The Ser Cβ and Tyr Cβ peaks increased significantly by 13C labeling. Especially the Tyr Cβ peak was newly observed in these spectra compared with the 13C non-labeled SF spectra in Figure 1. Further the intensity of the Ala Cβ peak became stronger due to the transamination from 13C labeled Ser Cβ carbon in silkworm.45,46 The 13C chemical shifts of three Cβ peaks of Ser, Tyr and Ala residues of S1 in Figure 2A-a indicate that these residues take exclusively random coil.53 On the other hand, only very small peaks were observed in the 13C CP/MAS NMR spectrum in the hydrated state (Figure 2A-b), which means that S1 sample is almost dissolved in water and significant loss of CP signals in the spectrum. 26–30 Actually, only very sharp peaks were observed in both 13C DD/MAS NMR and 13C r-INEPT NMR spectra as shown in Figures 2A-c and 2A-d, respectively. Figure 2B shows expanded four 13C solid-state NMR spectra of S2. The Ser Cβ peak splits into three peaks in the 13C CP/MAS NMR spectrum in the hydrated state as shown in Figure 2B-b. We will start with the assignment of this Ser Cβ peak. The lowest field broad peak is assigned to β-sheet structure, the central field sharp peak to hydrated random coil and the highest field sharp peak to Silk I* structure as judged from the chemical shift value as reported previously.21,24,38,54–57 The structural model of Silk I* is shown in Supporting Information (Figure 2S) and will be explained more in DISCUSSION. The Ala Cβ peak in Figure 2B-b, splits into two peaks which are assigned to β-sheet (lower field) and mixture of random coil and Silk I* structures (higher field) where the 13C chemical shifts of Ala Cβ peaks are in agreement between random coil and Silk I* structures.24,31,54,55 As will be explained in DISCUSSION, the β-sheet Ala Cβ and Ser Cβ peaks are further separated into two β-sheet peaks due to difference in the intermolecular arrangements.21–24,58–64 The Tyr Cβ peak consists of only β-sheet and random coil because Tyr residues in SF do not contribute to the Silk I* formation because Silk I* structure consists of the motif, (Ala-Gly-Ser-Gly-Ala-Gly)n.38,65,66 The Ser Cβ peak in Figure 2B-a should involve Silk I* peak by judging from Figure 2B-b, but two peaks were overlapped because of the peak broadening. The Ala Cβ and Ala Cα peaks in Figure 2B-a gave a similar pattern as those in Figure 2B-b although the peaks were a little broader. The significantly larger Tyr Cβ peak with random coil chemical shift was observed in Figure 2B-a compared with Figure 2B-b, which is due to a loss of the CP signal of Tyr Cβ peak by hydration rather than the structural change from random coil to β-sheet. By the peak deconvolution of the 13C DD/MAS NMR spectrum (Figure 2B-c) in the hydrated state, it is possible to determine the fraction of several conformations quantitatively as will be discussed later.21–24 It is noted that the intensity of Tyr Cβ peak in the 13C r-INEPT spectrum shown in Figure 2B-d is relatively small as compared with the relative intensity in Figure 2A-d, indicating that the fraction of mobile Tyr Cβ carbon decreases. Namely, the fraction of Tyr residues located in the hydrophobic domains increases in S2. Figure 2C shows four 13C solid-state NMR spectra of S3. As reported previously,15,48,49 the fraction of β-sheet structure increases due to structural change from random coil to β-sheet by methanol, which was clearly observed in 13C CP/MAS NMR spectrum in the dry state shown in Figure 2C-a. The increase of βsheet structure was also observed in the Tyr Cβ peak other than the Cα and Cβ peaks of Ala and Ser residues. By hydration, the relative intensities of random coil peaks decreased clearly for the Cβ peaks of Ala, Ser and Tyr residues in Figure 2C-b. This is due to a loss of CP signals as described above. Actually, the fraction of random coil increased again for these peaks in the 13C DD/MAS NMR spectrum in the hydrated state (Figure 2C-c) and only hydrated random coil peaks with high mobilities were observed in the 13C r-INEPT spectrum (Figure 2C-d). It is also noted that the intensity of Tyr Cβ peak is relatively small compared with the relative intensity in Figure 2A-d, which is a similar tendency as S2.



