Structural Comparison of Various Silkworm Silks: An Insight into the

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Structural Comparison of Various Silkworm Silks: An Insight into the Structure-Property Relationship Chengchen Guo, Jin Zhang, Jacob S. Jordan, Xungai Wang, Robert W. Henning, and Jeffery L. Yarger Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01687 • Publication Date (Web): 09 Feb 2018 Downloaded from http://pubs.acs.org on February 10, 2018

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Biomacromolecules

Structural Comparison of Various Silkworm Silks: An Insight into the Structure-Property Relationship Chengchen Guo†*, Jin Zhang‡, Jacob S. Jordan†, Xungai Wang‡, Robert W. Henning¶, Jeffery L. Yarger†* †

School of Molecular Sciences, Magnetic Resonance Research Center, Arizona State University, Tempe, Arizona 85287-

1604, USA ‡

Australian Future Fibres Research and Innovation Centre, Institute for Frontier Materials, Deakin University, VIC 3216,

Australia ¶

Center for Advanced Radiation Sources, The University of Chicago, Chicago, Illinois 60637, USA

Abstract Silkworm silk has attracted considerable attention in recent years due to its excellent mechanical properties, biocompatibility, and promising applications in biomedical sector. However, a clear understanding of the molecular structure and the relationship between the excellent mechanical properties and the silk protein sequences are still lacking. This study carries out a thorough comparative structural analysis of silk fibers of four silkworm species (Bombyx mori, Antheraea pernyi, Samia cynthia ricini, and Antheraea assamensis). A combination of characterization techniques including scanning electron microscopy, mechanical test, synchrotron X-ray diffraction, Fourier transform infrared spectroscopy (FTIR), and NMR spectroscopy was applied to investigate the morphologies, mechanical properties, amino acid compositions, nanoscale organizations, and molecular structures of various silkworm silks. Furthermore, the structure-property relationship is discussed by correlating the molecular structural features of silks with their mechanical properties. The results show that a high content of β-sheet structures and a high crystallinity would result in a high Young’s modulus for silkworm silk fibers. Additionally, a low content of β-sheet structures would result in a high extensibility.

INTRODUCTION Structure-property relationship of silk is one of the most intriguing topics in silk-based biomaterials research since it is closely related to biomimicking of natural silks with excellent mechanical properties.1,2 Over the past decades, considerable effort has been made to understand the structure-property relationship and some significant progress has been achieved. For example, X-ray fiber diffraction studies have demonstrated that silks including both silkworm silks and spider silks are ACS Paragon Plus Environment

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composed of heterogeneous structures, where both nanocrystalline domain and amorphous domain are identified within the fiber.3-6 A combination of spectroscopic studies such as solid-state NMR spectroscopy and Raman spectroscopy has further indicated that the nanocrystalline domain comprises pleated β-sheets structures that formed by the repetitive domains in protein sequences.7-9 More than that, theoretical modeling using molecular dynamics simulation has provided more insight into the structureproperty relationship, indicating the high mechanical performance of silk fibers is primarily due to the unique heterogeneous structure.10 However, to obtain a clear understanding of the structure-property of silk materials, structural characterization and analysis at molecular/atomic level are required. To date, a majority of such studies focuses on the silks produced by domesticated Bombyx mori (B. mori) silkworm and the Nephila clavipes (N. clavipes) spider. For instance, solid-state NMR spectroscopic studies have illustrated that the GAGSGA motif in B. mori silk and poly(Ala) motif and poly(Gly-Ala) motif flanking the poly(Ala) runs in N. clavipes dragline silk form β-sheet structures.8,11-13 Additionally, for N. clavipes dragline silk, it is shown that the Gly-Gly-Ala motif primarily in the major ampullate spidroin 1 (MaSp1) and the Gly-Pro-Gly-X-X motif in the major ampullate spidroin 2 (MaSp2) take on 31-helical structures and elastin-like type II β-turn structures, respectively.13,14 Besides those two most studied silks, limited studies of structural characterizations on other types of silks have been carried out. It is known that silks of different silkworm species have distinct mechanical properties, especially between mulberry (Bombyx mori) and non-mulberry (Antheraea pernyi et al.) silkworms.15,16 Previous studies have shown that the silk fibers produced by non-mulberry silkworms have a relatively high extensibility as compared to those from mulberry silkworms.15-18 The differing mechanical properties are believed to arise from variation in the protein sequences and molecular structures including secondary structures and hierarchical structures.16,18 However, no clear understanding has been obtained yet regarding the relationship between the molecular structures and mechanical property of silk fibers across silkworm species. In this work, a comparative study, primarily focusing on the molecular structure characterization, has been carried out on silk fibers of four silkworm species: Bombyx mori (B. mori), Antheraea pernyi (A. pernyi), Samia cynthia ricini (S. c. ricini), and Antheraea assamensis (A. assamensis). B. mori belongs to family of Bombycidae while A. pernyi, S. c. ricini, and A. assamensis belong to family of Saturniidae. Among these species, the silk fibers of A. pernyi and S. c. ricini have been characterized by Fourier transform infrared spectroscopy (FTIR) and solid-state NMR spectroscopy in previous studies, but with limited quantitative structural analysis.19-25 Comparing to that, the present study carries out a thorough quantitative structural analysis on four different types of silkworm silks with a combination of structural characterization techniques including FTIR spectroscopy, synchrotron X-ray fiber diffraction, and

13

C solid-state NMR spectroscopy.

