Understanding the Mechanical Properties and Structure Transition of

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Understanding the mechanical properties and structure transition of Antheraea pernyi silk fibre induced by its contraction Yu Wang, Jianchuan Wen, Bo Peng, Bingwen Hu, Xin Chen, and Zhengzhong Shao Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01691 • Publication Date (Web): 05 Feb 2018 Downloaded from http://pubs.acs.org on February 6, 2018

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Biomacromolecules

Understanding the mechanical properties and structure transition of Antheraea pernyi silk fibre induced by its contraction Yu Wang†§, Jianchuan Wen†§, Bo Peng‡, Bingwen Hu‡, Xin Chen†, Zhengzhong Shao†* †

State Key Laboratory of Molecular Engineering of Polymers, Laboratory of Advanced Materials and Department of Macromolecular Science, Fudan University, Shanghai 200433, People’s Republic of China. ‡

Engineering Research Center for Nanophotonics & Advanced Instrument, Ministry of

Education, Shanghai Key Laboratory of Magnetic Resonance, Department of Physics, East China Normal University, Shanghai 200062, People’s Republic of China.

ABSTRACT: Like most major ampullate silks of spider, the length of Antheraea pernyi (A. pernyi) silkworm silk can shrink to a certain degree when the fiber is in contact with water. However, what happens in terms of molecule chain level and how it correlates to the mechanical properties of the silk during its contraction is not yet being fully understood. Here, we investigate the water induced mechanical property changes as well as structure transition of two kinds of A. pernyi silk fibre, which are forcibly reeled from two different individuals (silkworm a and

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silkworm b, the silk fibre from either one represents the lower and upper limit of the distribution of mechanical properties, respectively). The tensile test results present that most of the mechanical parameters except the post-yield modulus and breaking strain for both silk fibres have the same variation trend before and after their water contraction. Synchrotron FTIR and Raman spectra show that the native filament from silkworm a contains more α-helix structures than that in silkworm b filament, and these α-helices are partially converted to β-sheet structures after the contraction of the fibres while the order of both β-sheet and α-helix slightly increase. On the other side, the content and orientation of both secondary structural components in silkworm b fibre keep unchanged, no matter it is native or contracted.

13

C CP/MAS NMR results further

indicate that the α-helix/random coil to β-sheet conformational transition occurred in the silk of silkworm a corresponds the Ala residues. Based upon these results, the detailed structure transition models of both as-reeled A.pernyi silk fibres during water contraction are proposed finally to interpret their properties transformation.

KEYWORDS: A. pernyi silk; supercontraction; tensile performance; structure transition.


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1. INTRODUCTION Animal silks are unusual materials compared to other fibrous materials not only because they are born with comprehensive mechanical properties as indicated by an exceptional combination of strength, extensibility, and toughness that are inapproachable by most biomimetic and man-made fibres,1-6 but also because they are discovered to be equipped with other extraordinary properties, such as supercontraction,7,8 shape memory,9 thermal conductivity,10 optical11,12 and sonic13 properties, etc. On the other hand, animal silks are usual materials to scientists because their unusual properties are understandable from the point of view of macromolecule, in terms of (micro)structure and morphologies. Therefore, many attempts have been made to understand the contributions of hierarchical structural features of silks to their various properties,4,6,14-19 which will lead the design of bio-inspired structural materials that can totally match their natural counterparts in terms of both structure and property characteristics and also guide the fabrication of high performance synthetic materials. Among the most studied silk categories, the Chinese wild silkworm Antheraea pernyi (A. pernyi) silk has been the focus of attention in recent years, mainly owing to its peculiar role played in the whole family of animal silks.15,20-24 Specifically, it is used to construct a cocoon, like domesticated commercial Bombyx mori (B. mori) silk. Its amino acid sequence,25 especially the motifs, however, is much resembles the major ampullate silk (dragline silk) of spider,26 with repetitive poly(alanine) regions of (Ala)n, where n=10-14, embedded into a semi-amorphous matrix that consists predominantly of glycine residues. Up to now, a variety of experimental techniques, such as FTIR spectroscopy,20,27 Raman spectroscopy,28 as well as solid-state NMR,29,30 have been employed to investigate the structure of A. pernyi silks or their analogues. Nevertheless, most of these studies are based on degummed silk fibres, which neglect the effect