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Figure 2. A., B. and C. are the expanded a. 13C CP/MAS NMR spectra of 13C labeled SF sponges, S1, S2 and S3 in the dry state, and b. 13C CP/MAS, c. 13C DD/MAS, and d. 13C r-INEPT NMR spectra of the sponges in the hydrated states, respectively. The definitions of S1, S2 and S3 are given in the text.

3.13C solid-state NMR spectra of three kinds of SF fibers in the dry and hydrated states Figures 3A, 3B and 3C show expanded four 13C solid state NMR spectra of each [3-13C] Ser-, [3-13C] Tyr- and [3-13C] Alalabeled SF fiber of F1, F2 and NF, respectively. The difference in the spectra is very small among three fiber samples compared with SF sponges. However, there are still differences when we compare the spectra carefully. In the 13C CP/MAS NMR spectra of the dry samples, the fraction of the random coil peak increased slightly in the Ser, Tyr and Ala Cβ peaks for two regenerated fibers, F1 and F2 (Figures 3A-a and 3B-a) compared with that of NF (Figure 3C-a). On the other hand, the difference in the shape of the peaks tends to be smaller for Ser Cβ and Ala Cβ carbons among the 13C CP/MAS NMR spectra of the hydrated three SF fibers (Figures 3A-b, 3B-b and 3C-b). However, there are significant differences in the shapes of Tyr Cβ peaks observed at the higher field of the Gly Cα peak. Namely decreases in the intensities of the Tyr Cβ peaks of F1 and F2 are larger than that of NF. This is due to increase in the mobility of the Tyr Cβ carbons of the former two fibers and as a result, a loss of the CP signal by hydration. The sharp peaks with high mobilities were observed for Ser, Tyr and Ala carbons in the 13C DD/MAS NMR spectra of three SF fibers (Figures 3A-c, 3B-c and 3C-c). This was assigned to random coils hydrated by penetrating water molecules although there was still non-hydrated random coil domain. In the 13C r-INEPT spectra, the spectral patterns are almost the same among three SF fibers except for absence of Tyr Cβ peak in the spectrum of F2 (Figures 3A-d, 3B-d and 3C-d).


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Biomacromolecules

Figure 3. A., B. and C. are the expanded a. 13C CP/MAS NMR spectra of 13C labeled SF fibers, F1, F2 and NF in the dry state, and b.13C CP/MAS, c. 13C DD/MAS, and d.13C r-INEPT NMR spectra of the fibers in the hydrated states, respectively. The definitions of F1, F2 and NF are given in the text.



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4. 13C solid-state NMR spectra of the chemically modified SF fiber, F3. The chemical modifications of SF structure have been frequently reported in order to add more useful function to SF or improve the defect when SF is used as biomaterials.1,4–6 Thus, it is interesting to know the change in the structure and dynamics of SF in the dry and hydrated states by chemical modification. In this paper, the modification of the regenerated SF fiber (stretching x3 times length) is introduction of hydrophobic side chain after blocking the OH group of Tyr residue by chemical reaction.42–44 Detail of the chemical reaction was described in EXPERIMENTAL SECTION. Figure 4A shows expanded four 13C solid state NMR spectra of [3-13C] Ser-, [3-13C] Tyr- and [3-13C] Ala-labeled F3 where those of F2 (broken red line) are also shown as a comparison. The expanded aromatic regions (110-160 ppm) of Tyr residues in the 13C CP/MAS NMR spectra of F3 and F2 in the dry state are also shown in Figure 4B and the fraction of the DMT-MM introduced in the Tyr OH group was determined to be 27±5 % by calculating the ratio of the area of Tyr Cζ peak for the chemically modified Tyr (150 ppm) to the total area of Tyr Cζ peaks (155 ppm and 150 ppm) of 13C CP/MAS NMR spectrum of F3. There is no change in the 13C CP/MAS NMR spectrum between F3 and F2 in the dry states as shown in Figure 4A-a. However, the shapes of Tyr Cβ peaks in the 13C CP/MAS NMR spectra are similar between the dry and hydrated states (Figures 4A-a and 4A-b), which is in contrast to the case of F2. This indicates that the environment of Tyr side chain in F3 became hydrophobic due to introduction of hydrophobic group into the OH group and as a result, a loss of CP of the Tyr Cβ peak decreased in F3. A more significant difference was observed in the 13C DD/MAS NMR and r-INEPT spectra between F3 and F2. In r-INEPT spectrum of F3 in the hydrated state (Figure 4A-d), there are essentially no sharp peaks. This means that no mobile carbons were observed in the r-INEPT spectrum due to the chemical modification of Tyr sites. Namely, the hydrophobic effect of the chemical modification at Tyr OH group extends to whole of the F3 chains. No sharp peaks were also observed in the 13C DD/MAS NMR spectrum of F3 in the hydrated state (Figure 4A-c).