Furthermore, with the aim of understanding the structure-property relationship, the correlation between the obtained structural features and the mechanical properties of silks are discussed in detail. ACS Paragon Plus Environment

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EXPERIMENTAL SECTION Degumming Silkworm Silks. Silkworm cocoons were cut into small pieces and washed with DI water. A solution of 0.5 wt% of Na2CO3 and 1 wt% of Marseille Soap in distilled water was made. The solution was heated until boiling and the B. mori cocoons were placed in the solution for 30 min. After 30 min, the B. mori silks were washed with DI water three times until all soap was removed and allowed to sit in a water bath for 5 min. This process was repeated once more for degumming the B. mori cocoons. For the other three types of non-mulberry silkworm cocoons, the solution with the same composition was used to boil the cocoons with fresh solution changed every 30 min. The S. c. ricini cocoons were boiled for 1 h and the A. pernyi and A. assamensis cocoons were boiled for 1.5 h. The non-mulberry silks were then washed with DI water three times until all soap was removed and remained in water bath for 5 min before dried at ambient temperature. Scanning Electron Microscopy (SEM). The degummed silk fibers were carbon coated with 2 nm thickness using Leica EM ACE600 before SEM observation using a Zeiss Supra 55VP Field Emission SEM. The morphological images were obtained at EHT level of 2kV by the SE2 signal. Mechanical Test. The degummed silk fibers were tissue dried firstly before dried at room temperature for 48 hr. Tensile testing of single silk fibers was performed on an Instron MicroTester with a 0.5N load cell (Model 5848; force resolution, 0.5% of the indicated load; position resolution 0.02 µm; strain rate, 50% per min) at 22.3 °C with relative humidity 50%. The gauge length of the tensile samples was 20 mm and the diameters of the fibers were determined by SEM. For each type of silks, 40 samples were measured to obtain the average data and standard deviation. FTIR Spectroscopy. FTIR Spectra of silk materials were recorded using a PerkinElmer FTIR spectrometer with a diamond ATR accessory. The instrument carries a MIR light source of 300-8000 cm-1, an OptKBr beam splitter (400-7800 cm-1) and a LiTaO3 detector (370-15700 cm-1). The spectra were collected with a spectral window of 400-4000 cm-1, a resolution of 4 cm-1, and 32 scans. Spectral corrections and decomposition were performed using a home-developed Matlab packages. The spectra were first smoothed with a 5-point triangle smoothing method and then baseline corrected prior to peak deconvolution analysis. Solid-state NMR Spectroscopy. NMR Spectra of natural degummed silkworm silks without isotope labeling were collected a Varian VNMRS 400 MHz spectrometer equipped with a 3.2 mm tripleresonance probe operating in double-resonance (1H/13C) mode. 1H13C cross-polarization magic-anglespinning (CP-MAS) experiments were performed in this work. The CP condition for 1H13C CP-MAS experiments consisted of a 2.25 µs 1H π/2 pulse, followed by a 1.0 ms ramped (3 %) 1H spin-lock pulse of 70 kHz radio frequency (rf) field strength. The experiments were performed with a 25 kHz sweep ACS Paragon Plus Environment


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width, a recycle delay of 3.0 s, 8192 scans and a two-pulse phase-modulated (TPPM) 1H decoupling level of 91 kHz at a MAS speed of 20 kHz for all samples. Synchrotron X-ray Diffraction. X-ray fiber diffraction (XRFD) was performed on the BioCars 14BMC beamline at the Advanced Photon Source at Argonne National Laboratory, Argonne, IL, U.S.A. The wavelength of the X-ray beam was 0.979 Å, with a fixed energy of 12.668 keV and the beam size on the sample was 130 × 340 µm (horizontal × vertical). Data were recorded using an ADSC Quantum-315r detector. Bundles of aligned fibers were wound around washers, with the fiber axis perpendicular to the X-ray beam. The sample-to-detector distance is 200 mm and an exposure time of 120 s was used in the experiment.

RESULTS AND DISCUSSION The Morphologies of Silkworm Silks. The morphologies of the pristine and degummed silk fibers were characterized by scanning electron microscopy (Figure 1). The pristine silks are twin silk fibers (bave) covered by sericin and become single fibers (brins) after degumming. There are deposited cubic crystals on the surface of the pristine A. pernyi fibers, as shown in Figure1b and Figure S1, indicating the fiber was selected from the outer surface of the cocoon. After degumming, all fibers show smooth surface indicating the complete removal of sericin. The width of degummed silk fiber is different across species, ranging from 12 µm (B. mori) to 36 µm (A. pernyi).

Figure 1. SEM images of pristine and degummed silkworm silks: (a,e) B. mori; (b,f) A. pernyi; (c,g) S. c. ricini; (d,h) A. assamensis. Scale bars are 5 µm.

