Refined Crystal Structure of Samia cynthia ricini Silk Fibroin Revealed

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A Refined Crystal Structure of Samia cynthia ricini Silk Fibroin Revealed by Solid-State NMR Investigations Tetsuo Asakura, Akio Nishimura, Shunsuke Kametani, Shuto Kawanishi, Akihiro Aoki, Furitsu Suzuki, Hironori Kaji, and Akira Naito Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 15 May 2017 Downloaded from http://pubs.acs.org on May 16, 2017

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A Refined Crystal Structure of Samia cynthia ricini Silk Fibroin Revealed by Solid-State NMR Investigations Tetsuo Asakura,* †Akio Nishimura, † Shunsuke Kametani, † Shuto Kawanishi †Akihiro Aoki, †Furitsu Suzuki,§ Hironori Kaji§ and Akira Naito †

†

Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588 JAPAN

§

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, JAPAN

*Correspondence to: Tetsuo Asakura

Tel & FAX: +84-42-383-7733

Email: [email protected]

Key words: Silk Fibroin / Samia cynthia ricini / Solid state NMR/NMR chemical shift calculation / Poly-L-alanine

Abstract

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Samia cynthia ricini is one of the wild silkworms and its silk fibroin (SF) consists of alternatively repeating poly-L-alanine (PLA) sequences as crystalline domain and glycine-rich sequences as non-crystalline domain; the structure is similar to those of spider silk and other wild silkworm silks. In this paper, we proposed a new staggered model for the packing arrangement of the PLA sequence through the use of the Cambridge Serial Total Energy Package program and a comparison of the observed and calculated chemical shifts of the PLA sequence with the Gauge Including Projector Augmented Wave method. The new model was supported by the inter-atomic distance information from the cross peaks of Ala Cβ dipolar-assisted rotational resonance (DARR) spectrum of the PLA sequences in S. c. ricini SF fiber. In addition, three

13

C

NMR peaks observed in the β-sheet region were assigned to the carbons with different environments in the same model, but not assigned to different β-sheet structures.


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Introduction There are a variety of silkworms and spiders, each producing silk with a unique primary and higher order structures.1,2 Their excellent properties such as high strength and high toughness have attracted researchers in diverse fields, such as biology, biochemistry, biophysics, analytical chemistry, polymer technology, textile technology and biomaterials.3-9 In order to optimize the information content of multidisciplinary approaches, it is useful to study the structure-property relationships in silks, particularly with respect to their primary and higher order structures. The most known silk, Bombyx mori (B. mori) silk ﬁbroin (SF) has been much studied, and ample knowledge has been gathered on its structure and dynamics, including structure-property relationships.4,5,9-11 The amino-acid composition of B. mori SF is known and comprises 42.9% Gly, 30.0% Ala, 12.2% Ser, 4.8% Tyr, and 2.5% Val.12,13 Its primary structure consists largely of a repeating sequence of six residues (GAGAGS)n which forms the crystal domains of the SF. The conformation of these sequences is mainly anti-parallel β-sheet structure, which was elucidated by X-ray diffraction analysis,14,15 infrared spectroscopy (IR),16-18 Raman17,19 and solid-state nuclear magnetic resonance (NMR).20-23 However, X-ray diffraction analysis gives only limited structural information because SF exists in the fiber form, not as a single crystal, and the packing structure is heterogeneous and not easy to study. IR and Raman experiments are also 3 ACS Paragon Plus Environment

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difficult to yield the packing structure. Instead, the combination of several solid-state NMR techniques can give detailed information on the packing structure of such heterogeneous SF samples. For example, the previous packing model of B. mori SF fiber after spinning (Silk II) initially proposed by Marsh and Pauling et al.14on the basis of X-ray diffraction analysis has been revised through the use of several solid state NMR techniques.23 In addition, a precise packing model of poly (Ala-Gly) as a model peptide for B. mori SF proposed by Takahashi et al.15 using X-ray diffraction data has also revised by us using solid-state NMR and conformational energy calculations.23 Recently much attention has been paid to the wild silkworm SF and spider silk as new biomaterials.24-26 Thus, in this paper, we aimed to determine the packing structure of the SF fiber from a wild silkworm, Samia cynthia ricini (S.c.ricini). The S. c. ricini SF typically contains polyalanine sequence Ala12-13 (PLA), embedded in a Gly-rich amorphous matrix, and the amino acid composition includes Ala (45.4%), Gly (31.7%), Ser (6.7%) and Tyr(5.8%).27 The primary structure is close to those of SF’s from other wild silkworms such as Antheraea pernyi (A. pernyi),

28

Antheraea yamamai (A.

yamamai) 29and Antheraea mylitta (A. mylitta),30 and also the major ampullate silk from the spider Nephila clavipes.31-34 The determination of the packing structure of the crystalline PLA domain can give packing information that are common for these silks. The PLA sequences in S. c. ricini SF fiber are considered to have anti-parallel β-sheet 4 ACS Paragon Plus Environment