Figure 4. A. is the expanded a. 13C CP/MAS NMR spectrum of 13C labeled SF fiber, F3 in the dry state, and b.13C CP/MAS, c. 13C DD/MAS, and d.13C r-INEPT NMR spectra in the hydrated states, respectively. The definition of F3 is given in the text. The broken lines are the spectra of F2. B. is the expanded aromatic regions of 13C CP/MAS NMR spectra of F2 and F3. The chemical reaction scheme of the introduction of DMT-MM at the Tyr side chain OH group is also shown.

5. The peak deconvolution of the 13C DD/MAS NMR spectra of SF samples in the hydrated state In order to compare the structure and structural change of these SF samples in the hydrated state more quantitatively, we tried the deconvolution of these peaks in the 13C DD/MAS NMR spectra of the samples, S2, S3, F1, F2, F3, and NF in the hydrated states. This can be done on the basis of conformation-dependent 13C NMR chemical shifts. Sum of Gaussian functions was used to fit the 13C DD/MAS NMR spectrum for each Cβ region of Ser, Tyr, and Ala residues (57-70 ppm, 30-46 ppm, and 12-25 ppm, respectively) by the Levenberg-Marquardt method implemented in Scipy67 which wraps MINPACK68. Each Gaussian function was attributed to a conformation of the residue judging by its chemical shift. Then the combination of the chemical shift, peak area, and full width of half maximum for each Gaussian function was obtained. As shown in Figure 5A, Ser Cβ peak consists of


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Biomacromolecules five peaks in 13C DD/MAS NMR spectrum of S2. The peaks at 61.5 ppm were assigned to non-hydrated random coil (broad peak) and hydrated random coil (sharp peak), the peaks at 64.0 ppm and 65.5 ppm to β-sheet A and β-sheet B, respectively.38,60 The presence of two β-sheet structures, A and B with different intermolecular arrangement has been discussed previously.60 In addition, an additional Ser Cβ peak at 60.5 ppm from Silk I* structure was observed as mentioned above.54–56 As for the Tyr Cβ carbon, the 36.1 ppm peak was assigned to non-hydrated random coil (broad) and hydrated random coil (sharp), and 40.3 ppm peak to β-sheet.21–24,38,66 Here the Tyr β-sheet peak was overlapped with the Gly Cα peak at 42.6 ppm (non-hydrated random coil, (broad) and hydrated random coil (sharp)) as reported previously. 21–24 Similar to the case of Ser Cβ peak, there are four peaks in Ala Cβ carbons. The peaks at 16.8 ppm, 19.6 ppm and 21.7 ppm were assigned to both non-hydrated random coil (broad) and hydrated random coil (sharp), β-sheet A and β-sheet B, respectively. 21–24,38 The additional Silk I* peak should be observed in Ala Cβ peak, but the chemical shift is the same as that of random coil and therefore the Silk I* peak was overlapped with the random coil peak at 16.8 ppm.21–24 All of the peak deconvolutions are shown in Figures 5A-G. The chemical shifts and full width of half maximum obtained from the deconvolution are summarized in Supporting Information Table 1S.