Mechanical Properties of Silkworm Silks. The tensile stress vs. strain curves of the four types of silkworm silk fibers are shown in Figure 2 and the calculated mechanical parameters are summarized in Table 1. It is known that silk fibers from single species present different stress-strain curves (type I, II, III, and IV), which results in the variability of the mechanical properties of silks.26,27 However, unambiguously, for the silks studied in this work (natural degummed silks), it is found that type II is predominantly for B. mori silk while type IV is more frequent for A. pernyi, S. c. ricini, and A. ACS Paragon Plus Environment

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assamensis silks. To obtain a general rule for the relationship between silk mechanical properties and molecular structures, averaged stress and strain values from 40 fibers for each silkworm species were used (Figure S2). The silks from A. pernyi, S. c. ricini, and A. assamensis have an average tensile stress ~ 400 MPa and breaking strains over 30 %, indicating the extensibility of the three silks are excellent. However, B. mori silk shows a lower breaking strain (~20 %) but the highest tensile strength (~635 MPa) among all silkworm silks. Comparing to spider silk, the overall tensile strength of silkworm silk fibers are much lower since spider dragline silk has an average tensile stress over 1200 MPa,28 but the silkworm silks have a comparable extensibility and toughness. In addition, it is noted that A. pernyi, S. c. ricini, and A. assamensis silks show similar stress vs. strain curves to spider dragline silks i.e. strain hardening occurred during the initial stage of deformation.29 In general, the results we obtained in this work agree well with the literature values though some variations due to the variable conditions for silk harvesting, degumming, and mechanical testing were found. To achieve a deep understanding of the origin of mechanical property variations across different types of silks and the structure-property relationship for silkworm silks, molecular structures of the silk fibers were carefully characterized and then a discussion correlating the molecular structures and the mechanical properties was carried out in the following paragraphs.

Figure 2. The tensile stress and strain curves for various degummed silkworm silk fibers.

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Table 1. Mechanical properties of various silkworm silks and spider silks. Tensile strength (MPa) Specie

Breaking strain (%)

Young’s Modulus (GPa)

Toughness (MJ/m3)

This work

Literature a

This work

Literature a

This work

Literature a

This work

Literature a

B. mori

635 ± 108

690 ± 20 460 ± 80

21.9 ± 4.9

38.5 ± 6.4 24.1 ± 7.7

11.7 ± 2.2

9.6 ± 0.6 7.2 ± 1.7

101 ±34

187 ± 33 81 ± 34

A. pernyi

426 ± 55

553 ± 64 430 ± 80

50.8 ± 7.0

45.0 ± 3.7 29.6 ± 10

5.0 ± 0.6

10.5 ± 0.8 4.8 ± 1.1

128 ±33

151 ± 25 79 ± 27

S. c. ricini

284 ± 88

560 ± 20 470 ± 110

34.2 ± 12.5

47.4 ± 6.1 29.4 ± 8.0

4.1 ± 0.6

6.3 ± 0.3 4.7 ± 1.3

61 ±33

156 ± 24 86 ± 30

A. assamensis

495 ± 48

490 ± 20 360 ± 100

51.1 ± 6.4

55.9 ± 5.3 29.2 ± 10.7

5.3 ± 0.4

4.5 ± 0.3 4.3 ± 1.2

141 ±28

148 ± 16 68 ± 31

-

1215 ± 60

-

20.0 ± 1.1

-

13.8 ± 0.8

-

111 ± 8

A. gemmoides

-

1376 ± 40

-

22.0 ± 1.0

-

8.3 ± 0.5

-

141 ± 1

L. Hesperusb

-

1441 ± 60

-

30.1 ± 1.8

-

10.2 ± 0.8

-

181 ± 10

N. clavipesb b

a

Literature values are obtained from ref. 18, 28, 30. b Spider dragline silks.

The Molecular Structures of Silkworm Silks. The morphologies and mechanical properties of silks are thought to originate from their molecular structures. First of all, amino acid compositions of all silks were analyzed based on their corresponding silk fibroin sequences and the results are shown in Figure 3A. It is found that all silkworm species show preponderance of Gly, Ala, Ser, and Tyr in their fibroins while the relative contents of different amino acids vary across the species. Gly and Ala are dominant in the fibroin of B. mori with a content of 45.9 % and 30.3 % respectively. However, for the three Saturniidae species (A. pernyi, S. c. ricini, and A. assamensis), their amino acid compositions are very similar where Ala is dominant in the fibroins with a content of 43.10 %, 45.40 %, and 42.50 % for A. pernyi, S. c. ricini, and A. assamensis respectively. Furthermore, comparing to B. mori, the three Saturniidae species show relatively higher Asp and Arg, which may have some special structural roles due to their charged side-chain groups. Besides amino acid analysis, a detailed investigation on the protein sequences of different fibroins was then carried out. It has been well demonstrated in literature that fibroin sequences of different types of silks including silkworm silk, spider silk, and other silks produced by insects are all composed of highly repetitive domains.31-33 Figure 3B shows the sequences of the representative repetitive domains of fibroins of different silkworm species. The repetitive domains of B. mori features repetitive motifs of (GA)nGX (X=S, Y or V), forming large blocks that are separated by spacer sequences, e.g. TGSSGFGPYVANG-GYSGYEYAWSSESDFGT.31 In comparison, the repetitive domains of the Saturniidae species are composed of alternating tandem repeats of poly(A) (12~13 contiguous alanine residues) and non-poly(A) regions. The non-poly(A) regions feature various combinations of GX and GGX (X=A, S, Y, D) sequences.32,33 These primary sequence features are very similar to the major ampullate spidroins of spider silks.34,35 Additionally, previous studies have shown that the GAGAGS motif in B. mori fibroin sequence and poly(A) motif in Saturniidae fibroin sequences ACS Paragon Plus Environment

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primarily form β-sheet structures and make up the bulk of the crystalline/semi-crystalline regions in silk fibers.3,11,19,21 (A)

(B)

Figure 3. (A) Comparison of amino acid compositions between different silkworm silks. The analysis was carried out based on the full sequences of A. pernyi and S. c ricini and heavy chain sequences of B. mori and A. assamensis. (B) Representative repetitive sequences of the different silk fibroins are shown: B. mori heavy chain (GenBank AF226688), A. pernyi (GenBank AAC32606), S. c. ricini (GenBank BAQ55621), and A. assamensis heavy chain (GenBank AIN40502).