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of water penetration during degumming or softening process on the properties and structure of native silk fibres. When loosely immersed in water, spider dragline silk can shrink by up to about 50% of their stretched length, which is a phenomenon described as supercontraction.7 As a consequence, the dried-out supercontracted silk displays lower elastic modulus, breaking strength and energy but higher extensibility compared to the native silk, and the silk fibre comes back to a “reference state” that is mainly determined by its primary structure.8,31,32 Up to now, a number of studies have been conducted to understand supercontraction and the underlying mechanisms, and there is a consensus that supercontraction is a result of reorientation of both the aligned noncrystalline chains and the crystallites in the presence of polar solvents (usually water).33-36 B. mori silk, on the other hand, does not supercontract in water, and its mechanical properties remain unchanged after water treatment.37 Recently, Fu et al. found that forcibly reeled A. pernyi silk also can shrink when it is exposed to water and the shrinkages are just around 7% or even lower.15 However, the detailed properties and structure transition of the A. pernyi silk during this process remains unclear. Besides, though our recent work showed that the α-helix structures existed in forcibly reeled A. pernyi silk can be significantly decreased after water treatment,21 it is still an open question whether these disappeared α-helix structures are converted to β-sheet or random coil structure. On the other hand, forcibly reeled A. pernyi silks from different individuals display considerable structural variability,22 so it is interesting to find if these different structures have varied responses to water treatment. Based on these challenges, in present work, the properties and structure transition of as-reeled A. pernyi silks from two different silkworms (these two kinds of silks have been found to represent the limits of the distribution of properties, which will be detailed in the results and


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discussion section) induced by water was investigated in detail. We first compared the mechanical properties of two as-reeled A. pernyi silks before and after water contraction. Then, the corresponding structure change was monitored by using synchrotron FTIR (S-FTIR), Raman spectroscopy combined with 13C CP/MAS NMR. Finally, the structure transition mechanisms of as-reeled A. pernyi silk fibres induced by water were suggested.

2. EXPERIMENTAL SECTION 2.1. Silk Fibre Preparation. Forcibly reeled silk fibres used here were all obtained in the same reeling speed (8 mm s-1)2,38 in laboratory conditions (20-25 °C, 40%-50% RH) from the mature A. pernyi silkworms, which previously lived on oak trees in a tussah field of Shandong province, China. The silks from the same silkworm were collected in the same spinning season on the same day. Forcibly reeling silks from the spinneret of mature A. pernyi silkworm is so convenient and controllable that it generates nearly uniform silks and further results in highly repeatable mechanical properties. The silks were wound and restrained on a plastic cylinder of 21mm in diameter and then stored under lab conditions before being tested. To prepare supercontracted silk fibre, as-reeled A. pernyi silk fibre was first adhered to the jaws of a Vernier caliper, and then immersed in water without strain for at least 24 h. The shrinkage was calculated through dividing the difference between the lengths of the native fibre and contracted one (drying in air) by its initial length. With the help of dividers, segments of silk fibres (bave) with a gauge length of 9 mm were then transferred and adhered onto the sample frame made from hard cardboard with double-faced tape. The samples for cross-section measurement and tensile test were arranged according to the method described elsewhere15 and detailed below.


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2.2. Cross-sectional Area Measurement. To precisely measure the cross-sectional area of the native silk fibre, a length of silk fibre adhered onto our custom-built mould was sputtered with gold under 10 mA for 3 min, and then embedded in epoxy resin. After dried in air over night, the silk-embedded epoxy resin was fractured into six segments perpendicular to the silk fibre. The fracture cross section was sputtered with gold, and then observed by a Tescan 5136MM with secondary electrons at 10 kV. The gold layer between a silk fibre and resin effectively weakens the interface adhesion and gives an outline in the resin equal to the silk's original cross section after the fibre was pulled out during the fracture. From this outline, we estimated the crosssectional area of the silk fibre using the software Vega TC provided with the SEM. Usually, at least 5 well-defined cross-sectional images should be obtained from a silk fibre for area measurement. The average area was used as the cross-sectional area of adjacent as-reeled silk fibres. The area of the contracted silk fibre was calculated with the tested hypothesis that its volume was constant after contraction. We took no account of the effect of sericin coating on the cross-sectional area as well as the mechanical properties described below, because (i) the sericin coating on the fibre’s surface is uniform along the silk fibre and extremely thin (100-200 nm) compared to the greater thickness of a single brin of A. pernyi silk (15-17 µm);22 (ii) the sericin coating remained intact after water treatment.22,39 With the normalization of cross-sectional area, the load-strain curve was converted to stress-strain curve. 2.3. Tensile Testing. Tensile tests were performed on Instron 5565, around 20-25 °C and 4050% RH at a strain rate of 0.005 s-1. Before the test, the side support of the frame was simply cut away, so that the force was transmitted through the silk fibre. In loading-unloading tests, the sample was first loaded to a strain of 17% and immediately unloaded to zero strain. After 1 min