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structure, as revealed by X-ray diffraction35,36 and solid-state NMR

36-38

, and more

knowledge of this structure is useful for determining the backbone torsion angles of the SF fiber. In our previous paper,39 we did a systematic structural analysis of alanine oligomers with anti-parallel β-sheet structure as a model for the crystalline region of spider dragline silk and S. c. ricini SF, by using solid-state NMR spectroscopy and X-ray crystallography. The alanine oligomers pack into two different arrangements, depending on the lengths of the sequences. Short sequences (n = 6 or less) pack into a rectangular arrangement. Longer sequences pack in a staggered arrangement. Thus, the packing arrangement of PLA sequences in S. c. ricini SF seems to be staggered. In this paper we report the atomic co-ordinates of the packing structure of PLA sequences in S. c. ricini SF using solid-state NMR techniques, such as dipolar-assisted rotational resonance (DARR),40-47 1H-detection in the double CP 1H-13C correlation48 and 1H and

13

C NMR

chemical shift calculation. In particular, the chemical shift calculation methods [e.g., the geometry optimization under periodic boundary conditions using the Cambridge Serial Total Energy Package (CASTEP) program49 and the Gauge Including Projector Augmented Wave (GIPAW) method50] have been successfully used to determine the packing structures of B. mori SF before and after spinning23,51 and also applied to the determination of the packing structures of (Ala)n (n = 3,4).52,53 In order to obtain the 5 ACS Paragon Plus Environment

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observed 13C and 1H chemical shifts of S. c. ricini SF, 13C DARR and 1H-detection in the double CP 1H-13C correlation measurements have been performed for [U-13C] S. c. ricini SF and their crystalline fraction.54 Experimental Section Preparation of Alanine oligomers 13

C selectively labeled 34-mer peptide, GGAGGGYGGDGG(A)6[3-13C] A19(A)5GG-

AGDGYGAG as a typical tandem sequence of S. c. ricini SF was synthesized with Fmoc-Ala-PEG-PS resin (PE Biosystems) on a PioneerTM Peptide Synthesizer using Fmoc chemistry in our laboratory.55 After synthesis, the samples were dissolved in 9 M LiBr and then dialyzed against water for 4 days. The precipitate was obtained and dried. After this treatment, the peptide powder with β-sheet structure was obtained. The [3-13C] Ala (99%

13

C enrichment) was purchased from Cambridge Isotopes Laboratories,

Andover, MA, USA. Preparation of [U-13C] S. c. ricini SF fiber and the crystalline fraction 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. [U-13C] S. c. ricini SF fiber was prepared as follows. Artificial diet with a mixture of [U-13C] glucose (99%, Cambridge Isotopes Laboratories) was fed to the 5th instar larvae from 3 to 6 days old for 4 days twice per 6 ACS Paragon Plus Environment

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day (morning and evening). A total of approximately 600 mg of [U-13C] glucose was fed per silkworm. The cocoons obtained from these S. c. ricini silkworms were degummed three times with 0.1% (w/w) sodium peroxide (Na2O2) solution at 100˚C for 30 min and washed with distilled water in order to remove silk sericin, another silk protein, from the surface of the silk fibers.36 The SF fibers were dried at room temperature prior to

13

C

DARR NMR measurements. The crystalline fraction of [U-13C] S. c. ricini SF was prepared as before.54 Hydrochloric acid solution (20ml, 5 mol/l) was added to 200 mg of the degummed [U-13C] SF and kept for 5 h under 80°C. Then the reaction was stopped by adding aqueous sodium hydroxide. The solution was centrifuged at 8,500 rpm for 30 min at 4°C. Distilled water was added to the precipitate and then centrifuged again under the same condition. This treatment was repeated three times and then the precipitate was freeze-dried. The crystalline fraction of [U-13C] S. c. ricini SF was obtained as a powder form of the precipitate. The non-labeled S. c. ricini SF fiber was also used for

13

C

CP/MAS NMR observation. 13

C CP/MAS NMR and 13C DARR measurements.

The

13

C CP/MAS NMR spectra of S. c. ricini SF and the 34-mer peptide,

GGAGGGYGGDGG(A)6[3-13C] A19(A)5GGAGDGYGAG were recorded at room temperature on a Varian Unity Infinity 400 MHz spectrometer with a

13

C operating

frequency of 100.6 MHz. Samples were spun at a MAS frequency of 10 kHz. The 7 ACS Paragon Plus Environment

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number of acquisitions was 8,000, and the recycle delay was 5 s. Radio-frequency (rf) field strength at 50 kHz was used for 1H-decoupling during the acquisition period of 12.8 ms. A 90° pulse width of 5 µs and CP contact pulse of 1 ms were also employed. Phase cycling was used to minimize spectral artifacts. The chemical shifts were referenced to TMS, using adamantane as a secondary standard (13CH peak at 28.8ppm). The