FTIR spectroscopy and solid-state NMR spectroscopy were used as primary tools to further characterize the secondary structures of fibroins of different silkworm species. FTIR spectroscopy is a convenient and reliable technique for investigating the overall secondary structures of proteins since amide vibration depends on the secondary structure of proteins. In comparison, solid-state NMR spectroscopy is a superior tool for obtaining more detailed structural information at atomic/molecular level, for example, analyzing the structure of single amino acid residue in proteins. Here, FTIR results on the overall secondary structure of the fibroin proteins are discussed first, followed by a structural analysis characterized by solid-state NMR spectroscopy. FTIR spectra of degummed silks of all four species are shown in Figure 4A and vibration band assignments are summarized in Table 2. Three strong characteristic bands at 1510 cm-1 (amide II), 1624 cm-1 (amide I), and 1698 cm-1 (amide I) that are assigned to β-sheet structure indicate preponderance of β-sheet structure in the silks for all species. To further obtain a quantitative understanding on the compositions of different secondary structures including β-sheet, helix/turn, and random coil, peak deconvolution analysis was carried out on the amide I vibration band, near 1650 cm-1. The reason for choosing the amide I vibration band is because it is the primary amide vibration band that depends on the secondary structure of the protein’s backbone and is hardly affected by the nature of the residues’ side-chain among all amide vibration bands.36-39 The FTIR spectra of silks were deconvoluted following the procedure described in detail in the supplementary information and the analyzed results are shown in Figure 4B. The content of every individual conformation is summarized in Figure 4C. It is found that the B. mori silk has the largest content of β-sheet structures among all silks with a value of ~ 50.0 %, which is in good agreement with the reported values determined by Raman and FTIR spectroscopy.40,41 This ACS Paragon Plus Environment


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relative larger β-sheet content for B. mori compared to other silks is due to higher abundance of (GAGSGA)n repetitive sequences that are responsible for making up the β-sheet structures within the silk fibroin sequence. Quantification of the abundance of the (GAGSGA)n repetitive sequences reveals that approximate 53 % of the silk fibroin is composed of (GAGSGA)n repetitive sequences,40 which is close to value of β-sheet content obtained from FTIR study. However, for the Saturniidae silks, the βsheet structures in the silks primarily derives from the poly(A) repetitive sequences, similar to spider silks.42 FTIR results indicate that the β-sheet contents for A. pernyi, S. c. ricini, and A. assamensis are 44.9 %, 45.4 %, and 42.6 % respectively. Additionally, these results are consistent with the abundances of the poly(A) repetitive sequences quantified from the fibroin sequences with an approximate value ~40 % for the three species.16 One possible reason for the relatively higher level determined by FTIR for the Saturniidae silks is that some other repetitive motifs such as poly(GA) or poly(GS) in the fibroins other than poly(A) may be also involved in making up the β-sheet structures.

Figure 4. (A) FTIR spectra of various degummed silkworm silks: B. mori, A. pernyi, S. c. ricini, A. assamensis. (B) Deconvolution results of amide I band in IR spectra of various silkworm silks. (C) Summary of the deconvolution results.


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Biomacromolecules Table 2. Assignments of FTIR vibration bands for different silkworm silks41,43 Assignment

Wavenumber (cm-1)

Band

Conformation

1698

Amide I, C=O stretching, C-N stretching

β-sheet

1650


random coil/α-helix

1624


β-sheet

1529

Amide II, C=O in-plane bending, C-N stretching, N-H in-plane bending

random coil/α-helix

1515

Tyrosine side chain

-

1510

Amide II, C=O in-plane bending, C-N stretching, N-H in-plane bending

β-sheet

13

C solid-state NMR spectroscopy was further applied to investigate the conformations of

individual amino acid residues within the fibroins. Figure 5A shows the 13C CP-MAS NMR spectra of various silks and chemical shift assignments are summarized in Table 3. As seen in Figure 5A, B. mori shows a larger component of Gly compared to other species indicated by the strong resonances of Gly Cα at 43.5 ppm and carbonyl at 170.0 ppm for B. mori. Furthermore, the carbonyl resonance at 170.0 ppm indicates that the Gly residues are primarily in β-sheet structures. However, it is difficult to determine conformations of the Gly residues in other silks due to the severe resonance overlap in the carbonyl region and the fact that

