Quantitative Correlation between Primary Sequences and

Mar 30, 2017 - The conformation was mainly α-helix with no β-sheet structure prior to ... Chengchen GuoJin ZhangJacob S. JordanXungai WangRobert W...
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Quantitative Correlation between Primary Sequences and Conformations in 13C‑Labeled Samia cynthia ricini Silk Fibroin during Strain-Induced Conformational Transition by 13C Solid State NMR Tetsuo Asakura,* Akio Nishimura, and Yuya Sato Department of Biotechnology, Tokyo University of Agriculture and Technology Koganei, Tokyo 184-8588, Japan S Supporting Information *

ABSTRACT: A better understanding of the correlation between the primary sequences and the conformations of silk fibroins (SF) is necessary in order to use silk more effectively as a functional material. In this paper, 13C CP/MAS NMR was used to monitor separately the conformational transitions of [3-13C]Ser-, [3-13C]Tyr-, and [3-13C]Alalabeled Samia cynthia ricini SF induced by stretching. The conformation was mainly α-helix with no β-sheet structure prior to stretching. At a stretching ratio of ×5, three peaks assigned to β-sheet structure were observed, and the fraction of these peaks increased rapidly upon further stretching. In particular, a rapid increase of the fraction of β-sheet at more than ×5 stretch was observed for the Ser residues that were associated with the transition of the polyalanine chain. For the Tyr residues, the transition from random coil to β-sheet occurs independently of the transition of the crystalline domain. These correlations were quantified and may be useful in future designs of artificial silk structure.

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INTRODUCTION In nature, there are many kinds of silks produced by silkworms and spiders, and they have captured the fascination of biologist, chemists, and material scientists for more than a century.1−4 The excellent mechanical properties of these silks include high modulus, strength, and extensibility that surpass most manmade materials.2,5,6 These properties can be correlated to specific amino acid sequences in silk.6−8 Therefore, in order to use such materials more effectively and design new silks with improved properties, a better understanding of the correlation between the primary amino acid sequences and higher order structures is necessary at the molecular level. Samia cynthia ricini (S. c. ricini) is a wild silkworm that produces silk fibroin (SF), whose primary structure has recently been published as shown in Figure 1.9 The S. c. ricini SF consists of 2879 amino acid residues, and the overall composition entails Ala (45.4%), Gly (31.7%), Ser (6.7%), Tyr (5.8%), and other amino acids in lower amounts. The primary structure of SF mainly consists of tandemly repeated sequences of a poly(L-alanine) (PLA) region and a Gly-rich region, similar to the sequences in the silk of other wild silkworms, such as Antheraea pernyi and Antheraea yamamai,2,6−8,10,11 and the major ampullate silk from the spider Nephila clavipes.2,4,12,13 The solution structure of S. c. ricini SF stored in the silk gland has been studied in vivo and in vitro using 13C and 15N solution NMR.14−19 The fast exchange in the NMR time scale between α-helix and random coil forms has been observed in the PLA region, and the fractional amount of each form changes depending on the observed temperature.15,16,18 The α-helix content of the SF determined from both the peak position of the Ala carbonyl carbon assigned to © XXXX American Chemical Society

Figure 1. Primary structure of Samia cynthia ricini silk fibroin.9 The color red means Ala residue, blue the Ser residue, and green:Tyr residues.

PLA and the Ala contents in the SF decreases linearly with increasing temperature, but most of the Gly residues maintain the random coil conformation during the helix−coil transition of the PLA region. Recently, the solution structure of a typical tandem repeat sequence Tyr-Gly-Gly-Asp-Gly-Gly-(Ala)12-GlyGly-Ala-Gly of the SF obtained from the silk gland was Received: March 3, 2017 Revised: March 27, 2017