13

DARR spectrum of [U-13C] S.c.ricini SF fiber was obtained after 32 scans at a

13

C C

resonance frequency of 99.5 MHz, using a JEOL ECX400 spectrometer at a spinning speed of 8 kHz with a 4 mm OD rotor.47 The π/2 pulse was 3.8 µs for 13C, and 3.4 µs for 1

H. TPPM 1H decoupling was performed with a contact time of 2 ms. The mixing time

was changed every 100 ms from 100 ms to 500 ms, combined with a relaxation delay of 2 s. In addition, the RF field was set at 8 kHz. The indirect dimension consisted of 256 data points. 1

H-detection in the double CP 1H-13C correlation measurements

The crystalline fraction of [U-13C] S. c. ricini SF was used for 1H-detection in the double CP 1H-13C correlation measurements. This experiment was performed at a 1H resonance frequency of 920 MHz, using a JEOL JNM-ECA920 spectrometer equipped with a 1H-X double resonance and ultra-high speed MAS probe at the Institute for Molecular Science in Okazaki, Japan.23,51-53 The sample spinning speed was actively stabilized by a pneumatic solenoid valve such that the spinning fluctuations were less 8 ACS Paragon Plus Environment

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than ±10 Hz at a spinning rate of 70 kHz. The temperature of the samples increases due to friction under fast MAS was estimated to be around 333 K at 70 kHz MAS according to Pb(NO3)2 temperature calibration.56 The 1H rf field strength for the excitation π/2 pulse (1.29 µs) was 194 kHz. The 1H chemical shift was referenced to the peak of silicon rubber and set to 0.12 ppm from TMS. For 1H-detection in the double CP 1H-13C correlation measurements, the pulse sequence 90Hy-CPx- t1C-90Cφ-τd-90Cy-CPx-t2H was used.39 Here, 90° is a π/2 pulse, CP is a 4-ms cross-polarization period with a 10% (first) and -10% (second) ramp of dephasing of transverse

13

C, t1 is the evolution period, τd is a 5-ms period for

13

C magnetization and 1H magnetization suppression, and t2 is

the detection period. Superscripts H and C indicate 1H and 13C, and subscripts x, y, and φ indicate rf phases, with φ = x and y for quadrature detection in t1. The 1H decoupling amplitude during t1C was 27 kHz. The spectrum was obtained after 64 scans at each period in the y domain with 512 points. 13

C and 1H NMR chemical shift calculations of the packing structure of PLA

sequence in S. c. ricini SF fiber The

characteristic

torsion

angles

of

(φ,ϕ)

=

(-138.6°,

134.7°)

for

an

anti-parallel β-sheet structure were used for Ala residues in PLA chains.35,37,38 The initial packing model of PLA was prepared using the cell dimensions reported by Arnott et al.35; a = 6.890 Å, b =10.535 Å, c = 9.468 Å and α = 90.0, β = 90.0, γ = 90.0. 9 ACS Paragon Plus Environment

The

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packing model was energy-minimized using the pcff force field of Discover software (Dassault Systems Biovia Corp., San Diego, CA, USA). Then, the geometry optimization was performed under periodic boundary conditions using CASTEP program (Dassault Systems Biovia Corp., San Diego, CA, USA).49 We used the generalized gradient approximation (GGA) for the exchange correlation energy based on the Perdew, Bruke and Ernzerhof (PBE) functional and ultrasoft pseudopotentials with a plane-wave energy cutoff of 380 eV. A 4 × 2 × 3 Monkhorst- Pack k-point grid was used for Brillouin zone sampling. The

13

C and 1H chemical shifts were then calculated using the GIPAW

method.50 The PBE approximation and “on the fly” pseudo potentials were used. The energy cutoff of the plane wave was set to 610 eV and a 4 ×2 × 3 Monkhorst-Pack k-point grid was used as described above. All calculations were carried out using the NMR-CASTEP program. The calculated

13

C and 1H chemical shifts of the PLA model

with anti-parallel β-sheet structure were found to be consistent with the observed Ala Cβ carbon and Ala Hβ proton chemical shifts at the highest field of the β-sheet peaks respectively without changing the relative chemical shift difference among all peaks. Thus, the references of the calculated smallest chemical shift of Ala Cβ carbon will be 19.7 ppm and 1.00 ppm for Ala 1Hβ proton. Results


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1. Expanded Ala Cβ β peaks in the 13C CP/MAS NMR spectra of (a) S. c. ricini SF fiber

and

(b)

13

C

selectively

labeled

34-mer

peptide,

GGAGGGYGGDGG(A)6[3-13C]A19 (A)5GGAGDGYGAG In our previous papers,20-23,36,57 we reported that the packing effect was clearly observed for Ala Cβ peaks in the 13C CP/MAS NMR spectra of B. mori and S. c. ricini SF fibers.