Figure 5. Deconvolutions of Ser, Tyr and Ala Cβ peaks in the 13C DD/MAS spectra of A: S2, B: S3, C: F1, D: F2, E: NF, and F: F3 in the hydrated states. The parameters, chemical shifts and half-height-widths obtained from the deconvolution were listed in Supporting information, Table 1S. These chemical shifts in the peak deconvolution were adjusted by ± 0.2 ppm to obtain a good fitting. The symbols are ①-① for Ser Cβ peak, ①-① for Gly Cα peak, ①-① for Tyr Cβ peak and ①-① for Ala Cβ peaks, respectively. The conformations are as follows: ① β-sheet B, ① β-sheet A, ① non-hydrated random coil, ① hydrated random coil, ① Silk I*, ①non-hydrated conformations, ① hydrated random coil, ① β-sheet, ① non-hydrated random coil, ① hydrated random coil, ① β-sheet B, ① β-sheet A, ① non-hydrated conformations and ①hydrated random coil including Silk I*conformation.

6. The fraction of different conformations of SF samples in the hydrated state The fractions determined by the deconvolution analyses of the 13C DD/MAS NMR spectra in the hydrated state are listed in Table 1 and shown as pie charts in Figures 6A-F for Ser, Tyr and Ala residues. The results of Gly Cα peaks are also shown in Figures 7A-G. The fractions of several conformations were compared between two SF sponges, S2 and S3 in the hydrated states as shown in Figures 6A and 6B, respectively. The fractions of β-sheet increase more than 10% for Tyr and Ala residues in S3 by methanol treatment compared with S2 as is expected. However, the increase in the fraction of β-sheet is very small for Ser residue by the treatment. This seems to be related with the presence of Silk I* structure in S2, which interfere with the conformational change to β-sheet by methanol31. The fraction of the hydrated random coil for Ser, Tyr and Ala residues decreases in S3. As shown in Figures 6B, 6C and 6D, the fractions of β-sheet increase and those of hydrated random coil decrease slightly in the order of S3, F1 and F2 for Ser and Ala residues. On the other hand, there are no significant changes among S3, F1 and F2 for Tyr residue. There is no significant change between F2 and NF, and only the fractions of β-sheet A and β-sheet B changes slightly as shown in Figures 6D and 6E, respectively. The fractions of several conformations among three regenerated SF fibers, F2 and F3 are compared as shown in Figures 6D and 6F, respectively. The fraction of hydrated random coil decreases significantly and conversely, those of non-hydrated random coil increase in F3 compared with F2. This is clearly due to the blocking of Tyr OH groups by DMT-MM reagent.



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Table 1. Fraction of different conformations of Ser, Tyr, and Ala residues determined by the deconvolutions of 13C DD/MAS spectra of the samples, A to F in the hydrated states shown in Figure 6. Ser Cβ

β-sheet B β-sheet A

nh.r.c.

h.r.c.

Silk I* h.r.c./(h.r.c.+nh.r.c.)

A. S2

12.1

25.8

42.8

11.0

8.3

20.4

B. S3

13.2

27.2

50.9

8.7

-

14.6

C. F1

18.2

32.6

41.7

7.5

-

15.2

D. F2

22.3

37.6

35.1

5.0

-

12.4

E. NF

27.6

32.3

35.3

4.8

-

11.9

F. F3

19.5

40.6

39.1

0.8

-

2.0

Tyr Cβ

β-sheet

nh.r.c.

h.r.c.

h.r.c./(h.r.c.+nh.r.c.)

A. S2

37.0

50.8

12.2

19.3

B. S3

50.8

46.1

3.1

6.3

C. F1

50.2

45.8

4.0

8.1

D. F2

54.4

42.4

3.3

7.2

E. NF

54.7

42.7

2.6

5.7

F. F3

49.6

48.8

1.6

3.2

Ala Cβ

β-sheet B β-sheet A

nh.r.c.

h.r.c.a

h.r.c./(h.r.c.+nh.r.c.)

A. S2

8.0

31.9

42.4

17.6

29.3

B. S3

14.3

43.5

33.2

9.1

21.5

C. F1

11.9

51.3

27.6

9.1

24.8

D. F2

16.9

51.7

25.2

6.2

19.8

E. NF

27.4

44.7

20.7

7.2

25.9

F. F3

19.3

41.2

37.7

1.8

4.6

h.r.c.: hydrated random coil and nh.r.c.: non-hydrated random coil. The h.r.ca in Ala Cβ peak includes both hydrated random coil and Silk I* structure.