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Macromolecules determined by solution NMR at the atomic level.19 Several solid state NMR techniques were also used to determine the detailed structure of the SF before and after spinning by determining the torsion angles of the backbone amino acids and intra- and intermolecular atomic distances within the motifs of tandemly repeated sequences of the S. c. ricini SF, using selectively stable isotope-labeled model peptides in the solid state.13,20−24 In general, three different analytical methods (13C CP/MAS NMR, X-ray diffraction, and Raman spectroscopy) have been used to monitor the conformational transition of S. c. ricini SF induced by stretching.25 All of the data indicated that the transition from α-helix to β-sheet occurred in the SF between the stretching ratios ×4 and ×6. Molecular dynamics (MD) simulation of the sequential model peptide of the S. c. ricini SF could reproduce the stretch-induced conformational change from the torsion angle change of Ala residues in the peptide.26 The conformational transition was also performed for A. perni SF9,10 where the primary structure is similar to that of S. c. ricini SF using 13C CP/MAS NMR11and synchrotron FTIR microspectroscopy.27 Recently, the conformational transition of 13C selectively labeled SF prepared from B. mori silk gland induced by stretching was monitored by 13C CP/MAS NMR to correlate quantitatively the primary sequence and conformation of the stretch-induced conformational transition.28,29 This conformational analysis is based on the conformation-dependent 13C NMR chemical shift coupled with selective 13C labeling of the SF samples involving Ser, Tyr, and Ala Cβ carbons.30−32 Since the Ser residues are present predominantly in the crystalline domains, Tyr residues predominantly in the noncrystalline domains, and Ala residues in both domains of the SF,33,34 we could discern the conformations and conformational changes of these residues in the crystalline and noncrystalline domains independently. The 13C conformation-dependent chemical shift maps of these three 13C-labeled carbons reported previously31,32 were handy for this purpose. In this paper, we applied the same analytical technique to study the conformational transition of S. c. ricini SF prepared from silk gland by monitoring the stretch-induced transition with 13C CP/MAS NMR. The 13C labeling of the SF was performed on the Cβ carbons of the same Ser and Tyr residues as those of B. mori SF.29 Although both Ser and Tyr residues are located in the Gly-rich region of S. c. ricini SF, the environment of these amino acid residues in the primary structure is quite different; in particular, 75.8% of Ser residues are present within five amino acid residues from both ends of the PLA sequence, while 90.5% of Tyr residues are present at more than six amino acid residues away from both ends of the PLA sequence as shown in Figure 1.9 Thus, the conformational transition in the Gly-rich region can be monitored by the spectral changes of the Ser and Tyr residues that serve as markers.35−37 In contrast, 90.0% of the Ala residues are located in the PLA region as the crystalline domain. The conformation and conformational transition of the crystalline domain can be monitored by the Ala Cβ peak. The Ala Cβ carbons are expected to be partially 13C labeled by transamination from [3-13C]Ala to [3-13C]Ser carbons as observed for 13C labeling of B. mori SF.29,38,39 This increase in the peak intensity is useful in the conformational analysis of Ala residues. The results obtained by NMR coupled with selective 13C labeling in the PLA and Gly-rich regions of SF will give important information

on the future design of the man-made artificial SF with targeted physical properties.25,40

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MATERIALS AND METHODS

Preparation of 13C Selectively Labeled S. c. ricini SF Samples. Eggs of S. c. ricini silkworms were kindly given by Prof. Saito (Kyoto Institute of Technology, Japan), and the silkworms were reared with an artificial diet, silk mate L4M (Nosan Co., Japan), in our laboratory. The 13C labeling of the 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.29,41 Briefly, the supplementary Tyr and Ser were mixed with 2.0 g of an artificial diet per day. The labeled Tyr and Ser were fed to larvae at 10 mg each on the fourth and fifth days of the fifth larval stage. To prevent scrambling of Ser into Gly, an amount of 20 mg of nonlabeled Gly was also mixed with the artificial diet per day. Thus, the total amount of Tyr and Ser was 20 mg per silkworm. The 13C-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. The fifth instar larvae that were older than 5 days were anesthetized in the ice-cold water for 10 min. The posterior divisions of silk glands were pulled out with forceps from a small incision on the abdominal side of the bead-thorax intersegment. The silk glands were washed repeatedly in distilled water. After immersing them in dilute acetic acid (0.1 wt %) for 10 min, the silk glands were washed carefully by gently agitating the acid solution to remove contaminants such as gland epithelium.25 The SF samples stored in the silk glands were stretched to the required stretching ratio, ×1, ×3, ×5, ×7, ×9, and ×11, at room temperature by using a stretching rate of 5−10 cm/s. The ends of the stretched SF samples were then fixed on the stretching apparatus to prevent relaxation and dried at room temperature for 1 day prior to NMR observation. The cocoons were degummed three times with 0.1% w/w sodium peroxide solution at 80 °C for 1 h and washed with distilled water in order to remove silk sericin, another silk protein, from the surface of silk fibers.25 The latter SF fibers prepared from cocoons directly were distinguished from the former stretched SF samples and named as natural SF fibers in this paper. The natural SF fibers were dried at 37 °C overnight prior to the next experiment. 13 C CP/MAS NMR Measurement and Spectral Deconvolution Analysis. 13C CP/MAS spectra were acquired on a Bruker DSX-400 AVANCE spectrometer operating at 100.4 MHz, with a CP contact time of 1 ms, TPPM decoupling, and magic angle spinning at 7 kHz. A total of 8192 scans were collected over a spectral width of 60 kHz, with a recycle delay of 3 s. The line broadening factor was 0 Hz to obtain NMR spectra. The 13C NMR chemical shifts were calibrated indirectly through the methylene peak of adamantane observed at 28.8 ppm relative to tetramethylsilane at 0 ppm.41 The stretching ratio was varied between 1 and 11 times (×1, ×3, ×5, ×7, ×9, and ×11) for both 13 C-labeled and nonlabeled silk samples with the same stretching ratio.29 The difference spectrum for each stretching ratio was obtained by subtracting the nonlabeled spectrum from the 13C-labeled spectrum and then deconvoluted by assuming Gaussian line shapes.29 In the deconvolution of the spectra, the half-height widths for the individual components of the peaks were changed while the chemical shifts were fixed. The experimental errors for the fraction values in the deconvolution analysis were about ±1%.