Fig. 1 shows expanded Ala Cβ peaks of the 13C CP/MAS NMR spectra of (a)

non-labeled S. c. ricini SF fiber and (b)13C single-labeled [3-13C] A19- 34-mer peptide, GGAGGGYGGDGG(A)6[3-13C] A19(A)5GGAGDGYGAG as the model for a typical tandem sequence of S. c. ricini SF. The broad peak observed at 16.6 ppm was assigned to random coil conformation, and other lower field peaks from 19.7 to 22.7 ppm were assigned to β-sheet structure.36,39,57 The peak pattern looked essentially the same between the spectra (a) and (b) although the latter peak was slightly sharper. The

13

C

single-labeled 34-mer peptides with the same molecular structure but with different 13C labeling sites were also synthesized and the 13C CP/MAS NMR spectra observed.58 The Ala Cβ peak patterns of the

13

C CP/MAS NMR spectra were essentially the same as

reported previously. This was an important starting point in the NMR structural analysis of the SF fiber. We must first judge whether the multiplet observed in the lower field Ala Cβ peak came from (a) inter-molecular packing effect of the PLA sequences or (b) intra-molecular heterogenous distribution of the local conformations of the PLA 11 ACS Paragon Plus Environment

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sequences along one SF chain. The essentially same pattern between them meant that the multiplet clearly came from the packing effect. It is noted that a particular residue might exist in multiple packing sites in the entire system. The chemical shifts of the highest field peak A and the lowest field peak C in the β-sheet region were easily determined to be 19.7 ppm and 22.7 ppm, respectively. However, the central peak B was slightly broader than the other peaks.

Fig. 1 The expanded Ala Cβ regions in 13C CP/MAS NMR spectra of non-labeled Samia cynthia

ricini

silk

fibroin

fiber

(a)

and

GGAGGGYGGDGG(A)6[3-13C]

A19(A)5GGAGDGYGAG (b).

2. Determination of Ala Cα α and C=O chemical shifts of [U-13C] S. c. ricini SF fiber Although the Ala Cα and Ala C=O peaks looked like a single peak (Fig. 2), it was necessary to examine the chemical shift difference for each peak which corresponded to the multiplet in the β-sheet region of the Ala Cβ peak. Fig.2 shows the

13

C DARR

spectrum of [U-13C] S. c. ricini SF fiber. However, there were no splits in the Ala Cα and 12 ACS Paragon Plus Environment

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Ala C=O peaks. Thus, the packing effect of the SF chains could be observed exclusively in the Ala Cβ peak. The Ala Cβ carbons are located outside of β-sheet planes of SF molecule and therefore only these Cβ carbons seem to be sensitive to the arrangement of the adjacent SF molecules, namely, the packing effect in the solid state. The observed 13C chemical shifts of PLA carbons of S. c. ricini SF fiber are summarized in Table 1 and compared with the calculated chemical shifts.

Fig. 2 13C DARR spectrum of [U-13C] Samia cynthia ricini silk fibroin fiber.

The correlations between Ala Cβ and Ala C=O carbons, and between Ala Cβ and Ala Cα carbons are also shown as the inserted Figs.

Table 1. Observed chemical shifts of Samia cynthia ricini silk fibroin fiber (13C) and their crystalline fraction (1H). The calculated

13

C and 1H chemical shifts of the PLA

model with anti-parallel β-sheet structure are also listed and consistent with the observed


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Ala Cβ carbon and Ala Hβ proton chemical shifts at the highest field of the β-sheet peaks without changing the relative chemical shift difference among all peaks. Thus, the calculated smallest chemical shift will be 19.7 ppm for Ala Cβ carbon and 1.0 ppm for Ala 1Hβ proton.

13

C

S. c. ricini fiber

Calc.

1

H

S. c. ricini crystal

Calc.

Ala

Ala

C=O

Cα

175.5

50.0

Ala Cβ

r.c.

16.6

A

19.7

B

21.2

C

22.9

176.6

52.6

1 ○

19.7

177.4

50.4

2 ○

20.7

177.2

51.1

3 ○

21.6

178.9

50.5

4 ○

24.4

Ala

Ala

HN

Hα

8.95

5.00

Ala Hβ

A

1.00

B

1.20

C

1.40

10.48

5.62

1 ○

1.01

10.14

5.93

2 ○

0.99


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10.80

5.88

3 ○

1.53

10.82

5.82

4 ○

1.40

3. Determination of Ala Hβ β, Hα α and NH chemical shifts of the crystalline fraction of [U-13C] S. c. ricini SF The resolution of one-dimensional 1H solid state NMR was generally poor, but the use of higher field magnet like 920 MHz equipped with ultra-high speed MAS probe at 70 kHz could attain remarkably well-resolved1H solid-state NMR spectrum.10,23,51-53 The latter ultra-high speed MAS probe for solid-state NMR that needs a small amount of sample was developed by us and the details were already reported elsewhere.52,59-61 The 1