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Biomacromolecules

Figure 6. Pie charts of fractions of conformations of Ser, Tyr and Ala Cβ peaks in the 13C DD/MAS NMR spectra of A: S2, B: S3, C: F1, D: F2, E: NF, and F: F3 in the hydrated states after peak deconvolution. The colors correspond to yellow: non-hydrated random coil, sky blue: hydrated random coil, poppy: β−sheet or β-sheet A, pink: β-sheet B, light green: Silk I*, and aqua marine: Silk I* and hydrated random coil.

Figures 7A-F shows change in the fraction of Gly Cα peak obtained from the peak deconvolution of all samples. Because of overlapping with 13C labeled Tyr Cβ peaks, the error in the fraction seems relatively large. Nevertheless, the fraction of the hydrated random coil is clearly larger for the sponges, S1 and S2 compared with the fibers. In addition, the fraction is the smallest for the chemically modified fiber, F3 as well as the case of Ser Cβ, Tyr Cβ and Ala Cβ peaks.



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Figure 7. Pie charts of fractions of conformations of Gly Cα random coil peaks in the 13C DD/MAS NMR spectra of A: S2, B: S3, C: F1, D: F2, E: NF, and F: F3 in the hydrated states after peak deconvolution. The colors correspond to gold: non-hydrated random coil and βsheet, and sky blue: hydrated random coil.

7. Mechanical properties of four kinds of SF fibers in the dry state The stress-strain curves of four kinds of SF fibers were observed in the dry state as shown in Figures 8A-D. The values of diameter (µm), breaking strength (MPa) and elongation at break (%) are summarized in Table 2. In general, thinner SF fibers are closely linked to increased fiber strength69 and therefore it is difficult to compare the mechanical properties simply among these fibers. However, the breaking strength of F1 was 149±10 MPa which is almost half of the strength of F2, 368±6 MPa. Thus, the strength increased significantly by stretching.51,52 The elongation at break decreased in the order of F1, F2 and NF.69 The larger diameter was obtained for the chemical modified SF fiber, F3. By the chemical modification of SF with DMT-MM, the hydroxyl of Tyr residue located in the amorphous region was substituted by hydrophobic triazine derivative by 27±5 %. The increase of the diameter could happen in the process of the chemical modification with DMT-MM, in which F2 was immersed in Na2CO3 solution. Thus, the strength decreased significantly. As seen in the deconvolutions of 13C DD/MAS NMR spectra shown in Figure 5, the fractions of hydrated random coil were very small in F3 because of increase in the hydrophobic character of the SF fiber. Actually, there are no peaks in the 13C r-INEPT spectrum. Thus, the hydrophobic effect extends to the whole domain as well as Tyr residues.


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Biomacromolecules

Figure 8. The stress-strain curves of the SF fibers, A: F1, B: F2, C: NF and D: F3. The mechanical property data are summarized in Table 1.

Table 2. Mechanical properties of SF fibers, F1, F2, NF, and F3 under dried condition. Fiber

Diameter/µm

Breaking strength/MPa

Elongation at break/%

F1

94.8±0.8

149±10

48±13

F2

50.5±1.0

368±6

21±8

NF

12.7±3.5

437±14

14±7

F3

130±5.0

148±10

27±5

DISCUSSION. The physical properties of SF change significantly by the interaction with water molecules and therefore it is important to clarify the effects of water on the structure and dynamics of SF in molecular level as well as the interaction between water and SF.2,70 Many analytical methods have been used. However, the interaction with water molecules and SF as well as a complete picture of the structure and dynamics of SF interacted with water molecules are still missing at molecular level. In general, water in the silk–water system can be divided into three categories: free water, freezing bound water, and non-freezing bound water.7–12,16–18 NMR is possible to study these problems from both sides of SF and water by selecting an NMR technique including species of nuclei appropriate to the particular issue. Previously, we used 1H pulse NMR to study the dynamics of the non-freezing bound water with SF fiber, film and powder.16 Recently, we could identify distinct components of the freezing bound water interacted with SF fiber and films from the 2H solution NMR relaxation measurements for water in SF-water system.22,23