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RESULTS [3-13C]Ser and [3-13C]Tyr Labeling of S. c. ricini SF. In order to study in detail the conformational transition of Ser and Tyr residues in S. c. ricini SF using 13C solid state NMR, selective 13C labeling was required because of the low S/N ratio of the 13C peaks for these residues due to their low amino acid content (Ser, 6.7%; Tyr, 5.8%). The 13C labeling could be attained by oral administration of an artificial diet with these 13 C-enriched amino acids to larvae of the fifth instar, as described in the Materials and Methods section. Figure 2 shows the expanded 13C CP/MAS NMR spectra (10−70 ppm) of (A) B

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Figure 2. Expanded region (10−70 ppm) of the 13C CP/MAS NMR spectra of S. c. ricini silk fibroin: (A) [3-13C]Ser-, [3-13C]Tyr-, and [3-13C]Ala-labeled silk fibroin before stretching, (B) [3-13C]Ser-, [3-13C]Tyr-, and [3-13C]Ala-labeled natural silk fibroin fiber, and (C) nonlabeled natural silk fibroin fiber. The line broadening factor was 0 Hz.

both [3-13C]Ser- and [3-13C]Tyr-labeled S. c. ricini SF film before stretching, (B) 13C-labeled natural SF fiber, and (C) nonlabeled natural SF fiber. Thus, we could observe the Ser and Tyr Cβ peaks of both [3-13C]Tyr- and [3-13C]Ser-labeled SF samples clearly and could discuss the conformational transitions of these residues in detail by paying attention to the conformation-dependent 13C chemical shifts and the changes in the line shape. The area of the Ala Cβ peak is considerably larger than that of the Ala Cα peak, which is due to transamination from [3-13C]Ser to [3-13C]Ala residue as observed for the 13C labeling of B. mori SF.38,39 In Figure 2B, the Tyr Cβ peak overlaps with the naturally abundant Gly Cα peak, but these two peaks can be separated by peak deconvolution.29 13 C CP/MAS NMR Spectral Change of S. c. ricini SF Induced by Stretching. A series of the 13C CP/MAS NMR spectra of 13C labeled S. c. ricini SF samples with different stretching ratio are shown in Figure 3 as a function of stretching ratio together with that of the natural SF fiber. The difference spectrum obtained by subtracting the nonlabeled spectrum from the 13C-labeled spectrum is also shown in Figure 4 for monitoring the conformational transitions of the SF from the 13 C labeling portion selectively by the peak deconvolution.29 The conformational transition with stretching can be easily monitored by the spectral change. As reported previously,20−25,42 the SF sample without stretching contains αhelix, which is shown by the sharp single peaks of Ala Cβ and Ala Cα carbons with the chemical shifts, 15.7 and 52.5 ppm, respectively. The Ala CO peak at 176.2 ppm indicates that the Ala residue is also part of an α-helix. With increasing stretching ratio (e.g., ratio of 5×), other peaks assigned to βsheet can be observed clearly in Ala Cα (49 ppm) and Ala Cβ (about 21 ppm) region. Thus, the conformational transition from α-helix to β-sheet occurs clearly for the Ala residues with increasing stretching ratio. For the natural SF fiber, most of the Ala Cα and Cβ peaks were observed as β-sheet structure.20−25 In contrast, the Cβ carbons of Ser and Tyr residues do not give clear-cut spectral information on the conformational transition. Thus, peak deconvolution analysis is required, which will be discussed in the next section. Determination of the Fraction of Several Conformations of Ala, Ser, and Tyr Residues of SF with Different Stretching Ratio by Peak Deconvolution Analysis. Ala