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recovery, the silk fibre was reloaded to its breakage. For the tensile test in water, a custom-built accessory,21 which can sustain the aqueous environment, was integrated into the Instron. 2.4. S-FTIR Microspectroscopy. The S-FTIR measurements were recorded at Beamline (BL01B) in the Shanghai Synchrotron Radiation Facility (SSRF). All the FTIR microspectra were acquired at 4 cm-1 resolution over the range of 4000-800 cm-1. A relatively large 20 × 20 µm square aperture was selected here due to the large width of flat as-reeled A. pernyi silk (≥30 µm) and 256 scans were co-added. The square aperture was placed in the center of the fibre to avoid the possible diffraction and scattering of the infrared light. Before each test of silk fibre, a background spectrum was collected from an area free of sample. To minimize IR absorption by CO2 and water vapor in the ambient air, the sample was purged using dry N2 during measurement. For each silk sample (1 cm long), at least three different positions were analyzed, and if there is no variation in these three S-FTIR spectra, we took it as one effective datum. For the polarization experiment, a KRS-5 IR polarizer was inserted in the infrared beam to study the dichroism of certain absorption bands. The data of all FTIR spectra were analyzed according to the method described in detail elsewhere.40 Briefly, the spectra were normalized using the band at about 1445-1455 cm-1 (variation along with the polarization angles) which is assigned to the CH3 in-plane bending vibration before data treatment. Deconvolution of amide III band (1300-1180 cm-1) was carried out using PeakFit v4.12. The number of peaks and their positions obtained from the second derivative spectra were fixed during the succeeding deconvolution process. A Gaussian model was selected for the band shape, and the bandwidth was automatically adjusted by the software. It should be pointed out that the data extracted from the deconvolutions (e.g., β-sheet content,


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etc.) were the average of more than five separate results from different positions and samples, although each spectrum shown in this paper was just from a single experiment. The molecular order parameter (Smol) of the corresponding secondary structural component was calculated as follows.41,42

where Ap(ν) and An(ν) are the absorbance values at wavenumber ν of a certain band with light polarized parallel and perpendicular to the fibre axis, respectively. 2.5. Raman Spectroscopy. Polarized Raman spectra of as-reeled A. pernyi silk in native and contracted (wet and dry) state were measured using a Renishaw inVia Reflex spectrometer coupled to a Lieca microscope. For each sample, both the spectra of fibre axis parallel and perpendicular with to the vibration direction of the laser beam were collected. Spectra were obtained from ten acquisitions of 50 s using the 785 nm line of a semiconductor laser with energy of approximately 300 mW. There were no signs of structural damage during the measurement, which was indicated by the repeatable Raman results on the silk fiber samples. 2.6. Carbon-13 cross-polarization magic-angle spinning nuclear magnetic resonance (13C CP/MAS NMR) Measurement. The

13

C CP/MAS NMR experiments were performed on a

Bruker AVANCEⅢ spectrometer with a 13C frequency of 150.96 MHz. Commercial Bruker triple-resonance 4-mm MAS probes and 4 mm zirconia rotors were used for the experiments. The 13C chemical shifts were determined from the carbonyl carbon signal (diso =176.5 ppm) of glycine relative to tetramethylsilane (TMS). The spectra were recorded at 10 kHz spinning rate, the 90° pulse lengths were 4.0 ms. To obtain the sufficient silk samples for NMR test, mature A. pernyi silkworm a was placed on a PET sheet so that it would spin a silk mat on the sheet in the natural “Figure of Eight”, and the silk mat was removed from the plastic sheet carefully. For the