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In this paper, we used the combinations of 13C r-INEPT, 13C CP/MAS and 13C DD/MAS NMR experiments coupled with 13C selective labeling of SF to provide the information of structure and dynamics of the hydrated SF sponges and fibers from SF side.23,24 When we study the effect of water on the SF, it seems important to examine how the supply of water molecules to the SF. We thus showed here the effects of water on the structure and dynamics of NF were quite different between two cases, that is, setting the fiber under high humidity atmosphere or immersing it in water directly. As shown in the 13C DD/MAS NMR spectrum, the mobility of only side chains of Ala and Ser residues in NF increases selectively by water vapor under high humidity. However, when the fibers were immersed in water directly, both broad and sharp peaks were observed for both backbone and side chain carbons. Thus, water molecules are considered to penetrate only random coil region of NF partially in the heterogeneous manner. The 13C r-INEPT spectra show the difference in both cases clearly. Thus, it is noted that the effect of water is quite different on the fiber between two hydration methods. Mo et al.9 studied the effect of water on the conformational transition of SF under thermal perturbations using near-IR and DSC. In the SF film with water content ranging from 10 to 15%, the freezing bound water could not detect in the near-IR spectra. However, when the water content is above 20%, the freezing bound water was observed, which facilitated the movement of the SF chains and contributed to the conformational transition of SF film at 60°C. Thus, they speculated that it was the freezing bound water that played the role of plasticizer. Our results are basically in agreement with their reports. Yazawa et al.13 reported that the plasticizing effect of water was not detected in SF fibers even if the SF fibers were incubated at high relative humidity, owing to their lower amorphous content and mobility. Then such a plasticizing effect could be observed for SF film at the highest relative humidity of 97%. Actually, we determined the fractions of β-sheet and hydrated random coil for the Ala, Ser and Tyr residues of SF fiber and SF films immersed in water using 13 C DD/MAS NMR method previously.22 Remarkable decrease of the β-sheet fractions and conversely, significant increase of the hydrated random coil fractions were observed for Ala and Ser residues by changing from SF fiber to SF films although essentially no changes were observed for the Tyr residues. Next, we discuss change in the fractions of several conformations observed in SF sponges and several regenerated SF fibers including natural fiber in the dry and hydrated states. Before discussing the structure of SF, it seems better to explain the specific structures of SF, that is, Silk I* and two kinds of β-sheet structures briefly. The Silk I* structure is an essential and key structure in understanding the SF structure before spinning which has been studied in detail. 1,21,24,31,37,38,54–56 In short, the Silk I* is a repeated β-turn type II structure with the torsion angles, (φ, ψ) = (−62°, 125°) for Ala residues and (φ, ψ) = (77°, 10°) for Gly residues of the motif, (Ala-Gly-Ser-Gly-Ala-Gly)n, which is a typical crystalline fraction of SF (Supporting Information Figure 2S).55 The intra- and inter-molecular hydrogen bonding was formed alternatively along the chain. In addition, Ser residues stabilize the Silk I* structure by the hydrogen bonding formation between the side chain OH groups and the C=O groups of the backbone chain.65 In addition, two kinds of β-sheet structures in the crystalline domains of SF fiber were observed in the 13C CP/MAS NMR spectra.58,59 The structure of SF after spinning which is called as Silk II was first proposed by Marsh, Corey, and Pauling61 as a regular array of antiparallel β-sheets using X-ray fiber di①raction of the crystalline region of SF. Later, Lotz and Cesari62 and Takahashi et al.63 noted an irregular structure to be present in the natural SF fibers with Silk II form. Takahashi et al.63 proposed that a crystal site is statistically occupied by either of two antiparallel β-sheet chains with di①erent relative orientations, in a 2:1 ratio, based on X-ray di①raction analysis of SF fibers. The latter analysis is more detailed and based on better data than the model proposed by Marsh et al.61 However, the model proposed by Takahashi et al. was not consistent with the distances of the intermolecular hydrogen bonds between the NH···OC groups of Ala and Gly residues obtained experimentally and not stable from the energy calculations. We proposed a new comprehensive model which satisfied the distances of the intermolecular hydrogen bonds and which was also energetically stable.64 In short, two kinds of β-sheet structures of Silk II in the crystalline domains of SF fiber were explained with Figure 9 as follows. Both of two arrangements crystallize in space group P21, a rectangular unit cell with the parameters a = 9.38①, b = 9.49①, and c = 6.98①, but strands of βsheet A (Figures 9a, 9b and 9c) are aligned along the crystallographic c-axis while those of β-sheet B (Figures 9d, 9e and 9f) are aligned along b-axis. The 2:1 ratio of the two arrangements, β-sheets A and B, was confirmed by our solid-state NMR analysis of a model peptide (Ala-Gly)15.64 Thus, Silk I*, two kinds of β-sheet structures and random coil were indistinguishable for each 13C NMR peak of SF and the fractions of these conformations were determined by peak deconvolution, which enabled the detailed structural analysis of SF.