Figure 3. 13C CP/MAS NMR spectra of [3-13C]Ser-, [3-13C]Tyr-, and [3-13C]Ala-labeled S. c. ricini silk fibroin stretched in different ratios and natural 13C-labeled silk fibroin fiber. The line broadening factor was 0 Hz.

Figure 4. Difference 13C CP/MAS spectra of [3-13C]Ser-, [3-13C]Tyr-, and [3-13C]Ala-labeled S. c. ricini silk fibroin stretched in different ratios and 13C-labeled natural silk fibroin fiber obtained by subtracting the nonlabeled spectra from the 13C-labeled spectra.

C

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Macromolecules Residue. In the 13C CP/MAS NMR spectra of B. mori and S. c. ricini SF, the Cβ signal of Ala residues is split into several peaks which give detailed information on the conformation and intermolecular chain arrangements in SF, as reported previously.24,25,41 Accordingly, we analyze the Ala Cβ peak in detail through peak deconvolution of the difference spectra shown in Figure 4. The S. c. ricini SF consists of 93 tandem repeats of a PLA region flanked by Gly-rich regions as shown in Figure 1.9 The length of the PLA ranges from 10 to 14 but is found mainly 12−13, corresponding to a narrow length distribution. The fraction of Ala residues located in the crystalline region of PLA in S. c. ricini SF is 90%, and the other Ala residues are located in the Gly-rich sequences and the terminal domains. Figure 5 shows the expanded Ala Cβ peaks of SF samples with different stretching ratios. The five peaks in the fiber

Table 1. Fractions of Several Conformations of Ala, Ser, and Tyr Residues Determined from Deconvolution of Ala Cβ, Ser Cβ, and Tyr Cβ Peaks in the 13C CP/MAS NMR Spectra of [3-13C]Ser-, [3-13C]Tyr-, and [3-13C]Ala-Labeled Silk Fibroins with Different Stretching Ratios, Respectively; Data of the 13C-Labeled Natural S. c. ricini Silk Fibroin Fiber (Experimental Error Is about ±1)a Ala C β s.r. 1 3 5 7 9 11 fiber

a

α-helix (15.7 ppm)

r.c. (16.5 ppm)

β-sheet A (19.8 ppm)

24 24 17 20 8 8 10

0 0 14 16 30 34 42

76 76 62 57 40 26 2 Ser Cβ

β-sheet B (21.2 ppm) 0 0 3 3 9 18 21 Tyr Cβ

β-sheet C (22.9 ppm) 0 0 4 4 12 14 25

s.r.

r.c.(61.1 ppm)

β-sheet (63.6 ppm)

s.r.

r.c. (36.1 ppm)

β-sheet (40.3 ppm)

1 3 5 7 9 11 fiber

86 84 74 76 65 50 30

14 16 26 24 35 50 70

1 3 5 7 9 11 fiber

97 88 83 79 76 71 67

3 12 17 21 24 29 33

s.r.: stretching ratio; r.c.: random coil.

(the values are also listed in Supporting Information Table S1). For the dried SF sample extracted from the silk gland without stretching (i.e., stretching ratio ×1), the fraction of αhelix was determined to be 76% by the deconvolution analysis as shown in Figure 5. The helical fraction of the overall sample can be calculated to be 76% × 0.454 (the fraction of Ala versus all amino acids in the SF) or 34.5%, which is in agreement with the reported value obtained from 13C CP/MAS NMR25 and also the value (33 ± 2%) calculated from Raman microspectroscopy by Rousseau et al.42 However, the value of 76% (the fraction of Ala residues in α-helix conformation for all Ala residues) is significantly lower than 90%, the value obtained by assuming that all Ala residues in PLA region exist in the α-helix conformation (Table 2). Thus, the Ala residues located at or near the N- and/or C-terminals of a PLA chain are expected to be random coil in the solid state (Figure 1). Indeed, theoretical thermodynamics considerations on α-helix−coil transition in PLA chain developed by Bixon et al. predicted a decrease in αhelix content at the terminal parts of the PLA chain.16,43 As shown in Figures 5 and 6, there was no significant change in the spectra between the stretching ratios ×1 and ×3. At the stretching ratio ×5, the peaks for β-sheet structure started to appear at around 20 ppm. Simultaneously, the fraction of αhelix decreased, and thus the conformational transition from αhelix to β-sheet occurred. This is in agreement with our previous findings from X-ray diffraction and Raman spectroscopy25 and also with the results of Raman microspectroscopy reported by Rousseau et al.42 The β-sheet NMR signal of Ala Cβ carbon consisted of three peaks;24 among them, the β-sheet A peak at 19.8 ppm was dominant. With a further increase of the stretching ratio, the fraction of α-helix decreased linearly, being the smallest in the natural SF fiber (Table 1 and Figure