13

C chemical shifts of Gly residue are generally insensitive to the

conformation of the Gly residue in the solid-state NMR analysis. Based on the resonances of Gly Cα for Saturniidae silks (~43.1 ppm), it is very likely that Gly residues are primarily in random coil structures. Besides Gly residues, the resonances of Ser Cα and Ser Cβ of all silks indicate that the Ser residues are involved primarily in β-sheet structures since these values fall in the typical chemical shift ranges of βsheet structure determined from model peptide.44,45 From previous reports, around 82 % of Ser residues are found in β-sheet structures for B. mori silks while this value for Saturniidae silks is around 70 %.25,46 For Tyr residues, the chemical shift analysis illustrates that the Tyr residues are not predominantly present in β-sheet structures and very likely to be involved primarily in helical or random coil structures. According to the literature, around 40 % of Tyr residues are found in β-sheet structures for B. mori silks while this value for Saturniidae silks is around 33 %.25,47 For Ala residues, it is noted that Ala Cβ resonances are asymmetric and broad in all

13

C CP-MAS spectra of silks, indicating the structural

heterogeneity in the natural silk fibers. Furthermore, it also indicates that the methyl side chains of Ala residues are more sensitive to the intermolecular/intramolecular chain arrangement than the backbone carbons, showing well-resolved conformation-dependent resonances.11 This allows us to analyze the heterogeneous structure of natural silk fibers in more detail by deconvoluting the Ala Cβ resonances. In addition, since Ala residues play crucial roles in forming β-sheet structures within fibroins, elucidating Ala conformations would lead to a clear understanding of the silk protein structures. Deconvolutions with Gaussian peaks were performed to estimate the relative contents of different Ala conformations. ACS Paragon Plus Environment


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For B. mori, Ala Cβ resonance yields three isotropic chemical shifts of 16.7, 20.5, 22.5 ppm (Figure 5B).11,48 The broad component at ~16.7 ppm is assigned to distorted structures including turn, helix and random coil, which is dominant in lyophilized silk (Figure S4). Two components at ~20.5 and ~22.5 ppm are assigned to β-sheet structures with different packing of the methyl side chains.8 Specifically, the resonance at ~20.5 ppm is assigned to the β-sheet structures in which all methyl groups of Ala residues point toward the same direction. The resonance at ~22.5 ppm is assigned to the β-sheet structures in which all methyl groups of Ala residues alternatively point to opposite directions. These assignments have been confirmed in previous studies with NMR investigations on model peptides such as (GA)15 and (GAGSGA)5.11,49 Overall, the result indicates that 80.0 % of Ala residues in B. mori silks is present in βsheet structures. For the other three species, due to the complexity of packing arrangement of the poly(A) sequence, three β-sheet components with chemical shifts of 19.8 ppm, 21.2 ppm and 22.9 ppm were used to deconvolute the Ala Cβ resonance along with a broad component representing the disordered turn, helix and random coil structure.21,24,25As summarized in Table 4, the ratios of Ala residues in β-sheet structures for A. pernyi, S. c. ricini, and A. assamensis are estimated to be 87.6 %, 87.4 %, and 88.8 %, respectively. Comparing to B. mori, these three species possess larger values because Ala residues are present primarily in poly(A) motif that is responsible for forming β-sheet structures. For B. mori, Ala residues are also found frequently in GAGXGA (X=Y, A) motifs that are present in non-β-sheet structures. By combining the quantitative solid-state NMR results with the information obtained from amino acid analysis, it is possible to estimate the overall content of β-sheet structures for different types of silk fibers if only Ala, Gly, Ser, Tyr residues were considered to form βsheet structures (Table S1). Specifically, for B. mori, the ratio of (GA)n sequence in β-sheet was estimated first based on the Ala ratio in β-sheet structures and then combined with the ratios of Ser and Tyr in β-sheet structures. For A. pernyi, S. c. ricini, and A. assamensis, the ratio of poly(A) repetitive sequences in β-sheet structures was estimated first based on the Ala ratio in β-sheet structures and then combined with the ratios of Ser and Tyr in β-sheet structures. The result indicates that ~60.5 % of amino acid residues are present in β-sheet structures for B. mori, which is consistent with the β-sheet content determined by NMR in previous report.11 However, this value is larger than the β-sheet content determined by FTIR spectroscopy, which is probably due to the presence of some Gly residues in (GA)n sequences in random coil structure. For A. pernyi, S. c. ricini, and A. assamensis, the overall contents of β-sheet structures in silk fibers are estimated to be ~ 47.4 %, 46.3 %, and 46.5 %, respectively. These values are consistent with the β-sheet contents determined by FTIR spectra analysis. Overall, with a combination of amino acid analysis, FTIR spectroscopy and 13C solid-state NMR spectroscopy, the molecular structures, specifically secondary structures of silk fibroins of different silkworm species were well characterized and studied quantitatively. The results indicate B. mori has a ACS Paragon Plus Environment

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higher overall content of β-sheet structures in silk fibroin than the other species, whereas larger portions of alanine residues in A. pernyi, S. c. ricini, and A. assamensis silks are found in β-sheet structures.

Figure 5. (A) 13C CP-MAS spectra of degummed silkworm silks. (B) Deconvolution of Ala Cβ peaks in 13C CPMAS spectra for various silks.