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water contracted samples, a certain number of mats were immersed in water for at least 24 h, then they were dried completely for NMR test. 3. RESULTS AND DISCUSSION 3.1. Tensile Performance of As-reeled A. pernyi Silks in Native and Contracted State. In our recent work, we found that as-reeled A. pernyi silks from different silkworms (more than 10 silkworms) show significant variability of properties, and they can be grouped into four generic types of samples mainly according to their different tensile responses to methanol and ethanol.21 Later, the corresponding structure origin of variability of properties of these silks was studied in detail,22 and the results indicated that hydrogen bond stacking state in the amorphous regions and the overall ordered fraction are dramatically different for silks from different individuals. Here, silk fibres from two individuals (labelled artificially as silkworm a and silkworm b), which represent the lower limit (silk from silkworm a) and upper limit (silk from silkworm b) of the distribution of properties and then structures, were chosen to investigate their properties and structure changes induced by water. The stress-strain curves of those as-reeled A.pernyi silks before and after contraction in water are shown in Figure 1 and the detailed mechanical properties are illustrated in Table 1. In native state, the two kinds of silk fibre present prominently different mechanical properties. Specifically, silk fibre from silkworm b displays higher modulus, yield and breaking stress, resilience, and shrinkage, but lower extensibility than those from silkworm a, which indicate noticeable variability of properties and are consistent with our previous observations.21,22 After pre-immersed in water for 24h, similarly, the contracted silk fibres show largely decreased initial modulus, yield stress, breaking stress and resilience compared to those in native state. However, the change of post-yield modulus and breaking strain are obviously different for these two kinds


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of silk fibres. For silks from silkworm a, the post-yield modulus increases and the breaking strain decreases after contraction in water, but they are just the reverse for samples from silkworm b. Undoubtedly, these significantly different property changes during water contraction derive from the intrinsic distinct structures of these two silk fibres (for example, the content of different secondary structures and the orientation of molecular chains) and they should have different response to water, which is described and analyzed in the following section. 800

a

600

800

Silkworm a As-reeled silk Water contracted silk

Stress (MPa)

Stress (MPa)

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

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400

200

b

Silkworm b

600 1 3

2

400

200

0 0

20

40 Strain (%)

60

80

0 0

20

40 Strain (%)

60

80

Figure 1. Typical stress–strain curves of as-reeled A. pernyi silks from silkworm a (a) and silkworm b (b) before (black lines) and after (red lines) contraction in water (in dry state). The cross sections of the silk-embedded epoxy resin are inserted in (a) and (b). The formation of the cyclic curve is also inserted in (b). The sample was first stretched a strain of 17% (1) and then released to zero strain (2), after that, it was re-stretched to its breaking point (3). Legend is the same for both (a) and (b). Scale bars: 10 µm.


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Table 1. Mechanical properties of as-reeled A. pernyi silks from two different individuals in native and contracted state a Initial modulus (GPa)

Post-yield modulus (GPa)

Yield stress (MPa)

Breaking stress (MPa)

Breaking strain (%)

Resilience (%)

Shrinkage (%)

Native (n=10)

10.4 ± 0.4

0.20 ± 0.04

203 ± 14

509 ± 48

69.6 ± 5.2

35.6 ± 0.3

5.5 ± 0.2

Contracted (n=6)

8.8 ± 0.2

0.63 ± 0.04

148 ± 2

495 ± 32

61.1 ± 4.4

34.2 ± 0.3

Native (n=9)

12.4 ± 1.2

1.52 ± 0.15

220 ± 28

636 ± 41

30.7 ± 4.8

44.1 ± 0.8

Contracted (n=6)

9.1 ± 0.4

1.34 ± 0.05

155 ± 10

560 ± 28

38.7 ± 2.4

39.0 ± 0.4

Silkworm a

Samples

Silkworm b

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

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a

7.4 ± 0.4

All values measured were expressed as mean ± standard deviations (SD).