Figure 5. Expanded Ala Cβ peaks of 13C CP/MAS NMR spectra of [3-13C]Ser-, [3-13C]Tyr-, and [3-13C]Ala-labeled S. c. ricini silk fibroin stretched in different ratios and 13C-labeled natural silk fibroin fiber with the deconvolution spectra.

spectrum were assigned to 15.7 ppm for α-helix, 16.5 ppm for random coil, 19.8 ppm for β-sheet A, 21.2 ppm for β-sheet B, and 22.9 ppm for β-sheet C.13,24,25 Through deconvolution, the fractions of several conformations were determined and summarized in Table 1. The fractions are also plotted as a function of the stretching ratio in Figure 6a. The half-height widths used in the peak deconvolution are plotted in Figure 7a D

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Figure 7. Half-height widths of several conformations in the convolutions of Ala Cβ, Ser Cβ, and Tyr Cβ peaks of the 13C CP/ MAS NMR spectra of [3-13C]Ser-, [3-13C]Tyr-, and [3-13C]Alalabeled S. c. ricini silk fibroin plotted against the stretching ratio and 13 C-labeled natural silk fibroin fiber.

Figure 6. Fractions of several conformations determined from deconvolutions of Ala Cβ, Ser Cβ, and Tyr Cβ peaks in the 13C CP/MAS NMR spectra of [3-13C]Ser-, [3-13C]Tyr-, and [3-13C]Alalabeled S. c. ricini silk fibroin plotted against the stretching ratio and 13 C-labeled natural silk fibroin fiber.

Table 2. Fractions of Ala, Ser, and Tyr Residues in the Poly(L-alanine) Sequence (PLA), in Non-Poly(L-alanine) Sequence (NPLA), the N-Terminus Block (NTB), and the C-Terminus Block (CTB) in the Primary Structure of S. c. ricini Silk Fibroina

6a). At the same time, the fraction of three β-sheet peaks increased linearly. The fractions of both β-sheet B and C peaks increased remarkably at the stretching ratio ×9. The fraction of random coil decreased gradually with increasing stretching ratio. In the natural SF fiber, the fraction of random coil was 10% (Table 1), which is in agreement with the value, 10%, of the fraction of Ala residues which do not contribute to the PLA chain in the primary structure (Table 2). Thus, these Ala residues exist without being incorporated into the β-sheet structure of PLA chains. Conversely, the Ala residues at the terminal groups of the PLA chain which did not contribute to α-helix of the PLA chain of the SF samples before stretching were incorporated into the β-sheet structure of PLA chain in the fiber formation. Note that the fraction of α-helix is very small and almost negligible in the natural SF fiber, and therefore the conformational transition from α-helix to β-sheet

region

Ala

Ser

Tyr

PLA NPLA CTB NTB

90 8.6 0.2 1.1

0.5 87.6 3.1 8.8

0 95.8 0.6 3.6

a

Namely, NTB is the sequence M(1)RV...AG(144) and CTB the sequence S(2854)SA...VH(2880) in Figure 1.9

structure occurs completely in the process of fiber formation by the S. c. ricini silkworm. There is a significant difference in the fiber fraction between the SF sample with stretching ratio ×11 E