Table 3. 13C chemical shifts of silkworm silks and polypeptides with known secondary structures11,44,45,49-53 Residue

B. mori

A. pernyi

S. c. ricini

A. Assamensis

α-helix

β-sheet

random coila,1

Gly Cα Gly CO Ala Cα Ala Cβ

43.5 170.0 49.7 16.7 20.5 22.5

43.1 49.4

43.1 49.3

43.1 49.3

16.6 19.8 21.2 22.9

16.6 19.8 21.2 22.9

16.6 19.8 21.2 22.9

46.0 174.9 52.3-52.8 14.8-16.0

43.2-44.3 168.4-169.7 48.2-49.3 19.9-20.7

43.4 173.2 50.8 16.6

Ala CO Ser Cα Ser Cβ Tyr Cα Tyr Cβ Tyr Cγ Cδ Tyr Cε Tyr Cζ Phe Cγ

173.0 55.5 64.4 37.2

172.6 55.2 63.8 37.0

172.4 54.9 62.8 37.2

172.4 55.0 63.5 37.4

176.2-176.8 59.2 60.7 54.8-58.6 36.1

171.6-172.4 54.5-55.0 62.3-63.9 52.1 39.3

176.1 56.6 62.1 56.2 37.1

129.4

129.5

129.6

129.5

129.7

128.0

128.9

115.9 156.0 -

116.1 156.9 136.7

116.2 156.4 136.9

116.1 156.5 136.4

116.1 154.2 138.4

115.0 155.2 136.3

116.5 155.6 136.9



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Table 4. Summary of the peak deconvolution of Ala Cβ resonance for various silks Species B. mori

A. pernyi

S. c. ricini

A. assamensis

Peak position (ppm)

Conformation

16.7 20.5 22.5 16.6 19.8 21.2 22.9

turn, helix and random coil β-sheet β-sheet turn, helix and random coil β-sheet β-sheet β-sheet

16.6 19.8 21.2 22.9 16.6 19.8 21.2 22.9

turn, helix and random coil β-sheet β-sheet β-sheet turn, helix and random coil β-sheet β-sheet β-sheet

Ala in β-sheet

Estimated Overall β-sheet from NMR

80.0 %

60.5 %

87.6 %

47.4 %

87.4 %

46.3 %

88.8 %

46.5 %

The Nanoscale Organizations of Silks. X-ray fiber diffraction was first used in 1950s to investigate the hierarchical structure of silk fibers,3 and since then, this technique has been developed and used on fine structural analysis of silk fibers including silkworm silks and spider silks in several studies.54 The focus of structural analysis of silk fibers with X-ray fiber diffraction is determining the structure, size, and orientation of β-sheet nanocrystallites, and estimating the degree of crystallinity of silk fibers as well. Based on previous studies, it has been well demonstrated that the nanocrystallite in silk fibers is nearly orthorhombic composed of β-sheets structure and they are preferentially oriented with the molecular chains along the fiber axis (Figure 6A). In this work, synchrotron X-ray fiber diffraction was used to investigate in detail the structure and organization of the nanocrystallites at nanoscale including unit cell dimension, nanocrystallite size, degree of crystallinity, and orientation order in various natural silk fibers. Figure 6B shows the well-defined 2D WAXS patterns of various silks. All samples show strong and clearly identified reflections including (020), (210), (211), and (002). The d-spacing values of all reflections are summarized in Table S1. The unit cell sizes for different silks were determined based on the d-spacing values of reflection (020), (210), and (002) (Table 5). For estimating the nanocrystallite sizes, the 1D intensity profiles derived from azimuthal integration along the equator and meridian were fitted using Gaussian peaks (Figure S5, S6), and the resulting full width at half maximum (FWHM) of the (020), (210), (200) peaks were used to calculate the nanocrystallite size. The nanocrystallite size was determined by Scherrer’s equation, L=(0.9λ)/(Bcosθ), where B is the FWHM of the fitted Gaussian peak for a specific reflection. The results of the analysis were shown in Table 5. As summarized in Table 5, B. mori has a larger nanocrystallite size along the c-axis (fiber axis) with c ~ 13.66 nm while the other three species have similar nanocrystallite dimensions with a×b×c close to 3.8×3.2×5.0 nm. This ACS Paragon Plus Environment

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difference is very likely related to the composition of the nanocrystallites. It is known that the (B)

(A)

Figure 6. (A) Schematic representation of organization and structure of nanocrystallites in silk fibers. (B) 2D WAXD patterns of different degummed silkworm silk fibers orientated perpendicular to the incoming beam: (a) B. mori; (b) A. pernyi; (c) S. c. ricini; (d) A. assamensis.

nanocrystallite in silks is primary composed of β-sheet structures. For B. mori, the repetitive sequences responsible for forming the β-sheet structures in B. mori silk, (GAGSGA)n (n~5-7), is longer than the repetitive sequences, poly(A) or poly(GA) in the other three silkworm silks. Since the molecular chain direction is along the fiber axis, the formed nanocrystallite size is larger for B. mori along the fiber axis.