3.2. Structure Transition of As-reeled A. pernyi Silks with Water Treatment Monitored by S-FTIR and Raman Methods. The structure difference between these two as-reeled A. pernyi silks and their structure change induced by water were first investigated by the S-FTIR and Raman methods. S-FTIR spectra show that as-reeled A. pernyi silk from silkworm a (Figure 2a) contains more α-helix structures (peaks at 1332, 1306, 1265 and 1105 cm-1) than the silk fibre from silkworm b (Figure 2b). After contracted in water, the peaks assigned to α-helix structures notably decrease for silk fibre from silkworm a. However, the spectrum of contracted silk fibre from silkworm b is quite similar to that of native sample. These are consistent with the Raman spectra results (Figure 2c and 2d) that contracted silk fibre from silkworm a shows dramatically decreased α-helix structure with peaks at 1658 and 1105 cm−1 compared to the native silk, and there is no observable spectral difference between native and contracted silk


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fibres from silkworm b. Besides, S-FTIR spectra of silk fibre from silkworm a show that the βsheet structures (965 cm−1) increase slightly after water treatment. It is generally accepted that A. pernyi silk mainly contains three conformations: β-sheet, αhelix and random coil.20 In order to analyze in detail the content of these three different secondary structural components before and after water treatment, amide III bands in FTIR spectra were deconvoluted according to the peak assignment of these three conformations described elsewhere.20 Specifically, the peak at 1222 cm-1 is assigned to β-sheet conformation, while the peaks at 1241 and 1265 cm-1 are assigned to the random coil and α-helix, respectively. The detailed results of the peak recognition and deconvolution are shown in Figure S1 and the content of these different conformations in native and contracted states are summarized in Table 2. For the silk fibre from silkworm a, α-helix conformation declines and β-sheet conformation increases significantly after contracted in water, while the random coil conformation increases slightly. This indicates that transition from α-helix to β-sheet is induced by water treatment. However, these three conformations almost keep unchanged before and after water contraction for silk fibre from silkworm b, which means there is no definite conformational transition. It should be mentioned that we didn’t consider the contribution of A. pernyi sericin coating on the content of different conformations of silk fibre because of its slight content and uniform distribution on the surface of silk fibre, as described above. In fact, XRD result shows that A. pernyi sericin mainly take amorphous structure (Figure S2a), which fits in well with the XRD result reported by Yang et al.43 In addition, S-FTIR result further excludes the existence of αhelix structure in A. pernyi sericin (Figure S2b).


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965

1500

1400

1300 1200 1100 Wavenumbers (cm-1)

c

1000

900

Parallel 1669 Perpendicular 1658 1105 Native

905

Contracted

900

1000

1100 1600 Raman shift (cm-1)

1265 1332 1306

1105

Absorbance

1332 1306

b

Native silk Contracted silk

1105

Absorbance

1265

965

1500

1400

1300 1200 1100 Wavenumbers (cm-1)

1000

900

1669 1658

d 905

1105 Native

Intensity (a.u.)

a

Intensity (a.u.)

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Contracted

1700

900

1000

1100 1600 Raman shift (cm-1)

1700

Figure 2. S-FTIR microspectra (a, b) and Raman spectra (c, d) before and after contraction in water: as-reeled A. pernyi silks from silkworm a (a, c) and b (b, d). A silk fibre was aligned either parallel (solid line) or perpendicular (dashed line) to the vibration direction of the laser light for Raman characterization. The same legend for (a) and (b), (c) and (d).


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Table 2. Comparison of secondary structures of as-reeled A. pernyi silks from two different silkworms in native and contracted state a Content (%) Conformation

Silkworm a (n=5)

Silkworm b (n=5)