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Macromolecules and natural SF fiber. This means that stretching process alone is not enough to form the well-oriented fiber. In attempts to examine other factors, many papers have been published, especially on B. mori SF and spider silk. The factors proposed included the complex spinning system, changes in the environment (e.g., pH and concentrations of metal ions) along the spinning duct,26,44−53 and self-assembly of the SF molecules in the aqueous solution.37,54−56 The change in the half-height widths of Ala peaks in deconvoluted spectra is plotted against the stretching ratio in Figure 7a. With increasing stretching ratio, the half-height widths of three β-sheet peaks become smaller, indicating that the chemical shift distribution (attributed to the variation of the torsion angles of the Ala residues in the PLA chain with the βsheet structure) become smaller. This means that the SF samples crystallize with stretching. In particular, the chemical shift distribution became the smallest when the fiber and the assembles of PLA chain with β-sheet structure were most oriented.42 Because the fraction of α-helix and random coil is small, it is difficult to glean more information from the halfheight widths of these peaks, but these peaks did become broader both in the SF with high stretching ratio and in the natural SF fiber. Ser Residue. The Ser residues are mainly present in the crystalline region of the (AGSGAG)n sequences of B. mori SF,33,34 but these residues are present in the Gly-rich sequences of S. c. ricini SF. In fact, 14.9% of the Ser residues are located adjacent to the crystalline region of the PLA chain as shown in Table 3. Thus, it is expected that the conformational transition of these Ser residues occurs in conjunction with the α-helix−βsheet transition of the PLA chain.

Figure 8. Expanded Ser Cβ peaks of 13C CP/MAS NMR spectra of [3-13C]Ser-, [3-13C]Tyr-, and [3-13C]Ala-labeled S. c. ricini silk fibroin stretched in different ratios and 13C-labeled natural silk fibroin fiber with the deconvolution spectra.

Table 3. Fractions of Ala (Excluding Ones in PLA), Ser, and Tyr Residues Located from the Nearest Ends of Poly(Lalanine) Sequences distance

Ala

Ser

Tyr

1 2 3 4 5 6 7 8 9 10 11 ≥12

0 2.3 67.7 2.3 0 2.3 2.3 0 0 0 13.1 10

14.9 11.9 9.8 20.1 19.1 2.1 4.6 0 0.5 7.7 1 8.2

0 0 0 0 9.5 43.5 25.6 1.2 1.8 14.9 0 3.6

occurred during the SF sample extraction from the silk gland or during sample drying.29 In general, the Ser residues in Gly-rich region of S. c. ricini SF are considered to change the conformation from random coil to β-sheet when the conformational transition of α-helix to β-sheet in the PLA region occurred. This was indeed observed previously in the conformational change of B. mori SF.29 However, there are no β-sheet structure of the PLA chain in the SF sample before stretching as mentioned in the previous section. Moreover, αhelix conformation of the PLA chain in S. c. ricini SF seems stable, and the conformational transition from α-helix to βsheet should not have occurred by relatively weak external forces during sample extraction from the silk gland or sample drying. Yet, the Ser residues partly change the conformation from random coil to β-sheet as shown in Figure 8. Thus, the Ser residues which contribute to the conformational transition must be located at positions apart from the PLA chain, such as in the sequence Gly-Ser-Gly in Figure 1. As summarized in Figure 6b, the increase of β-sheet and the decrease of random coil is observed at stretching ratio ×5, where the conformational change from α-helix to β-sheet occurs in the PLA region. There are many Ser residues close to PLA region along the chain; 75.8% of Ser residues are present within five amino acid residues from both ends of the PLA sequence (Table 3). These Ser residues may undergo conformational change from random coil to β-sheet by following the conformational

Figure 8 shows the expanded Ser Cβ peaks of SF samples with different stretching ratios together with peak deconvolutions. The fractions of two conformations, random coil and βsheet, determined by the deconvolution were plotted as a function of the stretching ratio (Figure 6b). The half-height width of the β-sheet peak is almost constant and independent of the stretching ratio, although the half-height widths of the peak for random coil increases slightly with increasing stretching ratio as shown in Figure 7b. The interesting point is the presence of β-sheet (14%) even in the case of dried SF without stretching. Because there are no β-sheet in the 13C solution NMR spectrum of SF stored in living S. c. ricini silkworm,14−19 the random coil to β-sheet transition must have F

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Macromolecules transition of the PLA chain from α-helix to β-sheet. The Ser residues in β-sheet structure may contribute to strengthening the SF chain through intermolecular hydrogen bond formation with side chain OH groups, just like B. mori SF.41,57−60 Thus, the conformation and conformational transition of Ser residues in S. c. ricini SF are different depending on the location of serine along the chain. Tyr Residue. Figure 9 shows the expanded Cβ peaks of Tyr residues in Gly-rich region of SF samples. Like Ser residues,

residues seem to contribute to the solubility of S. c. ricini SF in water due to the large bulkiness of the aromatic ring.41,61,62