Table 5. Summary of unit cell sizes, nanocrystallite sizes, crystallinity, and orientation factor for various silks from analysis of the WAXS patterns Unit Cell Size (Å)

Nanocrystallite Size (nm)

Species a

b

c

a

b

c

Crystallinity Orientation factor (f) (%)

B. mori

9.68 ± 0.20 9.36 ± 0.18 7.02 ± 0.14 3.38 ± 0.07 3.06 ± 0.06 13.66 ± 0.27

45.6 ± 2.5

0.979 ± 0.02

A. pernyi

9.62 ± 0.19 10.60 ± 0.21 6.90 ± 0.13 3.82 ± 0.08 3.06 ± 0.06 4.26 ± 0.08

32.0 ± 1.6

0.945 ± 0.02

S. c. ricini

9.63 ± 0.19 10.54 ± 0.21 6.90 ± 0.13 3.74 ± 0.06 3.27 ± 0.06 4.92 ± 0.10

33.2 ± 1.6

0.955± 0.02

A. assamensis 9.57 ± 0.18 10.56 ± 0.21 6.88 ± 0.12 3.85 ± 0.07 3.27 ± 0.06 5.13 ± 0.10

31.3 ± 1.5

0.967± 0.02

The degrees of the crystallinity (xc) for silks can be estimated by analyzing the crystalline and amorphous components present in the azimuthal integration profiles. According to the literature, different methods have been developed and applied for such analysis, where a large variation between them was found.55 In this work, the degree of crystallinity was determined based on the radial intensity profile of reflection at the equator using the equation xc=Ic/(Ia+Ic), where Ic and Ia are integrated intensities of the crystalline peaks and the amorphous peaks, respectively (Figure S5). It has been demonstrated in previous related studies that the degree of crystallinity calculated from the whole pattern and that from the sequential method are in good agreement.4,56 According to the calculation, B. ACS Paragon Plus Environment


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mori silk has a crystallinity of 45.6 % while all other silks have similar crystallinity ~ 32 %, indicating a higher overall abundance of nanocrystalline component in B. mori silk. More than that, the crystallinity determined for A. pernyi, S. c. ricini, and A. assamensis silks are very close to the crystallinity of spider dragline silk.57

Figure 7. 1D azimuthal intensity profiles of the radially integrated (020) peak with Gaussian fits for various silks.

Orientation factor (f) is another key parameter highly related to the physical properties of silk fibers since it represents the axial orientation distribution of nanocrystallites. From WAXS pattern, radial integration of a narrow ring around the (020) reflection can be used to approximate the orientation factor of the nanocrystallites within the silk fibers using Herman’s orientation function: f=(31)/2, where σ is the angle between c-axis and fiber axis.58 f is 0 for no preferred orientation in fibers and 1 if all nanocrystallites are perfectly aligned with respect to the fiber axis. can be obtained from the width of the equatorial reflection (020) according to the procedure detailed in the supplementary. Figure 7 shows the 1D azimuthal intensity profiles of the radially integrated (020) reflection for various silks. Two Gaussian peaks were used to fit the strong peak according to the structural model used in previous studies on spider silks, where a narrower Gaussian peak corresponds to the crystalline component and a broader Gaussian peak corresponds to the amorphous component.4,5 The orientation factors of the nanocrystallites were determined for all silks and the results are summarized in Table 5. ACS Paragon Plus Environment

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All silks show high values of orientation factor for the nanocrystallites, indicating the nanocrystallites in silks have a high degree of order and show excellent alignment with respect to the fiber axis. Among the silks, B. mori silk has the highest value (0.979), further indicating the nanocrystallites in B. mori silk have a better alignment than those in other three silks. Additionally, it is also noted from the analysis that the orientation factor for the amorphous component in B. mori silk is also high with a value ~ 0.918, indicating a good alignment (Table S3). Comparing to B. mori silk, A. pernyi, S. c. ricini, and A. assamensis have lower degree of order for the amorphous component with corresponding orientation factors of 0.313, 0.111, and 0.112 respectively. Relationship between Molecular Structures and Mechanical Properties. As seen from the mechanical test, silkworm silks show some interesting mechanical properties such as high tensile strength and Young’s modulus for B. mori silk, excellent extensibility for A. pernyi, S. c. ricini, and A. assamensis silks, and high toughness for all silks. These interesting mechanical properties are thought to originate from the characteristic molecular structures of silk fibroins. Here the structure-mechanical property relationship for silks is discussed by correlating the structural features of silks with their mechanical properties. Since FTIR spectroscopy can provide direct analysis of the overall β-sheet content in silk fibers comparing to NMR spectroscopy, it is more reasonable to use the values obtained via FTIR spectra analysis. Figure 8 shows correlations between the β-sheet content within silks and the mechanical parameters including Young’s modulus, breaking strain, and toughness. It illustrates that the Young’s modulus of silks would be enhanced with increasing of the β-sheet content while the extensibility (breaking strain) of silks shows an opposite trend. To understand the contribution of β-sheet structure on the mechanical properties of silks, more investigations were carried out to provide insights from molecular structure to nanoscale assembling. First, the correlation between β-sheet content and crystallinity of the silks shows that increasing β-sheet content in silks would result in increased crystallinity, indicating the β-sheet structure is the primary component to form nanocrystallites in silks (Figure S7). With the increase of crystallinity, the strength of silk fibers increases while the extensibility of silk fibers decreases (Figure S8). This is because the strength and the extensibility of silks primarily originate from the nanocrystallites and amorphous domain respectively within silk fibers.10,59 For B. mori, it shows the largest proportion of β-sheet structures (~50%) and the highest crystallinity among all four silks, so it has the highest tensile strength and Young’s modulus. However, due to the low ratio of amorphous domain, it has the lowest extensibility. The amorphous domain is considered to be composed of secondary structures other than the β-sheet structures. Based on the fibroin sequences and quantitative secondary structure analysis, silks produced by A. pernyi, S. c. ricini, and A. assamensis have larger portions of amorphous domains primarily composed of Gly-rich sequences than B. mori silk. The Glyrich sequence has been demonstrated to be a major contribution for the elastomeric properties of the proteins.60 In addition, this feature bears the closest resemblance to the major ampullate spidroin ACS Paragon Plus Environment