H nuclei are located outside the SF molecules and therefore expected to be more

sensitive to the packing effect. In order to ensure higher resolution 1H solid-state NMR spectrum, we prepared the crystalline fraction of [U-13C] S. c. ricini SF with anti-parallel β-sheet structure.54 In addition, the powder form was suitable for filling the sample into the high speed MAS probe with a 1.5-mm outer diameter. Fig.3 shows 1H-detection in the double CP 1H-13C correlation NMR spectrum of the crystalline fraction. The correlations between Ala Cβ β−sheet Α peak and the top of the Ala Hβ peak and also between Ala Cβ β−sheet C peak and the Ala Hβ C peak were detected in the 2D spectrum, although the Ala Hβ C peak apparently did not show up. A clear correlation could not be observed between Ala Cβ β−sheet B peak and the Ala Hβ B peak in the 2D


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spectrum. There were no such correlations between Ala Cβ (Α and C) and Ala Hα peaks and also between Ala Cβ and Ala NH peaks. These results indicate that chemical shift difference between Ala Cα (A and C) is quite small. The observed 1H chemical shifts are summarized in Table 1 and compared with the calculated chemical shifts.

Fig.3 1H-detection in the double CP 1H-13C correlation spectrum of the crystalline fraction of Samia cynthia ricini silk fibroin.

4. Preparation of new packing structure The original Arnott model35 for PLA molecule is shown in Fig.4(a). It consists of interleaved anti-parallel β-sheets with neighboring sheets randomly displaced ±1/2 a (a being the interchain distance). Optimum values of the packing and conformational


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parameters for the statistical structure were determined by the linked-atom least-squares method for polymer crystalline fibers.62 In addition, the model claimed that there was essentially no significant difference between the unit cell dimensions of the PLA molecules obtained by Arnott and those of the crystalline region, i.e., the PLA sequences of tussah SF fiber. (Tussah silk is produced from larvae of several species of silkworms belonging to the moth genus Antheraea, including A. mylitta, A. pernyi and A. yamamai.) Thus, the primary structure of tussah SF consisted of polyalanine sequence of Ala12-13 (PLA), embedded in a Gly-rich amorphous matrix, which was similar to that of S. c. ricini SF.27-30

Earlier, we have already reported that the

13

C CP/MAS NMR spectra

were very similar between S. c. ricini SF and A. pernyi SF.63 Thus, the structural model of PLA molecules proposed by Arnott et al.

35

seemed to be suitable as an initial model

for the structural analysis of the crystal structure of S. c. ricini SF fiber. The energy optimization of the original Arnott model was done by CASTEP calculation, and a new staggered model was obtained as shown in Fig.4(b). The packing structures looked slightly different between two structural models.


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(a)

(b)

Fig.4 (a) Arnott model and (b) new staggered model. Details are described in the text. 1 to ○ 4 , and also The Ala Cβ carbons with different environments were noted as from ○

1 * to ○ 4 * in Fig.4(b) used in the peak assignments. The inter-molecular direct from ○

hydrogen bonding pairs of NH…O=C bonds were noted as from I to IV, and also from I* to IV* in Fig.4(b).

In order to discuss the difference of two models more quantitatively, the geometric parameters of the inter-molecular direct hydrogen bonding between the NH group in one 18 ACS Paragon Plus Environment

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molecule and the C=O group in another molecule were summarized in Table 2. Here the numbers I-IV and I’-IV’ correspond to the inter-molecular direct hydrogen bonding pairs of NH…O=C bonds in Fig.4(b). For Arnott model, the averaged distances of the NH…O=C bonds were 1.88 Å and the angles of N-H…O were 163°, while the corresponding averaged distances were 1.81 Å and the angles of N-H…O were 165° for the new staggered model. Thus, the hydrogen bonding networks of the latter model are more packed and the structure is expected to be more stable.57 From the atomic distance of the NH…O=C bond, it is possible to predict the chemical shift of NH protons.52 The predicted values are also listed in Table 2. Table 2. The distances (Å) of the inter-molecular direct hydrogen bonding between NH group in one molecule and C=O group in another molecule together with the related angles (degree) for the hydrogen bonding of both packing structures, i.e., Arnott model in Fig.4(a) and new staggered model in Fig.4(b). Predicted chemical shifts of NH protons (δNH) were evaluated from the hydrogen bond distances of NH…O=C. Arnott model NH-O

New staggered model

distance / Å

angle / deg

δNH / ppm

distance / Å

angle / deg

δNH / ppm

I

1.885

156.7

8.8

1.853

161.0

8.9

II

1.859

168.1

8.9

1.768

168.3

9.4

III

1.887

158.7

8.8

1.808

162.9

9.2

IV

1.905

168.7

8.7

1.817

166.3

9.1

III'