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DISCUSSION The structural analysis of solid state NMR of SF coupled with selective 13C labeling of the SF sample and the 13C conformation-dependent NMR chemical shifts is very effective and gives important information on the future design of manmade artificial silk fiber with specific physical properties.41,63−67 In our previous papers,28,29 the conformational transition of SF prepared from B. mori silk gland induced by stretching was monitored by 13C CP/MAS NMR, which allowed us to correlate the conformations to specific amino acid sequence motifs using the same analytical NMR technique.28,29,31,32,41 In both cases, three amino acids (Ser, Tyr, and Ala Cβ carbons) were 13C labeled. The difference between B. mori and S. c. ricini SF’s is the domain where the Ser residues are located. The Ser residues are predominantly located in the crystalline domain of the repeated Ala-Gly-Ser-Gly-Ala-Gly sequences in B. mori SF33,34 and in the Gly-rich region in S. c. ricini SF. It is known that Ser residues contribute to the strengthening of the Silk I* form (type II β-turn structure) of B. mori SF.57,58,68−70 This is due to the intramolecular hydrogen bond formation between the side chain OH group and backbone carbonyl groups of repeated Ala-Gly-Ser-Gly-Ala-Gly sequences.57−59 Moreover, in the B. mori SF fiber, Ser residues also contribute to the strengthening the fiber due to the intermolecular hydrogen bonding. Thus, Ser residues contribute to strengthen the structure of B. mori SF in both forms of Silk I* before spinning and Silk II after spinning. A different situation was found for the S. c. ricini SF without stretching where 14% of β-sheet was observed although there are no β-sheets detected in the Ala Cβ peak. The β-sheet observed in the Ser peak has to be produced during the sample extraction from the silk gland or sample drying.29 The α-helix conformation of the PLA chain in S. c. ricini SF seems stable, and therefore the conformational transition from α-helix to βsheet does not occur by relatively weak external forces in the sample extraction from the silk gland or drying, but the Ser residues located away from PLA sequences may form β-sheet through such weak external forces. Thus, the Ser residues close to PLA chain tend to change their conformation from random coil to β-sheet by following the conformational transition of the PLA chain from α-helix to β-sheet with increasing stretching ratio. In contrast, the conformational transition from random coil to β-sheet for Tyr residues with increasing stretching ratio occurs gradually as shown in Figure 6c. This change is independent of the conformational transition of α-helix to βsheet in PLA chain, that is, the remarkable increase of β-sheet at stretching ratio ×5 (Figure 6a). The fraction of β-sheet reaches to about 30% in S. c. ricini SF. Such a gradual conformational transition from random coil to β-sheet of Tyr residues was also observed in B. mori SF.29 One of the roles of Tyr residue in B. mori SF is a significant contribution to the solubility in water by destroying the β-sheet structure with alternate sequences of Ala and Gly residues. As reported previously,61,62 we synthesized a peptide (Gly-Ala)2-Gly-Tyr-Gly-(Ala-Gly)2 where the Tyr residue was present in the center of the sequence. Surprisingly, the peptide was easily soluble in water although the peptides (Gly-Ala)2-Gly-Ser-Gly-(Ala-Gly)2 or (Gly-Ala)5-Gly were insoluble in water. Thus, the presence of the repeated sequences ((Gly-Ala)2-Gly-Tyr)n or (Gly-Ala-Gly-Tyr-Gly-

Figure 9. Expanded Tyr Cβ peaks of 13C CP/MAS NMR spectra of [3-13C]Ser-, [3-13C]Tyr-, and [3-13C]Ala-labeled S. c. ricini silk fibroin stretched in different ratios and 13C-labeled natural silk fibroin fiber with the deconvolution spectra.

there are two peaks, random coil and β-sheet. The fractions of these two conformations determined by the deconvolution analysis are shown as a function of the stretching ratio in Figure 6c. In contrast to Ser residues, the Tyr residues take on mostly random coil conformations (Table 1) for unstretched SF samples, although a small amount of β-sheet (3%) is present. At the stretching ratio, ×3, 10% β-sheet was clearly observed. The Tyr residues are located mostly at the center of Gly-rich sequences; 90.5% of Tyr residues are located at more than six amino acid residues away from both ends of the PLA sequence (Table 3).9 Thus, the conformational transitions from random coil to β-sheet occur gradually and independent of the conformational transition of PLA chain. The extent of the conformational transition is relatively small compared with those of the Ser and Ala residues but still occurs. The Tyr G