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(MaSp1) of spider dragline silk, where the GGX (X=A, D, Y) motif is significantly frequent and repetitive. The GGX motif in MaSp1 has been reported to be very likely present in a 31-helical structure, which may possess a structural role of reinforcing the organization of the highly ordered β-sheet nanocrystallites made of poly(A) repetitive sequences.61 More than that, significant amount of β-sheet structures which would be likely to unfold upon stretching was found to be present in the amorphous domain for Saturniidae species (e.g. A pernyi).6 Due to these factors, silks of Saturniidae species show better extensibility than B. mori silk. An approximate correlation between the extensibility and the βsheet content was established for silkworm silks, where Strain (%) = -4.22 × β-sheet content (%) + 233.7. In comparison, the Young’s modulus of silks shows relatively less direct correlation to the βsheet content due to its complexity with a balance of β-sheet content, nanocrystallite size, nanocrystallite alignment, and the nanoscale/macroscale organization of crystalline domains. Molecular dynamics simulations in previous report has shown that silks with smaller β-sheet nanocrystallites confined to a few nanometers would achieve higher stiffness, strength and mechanical toughness than those with larger nanocrystallites.10 However, in the present work, B. mori silk has the largest nanocrystallites (3.38×3.06×13.66 nm), but it has the highest Young’s modulus and similar toughness compared with other three silks (Figure 8). This is probably due to the packing arrangement of β-sheet nanocrystallites across the species are different.24,48 Spider dragline silks have similar β-sheet content (~ 45%) and nanocrystallite dimensions (~ 2.7×4.0×7.1 nm) to A. pernyi, S. c. ricini, and A. assamensis silks.40,41,57 However, they show much higher tensile strength than all silkworm silks (Table 1). A sequencedependent study has shown that such high tensile strength of spider dragline silk may result from the presence of two proteins in the silk, MaSp1 and MaSp2, which could form unique hierarchal structures and further enhance the organization of nanocrystallites.62 So in order to get a better understanding the structure-property relationships for silk fibers, more investigation on hierarchal structures of silks are needed.

Figure 8. Correlations between the β-sheet content and the mechanical parameters including Young’s modulus (A), breaking strain (B) and toughness (C) for various silkworm silks.


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CONCLUSION Silk fibers of four silkworm species (B. mori, A. pernyi, S. c. ricini, and A. assamensis) were investigated using a combination of characterization techniques including scanning electron microscopy, synchrotron X-ray diffraction, Fourier transform infrared spectroscopy (FTIR), and NMR spectroscopy. The results show that the molecular structure of the B. mori silk is quite different from the other three species. Quantitative FTIR and 13C solid-state NMR results illustrate that B. mori silks have a higher βsheet content in the silk fibroin than the other three species. The structural parameters of nanocrystallites including size, crystallinity and orientation factor in different silks were analyzed using fiber X-ray diffraction. The result indicates the B. mori silk has the largest nanocrystallites size along the fiber axis and the highest orientation factor for the nanocrystallites organization in silk fibers as well. For the other three species, they are very similar between each other based on structural analysis and also show some similarity to spider silks such as the repetitive motifs in protein sequences and the dimension of nanocrystallites. Lastly, the correlations between the molecular structures and mechanical properties were discussed in detail in order to achieve a better understanding of the structure-property relationship for silk materials. It indicates that the β-sheet structure relates closely with the stiffness, extensibility of silk fibers. However, the toughness of silk fibers has less correlation with the β-sheet structure. The discovery of the correlation between the molecular structures and mechanical proprieties would facilitate the design of bioinspired materials that mimic the outstanding properties of natural silks.63-65

ASSOCIATED CONTENT Supporting Information SEM images of pristine A. pernyi silkworm silks; Multiple tensile stress and strain curves for each type of silk fibers; Preparation of lyophilized B. mori silk fibroin; Deconvolution of Amide I band in FTIR spectra of silks; Determination of orientation factor with fiber X-ray diffraction-calculation of ; IR spectra of degummed and lyophilized B. mori silks;

13

C CP-MAS spectra of degummed and

lyophilized B. mori silks; Summary of the estimation of the overall β-sheet for various silks; d-spacing values (in Å) of characteristic reflections for various silks; 1D intensity profiles for various silks derived from azimuthal integration along the equator with 15° on either side of the horizontal axis; 1D intensity profiles for various silks derived from azimuthal integration along the meridian with 15° on either side of the horizontal axis; Summary of the orientation factor (f) determined from reflection (020) for various silks; Correlations between the β-sheet content and the crystallinity for various silkworm silks; Correlations between the β-sheet content and the mechanical parameters including Young’s modulus, breaking strain, and toughness for various silkworm silks; FTIR spectra of B. mori, A. pernyi, S. c.



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ricini, and A. Assamensis silkworm silks. These materials are available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected], [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The research was supported by grants from the National Science Foundation (CHE-1011937, DMR1264801) and the Australian Research Council Discovery project (DP120100139). We thank Dr. Brian Cherry for help with NMR instrumentation, student training, and scientific discussion. Dr. Jin Zhang acknowledges the support from Endeavour Fellowship and Victoria Fellowship and the host organizations including Arizona State University and National University of Singapore.

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Biomacromolecules


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