Native

Contracted

Native

Contracted

β-sheet

20 ± 1

28 ± 2

28 ± 2

27 ± 1

Random coil

46 ± 2

48 ± 1

48 ± 3

48 ± 2

α-helix

34 ± 2

24 ± 1

24 ± 1

25 ± 3

a


We also compared the S-FTIR microspectra of as reeled A. pernyi silks in native and contracted states obtained from different polarization angles (0° to 90°). As for silk fibre from silkworm a, it shows that α-helices are nearly distributed evenly within native silk fibre (Figure S3a) and they all decrease dramatically after water treatment (Figure S3b). However, the absorption peaks at 1222 and 965 cm-1 assigned to β-sheet structure all become gradually smaller from 0° (parallel to the fibre axis) to 90° (perpendicular to the fibre axis) for both native and contracted silks, indicating parallel dichroism. Besides, we can clearly see from peak at 965 cm-1 that β-sheet structure of different angles all increase after water treatment, further indicating the conformational transition from α-helix to β-sheet. However, for sample from silkworm b, its SFTIR microspectra at different polarization angles are quite similar to those of contracted silk fibre from silkworm a (Figure S3b), and they keep unchanged after contraction in water. To further explore the variation of orientation of β-sheet structure during water treatment, we focused our analysis on the single peak at 965 cm-1 because it uniquely represents the β-sheet structure.20 As shown in Table 3, in native silk from silkworm a, the molecular order parameter


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Smol value is 0.83 ± 0.03, but it increases to 0.87 ± 0.02 after water contraction, suggesting that the β-sheet structure in native silk fibre from silkworm a is poorly oriented along the fibre axis and contraction slightly increases its order. Nevertheless, the order parameter Smol of β-sheet structure of native silk from silkworm b reaches 0.93 ± 0.01 and it keeps constant at 0.93 after water treatment. In addition, there is nearly no absorption appearing at 90° (Figure S4a and S4b). These facts indicate that the β-sheet structure in this kind of sample is almost perfectly orientated along the fibre axis and it is immune to water treatment. It should be noted that the influence of water treatment on the orientation of β-sheet structure of A. pernyi silk either from silkworm a or b is inconsistent with that of spider dragline silk (eg. Nephila edulis).42 The orientation of β-sheet structure of spider dragline silk is decreased after supercontraction in water, compared to that of native silk. This inconsistence may be attributed to the distinguishing ordered fractions of these two kinds of silks,15 A. pernyi silk shows higher ordered fraction (or degree of crystallinity) than spider dragline silk. As such, the β-sheet structure of A. pernyi silk is difficult to move to change its orientation during water treatment. For the silk from silkworm a, the increase of orientation of β-sheet structure should arise from the conformational transition during water treatment.


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Table 3. Molecular order parameter (Smol) of different conformations of as-reeled A. pernyi silks from two different silkworms in native and contracted state a Smol Conformation

Silkworm a (n=4)

Silkworm b (n=3)

Native

Contracted

Native

Contracted

β-sheet (965 cm-1)

0.83 ± 0.03

0.87 ± 0.02

0.93 ± 0.01

0.93 ± 0.02

β-sheet (Amide III)

0.45 ± 0.03

0.53 ± 0.02

0.48 ± 0.01

0.49 ± 0.01

Random coil (Amide III)

0.15 ± 0.02

0.10 ± 0.04

0.12 ± 0.01

0.13 ± 0.02

α-helix (Amide III)

0.02 ± 0.01

0.20 ± 0.02

0.23 ± 0.01

0.23 ± 0.02

a


Unlike β-sheet conformation, there is not a single and whole absorption peak in the S-FTIR spectrum of A. pernyi silk that only provide unambiguous information for either random coil or α-helix conformation. Therefore, we used amide III band to investigate the change of orientation of random coil and α-helix conformations induced by water, because this band can be deconvoluted into β-sheet, random coil and α-helix components, as mentioned above, and it shows remarkable dependence on polarization angle (Figure S3 and S4). The deconvolution results of amide III band acquired with the IR beam both parallel and perpendicular to the fibre axis are illustrated in Figure S5 and the calculated Smol value are summarized in Table 3. For sample from silkworm a, the order parameter for random coil conformation decreases slightly from 0.15 to 0.10 after water contraction, while it increases from 0.02 to 0.20 for α-helix. Besides, the order parameter of the β-sheet component increases slightly after water treatment, like that determined from 965 cm-1. We speculated that the change of Smol for silk fibre from silkworm a may arise from the structure transition induced by water. However, the Smol values of