DOI: 10.1021/acs.macromol.7b00454 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules Ala)n adjacent to the repeated sequences ((Gly-Ala)2-Gly-Ser)n with β-sheet structure33,34 destroys the β-sheet structure effectively and cause increase in the solubility of B. mori SF. On the other hand, in the S. c. ricini SF, the Tyr residues are situated as (Gly)2-Tyr-(Gly)2 (28%), Ser-Gly-Tyr-(Gly)2 (22%), and His-Gly-Tyr-Gly-Ser(16%) sequencesall Glyrich regionsapart from the PLA chain (Table 3), and therefore the contribution of the Tyr residues to the fiber’s solubility in water seems to be lower compared with B. mori SF. The dynamics of Tyr residue in natural S. c. ricini SF fiber was already studied using 2H solid state NMR coupled with 2H labeling of the sample, i.e., [3,3-2H2]Tyr- or the aromatic ring carbons, [3′,5′-2H2]Tyr-labeled SF.71 The 2H NMR spectra of the [3,3-2H2]Tyr-SF showed typical rigid powder patterns, indicating that there is essentially no motion about the Cα−Cβ bond axis independent of the backbone conformation of the Tyr residues. In contrast, the 2H solid state NMR line-shape analysis of [3′,5′-2H2]Tyr-SF indicated that 60% of the rings are engaged in fast motional averaging (107 Hz), while 40% undergo slow motion (104 Hz). Thus, the former Tyr residues with relatively higher motion of the aromatic ring are present in a loosely packed state, and the 60% fraction is almost the same as the fraction of random coil (67%) in the natural SF fiber. Most of Ala residues exist as α-helix in the S. c. ricini SF before spinning and β-sheet in the SF after spinning. The latter β-sheet structure is the origin of the strength of the SF fiber due to crystallinity. The α-helix form imparts good solubility to SF in water because the solidified SF sample with α-helix is still soluble in water if the sample is fresh. The Ala Cβ NMR signal consists of three peaks in the natural SF fiber spectrum. A further solid state NMR experiment including the 13C chemical shift calculation and spin−lattice relaxation times and T1 measurement13,24,60,72 is now in progress in our laboratory.

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AUTHOR INFORMATION

Corresponding Author

*(T.A.) Tel & Fax 81-42-383-7733; e-mail [email protected]. jp. ORCID

Tetsuo Asakura: 0000-0003-4472-6105 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS T.A. acknowledges support by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Supports of Japan (26248050) and Impulsing Paradigm Change through Disruptive Technologies Program (ImPACT). We also thank Dr. H. N. Cheng (Southern Regional Research Center, USDA Agricultural Research Service, New Orleans, LA 70124) for discussions.

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REFERENCES

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CONCLUSION The conformational transition of [3-13C]Ser-, [3-13C]Tyr-, and [3-13C]Ala-labeled Samia cynthia ricini silk fibroin induced by stretching was studied using 13C CP/MAS NMR. The analysis of the crystalline domain was performed on the Ala Cβ peaks, and the analysis of Gly-rich region on the Tyr [3-13C]Cβ and Ser [3- 13 C]Cβ peaks. In the crystalline domain, the conformation was mainly α-helix, and no β-sheet structure was found in the dried silk fibroin without stretching. At the stretching ratio ×5, three peaks of β-sheet structures were initially observed and the fractions of these structures increased with rapid further stretching. The transition from α-helix to βsheet in the crystalline domain was clearly observed. In contrast, 20% β-sheet was observed for the silk without stretching, and only random coil conformation was found for Ser residues. The transition from random coil to β-sheet for the Tyr residues in the Gly-rich region occurs gradually and independent of the transition of the crystalline domain. These results will be helpful to the future designs of new silk molecules with targeted physical properties.

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of the 13C CP/MAS NMR spectra of [3-13C]Ser-, [3-13C]Tyr-, and [3-13C]Ala-labeled S. c. ricini silk fibroin plotted against the stretching ratio and 13C-labeled natural silk fibroin fiber (PDF)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.7b00454. Table S-1: half-height widths of several conformations in the deconvolution of Ala Cβ, Ser Cβ, and Tyr Cβ peaks H

DOI: 10.1021/acs.macromol.7b00454 Macromolecules XXXX, XXX, XXX−XXX

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