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Biomacromolecules

Figure 9. The β-sheet model I (top row) and model II (bottom row) for the crystalline regions of SF fiber with Silk II form, shown from three orthogonal orientations.66 The β-sheet A and B in Ala Cβ peaks in Figure 6 are assigned to the models I and II, respectively. The arrows in (a) and (d) represent (AG)n sequences with the direction from the C-terminus to N-terminus. In the model I, the molecular axis is along the crystallographic axis c, and in the model II, the molecular axis is along the crystallographic axis b. The models I and II have different alternative antipolar arrangements for the four antiparallel β strands, a, b, and a’, b’, taking different directions of methyl groups (indicated by Me), as shown in (b) and (e). Distances between NH···OC groups in the models I and II are shown in (b) and (e), respectively. These images were visualized with VMD.71

We showed here that the conformations of silk sponges were significantly different by changing the preparation methods in the dry state when the immobile components were emphasized in the 13C CP/MAS NMR observation. These results are essentially the same as reported previously. 31,37,38,49,56,59–61 This difference is more emphasized for the sponges in the hydrated state as mentioned above. For example, when we use SF for biomaterials like bone generation or artificial graft, the sponge is one of the starting materials and use them in water.1,4,24,72 Thus, the characterization of the structure is important in controlling the strength, elasticity and biodegradation and so on. The relationship among the preparation method and structure and dynamics in SF sponges was clarified for each residue, Ser, Tyr and Ala which are present in predominantly crystalline domain, predominantly non-crystalline domain and in both domain, respectively. The quantitative evaluation of several conformation of both mobile and immobile components was performed from the peak deconvolution of the 13C DD/MAS NMR spectra in the hydrated state and therefore the further discussion is developed on the basis of the fraction (Figures 6 and 7). The observation of Silk I* structure in Ser and Ala residues is characteristic of the structure of the SF sponge, S2. This means that the state of SF similar to the state stored in the silk gland was reproduced partially by standing SF sponge under high humidity atmosphere. By methanol treatment, Silk I* structure in the sponge disappeared. In the sponges, S2 and S3, both β-sheet A and B appeared in Ala and Ser residues. The ratio of A and B is approximately 1:2 for Ser residue, but the fraction of B increases for Ala residue. Thus, the hydrated structures of the sponges change significantly depending on the methods of the preparation of the sponges. On the other hand, it is noted that MeOH affects the conformation of Tyr residue in the non-crystalline region significantly and increase β-sheet