1.887

158.7

8.8

1.809

162.9

9.2

IV'

1.905

168.7

8.7

1.817

166.2

9.1

I'

1.885

156.7

8.8

1.859

160.9

8.9


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II'

1.859

168.1

8.9

1.770

168.2

9.4

Average

1.884

163.1

8.8

1.813

164.6

9.1

5. 13C and 1H NMR chemical shift calculations The

13

C and 1H chemical shifts were calculated using GIPAW method for the new

staggered model obtained here. The calculated chemical shifts are listed in Table 1 1 to ○ 4 for different Ala together with the observed chemical shifts. Here the numbers ○

Cβ carbon correspond to the numbers in Figs. 4(b). The observed and calculated chemical shifts are also shown in Fig. 5 as stick spectra. In order to emphasize the assignments of the Ala Cβ and Ala Hβ peaks which are sensitive to the packing effect, the calculated smallest chemical shifts of Ala Cβ carbon and Hβ proton have been adjusted to be consistent with the observed highest field peaks in the β-sheet regions. These are 19.7 ppm for the 13C peaks and 1.00 ppm for the 1H peaks, respectively. Thus, the apparent difference between the calculated and observed chemical shift tends to be larger for Ala C=O and Ala NH peaks.


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Fig.5 Observed chemical shifts of Samia cynthia ricini silk fibroin fiber (13C) and their crystalline fraction(1H) together with the assignments. The calculated

13

C and 1H

chemical shifts of the PLA model were also shown which have been adjusted to be consistent with the respective observed Ala Cβ carbon and Ala Hβ proton chemical shifts at the highest field of the β-sheet peaks without changing the relative chemical shift difference among all peaks. Thus, the calculated smallest chemical shift will be 19.7 ppm


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for Ala Cβ carbon and 1.0 ppm for Ala 1Hβ proton. Details are described in the text. The chemical shifts are shown as stick spectra.

1 to ○ 4 , correspond to four carbons The four calculated Ala Cβ chemical shifts, ○

with different environments in the new staggered model shown in the Fig.4 (b). The four 1 * to ○ 4 *, are also shown in Fig.4(b), but the environments are essentially the carbons, ○

1 to ○ 4 , respectively. There are three observed peaks, from peak A to same as those of ○

peak C. The central peak, B, located between peaks A and C seems to be broad. Thus, we 1 and the lowest field peak C to carbon ○ 4 assigned the highest field peak A to carbon ○

2 to peak A . In view of the observed highest intensity of peak A, we assigned carbon ○

3 was assigned to peak B. As for Ala Cα and C=O too. Thus, the remaining carbon ○

1 -○ 4 were also slightly different. carbons, the calculated chemical shifts of the carbons ○

However, the calculated maximum difference was 2.2 ppm for both Ala Cα and Ala C=O carbons, which were less than half of the corresponding chemical shift variation of Ala Cβ carbon at 4.7 ppm (Table 1). Therefore, it seemed that the chemical shift distribution could not be observed in the 13C CP/MAS NMR spectra of Ala Cα and Ala C=O peaks, contrary to the case of Ala Cβ peak.

As shown in Fig.3, there are two

correlations between Ala Cβ β−sheet Α peak and the top of the Ala Hβ peak, and also between Ala Cβ β−sheet C peak and the Ala Hβ C peak in the observed double CP 1

H-13C correlation spectrum. From the comparison with the observed and calculated 22 ACS Paragon Plus Environment

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1 and ○ 2 in Fig.4(b) can be assigned to the Ala Hβ A chemical shifts, the protons ○

3 and ○ 4 are assigned to the lower field peak at 1.0 ppm, and other two protons ○

shoulder of the peak at 1.00 ppm, although the Ala Hα C peaks can be assigned to either one. The calculated peaks are slightly different among four carbons in both Ala Hα and Ala NH regions, but we have not tried to make further assignments. 6. Confirmation of the assignment of the peaks, A, B and C in Ala Cβ β region from the 13C DARR spectrum of S.c.ricini SF fiber Fig. 6(a) shows the expanded Ala Cβ region in the 13C-13C DARR spectrum of [U-13C] S. c. ricini SF fiber shown in Fig. 2. The correlation between the β-sheet peaks, A and C 1 and ○ 4 was clearly observed, indicating that the distance between the carbons ○

assigned to the peaks A and C were shorter than 5 Å under our NMR experimental condition

40,41,47

. In addition, the distance between the carbons assigned to the peaks A

and B was also shorter than 5 Å because the correlation between these two peaks was also observed. However, the correlation between the peaks B and C could not be observed. The inter-atomic distances between two Ala Cβ carbons in the new staggered model shown in Fig.4(b) were systematically calculated and listed in Table 3. The distances less 1 and ○ 1 * atoms, than 5 Å are marked in Table 3. The distances between the carbons ○

were less than 4 Å, but the correlation could not be observed because of the same 23 ACS Paragon Plus Environment

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1 and ○ 1 *. Similarly, the correlations between ○ 2 and chemical shifts of the carbons ○

2 * (and between ○ 3 and ○ 3 * and between ○ 4 and ○ 4 *) could not be observed. ○

1 (○ 1 *) Except for these, the distances less than 5 Å were observed between carbons ○

3 (○ 3 *) , and between the carbons ○ 2 (○ 2 *) and ○ 4 (○ 4 *). Judging from the and ○

calculated chemical shifts and peak intensities, we assigned the highest field peak, Ala 1 and ○ 2 . Thus, the observation of the correlation between Hβ β-sheet A, to carbons ○

Ala Hβ β-sheet A and Ala Hβ β-sheet C peaks indicates that the distance between 1 or ○ 2 and the carbon ○ 3 or ○ 4 is less than 5 Å. Similarly, the correlation carbons ○

observed between the Ala Hβ β-sheet A and Ala Hβ β-sheet C peaks indicates that the 1 or ○ 2 and the carbon ○ 3 or ○ 4 was less than 5 Å. Thus, distance between carbons ○

these observations support the assignment of the peaks, A, B and C, in the β-sheet region of the Ala Cβ peak.


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Fig. 6 (a) Expanded Ala Cβ region in 13C-13C DARR spectrum of [U-13C] Samia cynthia ricini silk fibroin fiber. The correlations between the Ala Cβ and Ala Cα carbons, and between the Ala Cβ and Ala C=O carbons are also shown in the figure. The distances 1 and ○ 2 ) and carbon ○ 4 , and also those less than 5 Å between the group (carbons ○

1 and ○ 2 ) and carbon ○ 3 in the new staged model are between the group (carbons ○

shown as a histogram in Fig. 6(b) used for interpretation of the observed correlations in Fig.6(a).

Table 3 The calculated distances (Å) between two Ala Cβ atoms together with the calculated chemical shifts of the marked carbons in Fig.4(b) in the new staggered model. 25 ACS Paragon Plus Environment

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chemical shift Ala Cβ

1 * ○

1 ○

2 * ○

2 ○

3 ○

3 * ○

4 ○

/ ppm 19.7

1 ○

19.8

1 * ○

3.94

20.7

2 * ○

5.98

7.17

20.7

2 ○

7.17

5.98

4.07

21.6

3 ○

4.22

4.45

5.15

5.62

21.6

3 * ○

4.44

4.23

5.62

5.15

3.68

24.4

4 ○

5.72

5.45

3.86

4.74

6.45

6.22

24.4

4 * ○

5.46

5.72

4.74

3.85

6.22

6.46

4.06

Discussion PLA sequences, (Ala)n (n= 12,13) with anti-parallel β-sheet structure are found in the crystalline fraction of S. c. ricini SF fiber.27 (Ala)n also occurs in the crystalline fraction of other wild silk worm silks

2,28-30

and the major ampullates of the silks from many

spiders7,31-34,42-46 although the length n is relatively shorter for the latter spider silks. Thus, the PLA sequences seem to be a key element in the structures of these silk fibers with high strength. In our previous paper,39 we determined the packing structures of a series of (Ala)n (n= 3,4,5,6,7,8,12) with anti-parallel β-sheet structure using 13C CP/MAS NMR and X-ray diffraction powder patterns. The (Ala)n peptides pack into two different arrangements, depending on the length of the sequence. Thus, short sequences (n=6 or 26 ACS Paragon Plus Environment

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less) pack into a rectangular arrangement, but longer sequences pack in a staggered arrangement. Both the line shapes of Ala Cβ carbons in the

13

C CP/MAS NMR spectra

and X-ray powder diffraction patterns show a similar pattern for the short, antiparallel oligopeptides (Ala)4, (Ala)5 and (Ala)6, indicating that these peptides have similar crystal structures to that of antiparallel (Ala)4 determined by us.39 using X-ray single crystal structural analysis (Supporting information Fig. S1). On the other hand, for (Ala)7, (Ala)8, (Ala)12 and S. c. ricini SF fiber with anti-parallel β-sheet structures, both the

13

C

CP/MAS NMR spectra and X-ray diffraction data are markedly different from those for short PLA sequences. Thus, if we can determine the latter packing arrangement, which is different from the former packing arrangement that is typical for (Ala)4, we can cover all of the packing structures of PLA sequences that appear in the crystalline domains of spider silks and wild silkworm silks. In this paper, the atomic co-ordinates of the packing structures of S. c. ricini SF fiber was reported. The line shapes of the Ala Cβ carbons were essentially the same between S. c. ricini SF and [3-13C] A19 34-mer peptide with anti-parallel β-sheet structure. This meant that the multiplet observed in the Ala Cβ peaks was due to short range packing effect along the PLA sequence of S. c. ricini SF. Indeed, this was also supported from the observation that the Ala Cβ spectral pattern was essentially the same among (Ala)n (n=7,8,12) and S. c. ricini silk fiber. In our previous paper,36 the structural transition from 27 ACS Paragon Plus Environment