Page 16 of 20

fraction. Conversely, for Ser residue in the crystalline region, the effect of MeOH is not so much compared with Tyr and Ala residues. On the other hand, the changes in the dry structure were not so much for several regenerated SF fibers including natural fiber although the mechanical properties of these fibers change remarkably. Thus, the mechanical properties depend on the factors reflecting longer range structure than the local structure detected by NMR. Namely, they depend on the amount of the intramolecular β-sheet crystallites, the size of β-sheet crystallites and orientation of β-sheet crystallites as well as non-crystalline structure in the SF fibers.5,51,52,69 In order to study the molecular orientations of SF fiber samples including the determination of the fraction of oriented components, it is effective to observe the solid-state NMR spectra of the aligned silk fiber samples by changing the angle between the fiber axis and the direction of magnetic field by focusing on the large anisotropies of the carbonyl carbons or nitrogen atoms or 2H solid-state NMR of stable isotope labeled silk fiber samples as reported previously.73–82 In this paper, we only pointed out change in the fractions of β-sheets A and B among three fiber samples because the fractions reflect the aspect of inter-molecular packing arrangement. Plaza et al.83 reported the effect of water on the mechanical properties of the regenerated and native SF fibers. The breaking strengths were observed for as spun, stretched (x 1,1, x2.0 and x3.8) and native SF fibers in the dry and in water. For both cases, the strengths increase in the order of as spun, stretched (x 1,1, x2.0 and x3.8) and native SF fibers although the absolute strengths decrease in water compared with those in the dry state. Thus, we believe that the structural analysis using 13C DD/MAS NMR of SF fibers in the hydrated states is applicable to interpret the difference in the mechanical properties among the regenerated and native SF fibers in the dry states qualitatively. The fraction of β-sheet B increases in the order of F1, F2. and NF for Ala and Ser residues, but the fraction of β-sheet A did not change a little. Thus, the intermolecular packing arrangements of the β-sheets A and B were examined carefully. The distances between the hydrogen bonds of NH···OC in the β-sheet models I (β-sheet A) and II (β-sheet B) are shown in Figures 9b, 9e, and Table 2S and their averages were calculated as 1.87 ① and 1.84 ① respectively. Thus, the β-sheet B seems slightly stable because of the shorter lengths of intermolecular hydrogen bonding and therefore increase in the fraction of β-sheet B might cause the higher strength. Although more experiments are required to confirm this assumption, it is interesting to monitor of the fraction of β-sheet B in 13C solid-state NMR spectra of SF fiber as one of the structural factors to prepare stronger regenerated SF fiber other than the fraction of β-sheet in the samples.

CONCLUSIONS The effects of water on the structure and dynamics of SF samples were studied using three kinds of 13C NMR techniques, rINEPT, CP/MAS, and DD/MAS spectroscopies. At first, the effects of water on the structure and dynamics of the natural SF fibers were compared for the fibers under high humidity atmosphere or immersed in water directly. In the former case, the relative intensities of the side chain carbons increased considerably compared with those of backbone carbons. On the other hand, new sharp peaks with highly mobile hydrated random coil were observed partly for both side chain and backbone carbons when the fibers were immersed in water directly. Subsequently, different types of SF materials, sponges and fibers, with different preparation methods were prepared and compared for their NMR spectra in the dry and in the water immersion states. By preparing [3-13C] Ser, [3-13C] Tyr and [3-13C] Ala labeled SF samples, we could obtain information of structure and dynamics of SF from the crystalline and non-crystalline domains independently. The fraction of β-sheet increased in the order of freeze-dried water-soluble sponges, water-insoluble sponges prepared by standing them under high humidity atmosphere and those by immersing in methanol. In the hydrated sponges, several conformations, that is, Silk I* and two kinds of β-sheet structures, A and B, random coil and hydrated random coil with high mobility were observed and the fractions of these conformations were determined from the 13C DD/MAS NMR spectra. The fractions were different remarkably among three sponges. On the other hand, there are only small differences among two kinds of regenerated 13C-labeled SF fibers and native fiber although the stressstrain curves are quite different. The increase in the fraction of β-sheet B in 13C solid-state NMR spectra of SF fiber might be as one of the structural factors to prepare stronger regenerated SF fiber other than the fraction of β-sheet in the samples. A chemically modified fiber was prepared by blocking the side chain OH sites of Tyr residues in the regenerated SF fiber with hydrophobic group partly, and 13C solid-state NMR and the stress-strain curves were also observed.

ASSOCIATED CONTENT SUPPORTING INFORMATION Figure 1S. Expanded 13C r-INEPT, 13C DD/MAS, and 13C CP/MAS NMR spectra of NSF in high humid state with 97% relative humidity. Figure 2S. Silk I * structural model of packing structure of (Ala-Gly)n chains with type II β-turn structure.54,55 Dotted lines denote the hydrogen bonds. Table 1S. 13C chemical shifts δ/ ppm and half-height-widths / Hz obtained from the peak deconvolution. Table 2S. Distances of hydrogen bonds between NH···OC groups in b-sheet model I and II.


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Biomacromolecules

AUTHOR INFORMATION Corresponding Authors *(T.A.) Tel & Fax 81-42-383-7733; e-mail [email protected].

ORCID Tetsuo Asakura: 0000-0003-4472-6105

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS T.A. acknowledges support by a JSPS KAKENHI, Grant-in-Aid for Scientific Research (A), Grant Number JP26248050 and Impulsing Paradigm Change through Disruptive Technologies Program (ImPACT).

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