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

α-helix (the structure before spinning) to β-sheet (the structure after spinning) of S. c. ricini SF film was monitored by stretching of the SF film using 13C CP/MAS NMR. The film was prepared from SF stored in the silk gland. The β-sheet peak was observed with increasing stretching ratio, starting at the x6 stretching ratio. At the initial stage of appearance of β-sheet, the relative intensity of the highest field peak at 19.7 ppm in Ala Cβ region was higher than in the spectrum of the SF fiber. Thus, the β-sheet structure observed at the initial stage of the stretching seems to be incomplete and different from the new staggered model. The structural model of PLA molecule with anti-parallel β-sheet structure reported by Arnott et al.35 using X-ray diffraction analysis was considered to be the initial structure for further structural analysis performed here. The energetically optimized model calculated by CASTEP program and GIPAW chemical shift calculation was effectively used to determine the new staggered model. The advantage of these approaches has been proved in the determination of the packing structures of (Ala)n (n=3,4) where the atomic co-ordinates were known 52,53 and also the packing structures of B. mori SF fibers before and after spinning.23,51 The packing structure as well as the conformation of silk fibers is considered to be the origin of high fiber strength and toughness1-6, and therefore it is very important to determine the packing structure. However, it is not easy to determine the


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packing structure because SF is heterogenerous10,20,36,39 and it is difficult to obtain single crystals from SF for single crystal X-ray analysis35,39. There are other advantages of these approaches. The new staggered model proposed here can explain the observed 13C and 1H spectra of S. c. ricini SF fiber, especially, the Ala methyl region that is sensitive to the packing structure. The model was also supported by the calculated distances. Although it is difficult to point out the origin of the difference between the observed and calculated chemical shifts correctly, we think that the use of the initial structural model of poly(L-alanine) determined by Arnott et al.35 might be one of the main origins of the discrepancies. Thus, there are Gly-rich regions other than (Ala)12 sequences in real S.c. ricini silk fibroin and the presence of such Gly-rich regions in the chain may modify the structure of (Ala)12 sequences. We believe that the solid state NMR is at present the best analytical method to address this complicated problem and to determine the inter-molecular arrangement of the native silk fibers at atomic level. The 13C DARR spectral pattern of the Cβ region of Ala residue in the PLA sequences of the silk fiber was also compatible with the DARR pattern for distances less than 5 Å predicted from the model. Thus, at present we can propose the packing structures for all PLA sequences that appear in the crystalline domains of spider silks and wild silkworm silks. Our structural analysis gives also an important lesson in the NMR analysis of SF 29 ACS Paragon Plus Environment

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fiber. In general, the observation of multiple peaks in the β-sheet region of Ala Cβ peak in the solid-state NMR spectrum of SF fiber may imply the presence of the corresponding number of different packing structure. However, our NMR study showed that multiple peaks were assigned to the carbons with different environments in the same packing structure. This was done by the combination of solid-state NMR and chemical shift calculation with CASTEP and GIPAW methods. Thus, peak assignments must be done very carefully, especially for the β-sheet structure where inter-molecular atomic distances are relatively close. Conclusions From the detailed studies conducted in this work, a new staggered model (Fig.7) could be proposed for the packing arrangement of the poly-L-alanine sequence of Samia cynthia ricini silk fibroin fiber using the Cambridge Serial Total Energy Package program and the chemical shift calculation with Gauge Including Projector Augmented Wave method. This model was supported by the 13C solid-state NMR results of the silk fiber.


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Fig. 7 A new staggered model proposed in this paper. The details are described in the text.

Supporting Information. Fig.S1 Atomic co-ordinate of (Ala)4 with anti-parallel β-sheet structure determined by single crystal X-ray diffraction analysis.

Acknowledgements

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). The calculations were supported by supercomputer system at ICR, Kyoto University. We also thank Dr H. N. Cheng (Southern Regional Research Center, USDA Agricultural Research Service, New Orleans, LA 70124, USA) for discussions.

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For Table of Contents Use Only

Table of Contents Graphic

A Refined Crystal Structure of Samia cynthia ricini Silk Fibroin Revealed by Solid-State NMR Investigations Tetsuo Asakura,* †Akio Nishimura, † Shunsuke Kametani, † Shuto Kawanishi †Akihiro Aoki, †Furitsu Suzuki,§ Hironori Kaji§ and Akira Naito †

†

Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588 JAPAN

§

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, JAPAN

*Correspondence to: Tetsuo Asakura


Refined Crystal Structure of Samia cynthia ricini Silk Fibroin Revealed

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