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these three conformations in contracted silk from silkworm b are nearly the same as those in native silk fibre, which strongly indicate that contraction has no detectable effect on the orientation of different conformations in silk fibres from silkworm b. Noting that the Smol value determined from amide III band is calculated using the relative intensity of each conformation (dividing the area of each conformation by the peak area at ∼1450 cm-1) at 0° and 90°.20 It also can be seen from the Raman spectra in Figure 2c that the band at 905 cm-1, which is associated with the poly(alanine) segments,28 exhibits stronger polarization dependence after water treatment, demonstrating that the poly(alanine) blocks have a higher orientation in contracted silk than that in native silk from silkworm a. However, the orientation of poly(alanine) blocks in silk fibre from silkworm b is already high and nearly remains unchanged after water contraction (Figure 2d). These facts agree well with the FTIR results mentioned above, because poly(alanine) blocks are mainly involved in the β-sheet and α-helix. 3.3. Structure Transition of A. pernyi Silks from Silkworm a with Water Treatment Monitored by

13

C CP/MAS NMR Method. We performed

13

C CP/MAS NMR test on the A.

pernyi silk fibres (silk mat) from silkworm a in both native and contracted states to investigate the structure transition associated with the amino acid residues, especially the alanine (Ala) residues. The change in

13

C CP/MAS NMR spectra is presented in Figure 3a and 3b, and the

specific chemical shifts as well as the corresponding local conformation are summarized in Table S1.30,44 It should be mentioned that the secondary structure and tensile properties of silk mat used here are quite similar to those of as-reeled silk fibres from silkworm a. From the Ala Cα and Cβ peaks shown in Figure 3a, we can see that the Ala residues in the poly(alanine) segments44 in native silk fibres almost equally take both β-sheet and α-helix structures. In addition, the chemical shifts values of two Ser carbons (Cα and Cβ) also indicate


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that some Ser residues in native silk fibres take α-helix structures. However, after water contraction, the Ala Cα, Cβ, and C=O peaks at 52.9, 15.7 and 176.4 ppm (α-helix), respectively, decrease dramatically, and the Ala residues in the silk fibre take mainly β-sheet (20.5, 22.8, 49 and 172.4 ppm) conformation (Figure 3b). The Ser Cα and Cβ peaks also shift from 59.6, 60.9 ppm (α-helix) to 54.8, 63.5 ppm (β-sheet), respectively, although these peaks were broad.44 These results undoubtedly suggest that α-helix is converted to β-sheet for both Ala and Ser residues after water treatment. The Ser residues in A. pernyi silk fibroin are located at the position adjacent or close to poly(alanine) segments, and thus Ser residues are incorporated into α-helix or β-sheet conformation, which is consistent with the results reported previously.44

a

Ala C=O Gly C=O

random-coil α-helix β-sheet

b

Ala Cα Ala Cβ

Ala C=O

Ser Cα Ser Cβ

Gly Cα

Ser Cα Ser Cβ

200

175

150 50 ppm from TMS

25

0

30

25

20 15 ppm from TMS

10

Figure 3. (a) 13C CP/MAS NMR spectra of native (black line) and water-contracted (red line) A. pernyi silks. (b) Spectral decomposition (broken lines) of Ala Cβ peaks of A. pernyi silk fibres before (top) and after (bottom) water-treatment.

We also collected the

13

C CP MAS NMR spectrum of contracted silk fibres in wet state and

found that it significantly differs from that in dry state (Figure S6). Particularly, the wet silks show a clear decrease in the intensity for most of the peaks, with an exception for Ala Cβ peak


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that associated with the β-sheet structure. This demonstrates that water mainly penetrates the glycine rich regions and the poly(alanine) regions that adopt α-helix and random coil conformations rather than the poly(alanine) β-sheet domains in A. pernyi silks. The regions attacked by water display an increase in molecular chain mobility, which leads to a loss in CP signal.45 However, there is a decrease in the intensity for Ala Cα peak that associated with the βsheet structure, which should be caused by the loss of intensity of its low and high ppm shoulder. To detail conformation transition related to Ala residues, the Ala Cβ peak was expanded and decomposed (Figure 3c and 3d) and the extracted results are summarized in Table 4. After water treatment, the fractions of α-helix and random coil conformations decrease, while the fraction of β-sheet conformation increases dramatically compared to those before water treatment (Table 4), indicating that conformation transition from α-helix and/or random coil to β-sheet is induced by water for Ala residues. We speculate that the conformation transition related to the Ala residues is focused on the poly(alanine) regions, because the Ala residues in the glycine rich domain always adopt random coil conformation.44 In fact, the fraction of random coil component in contracted silks is in agreement with the content of Ala residues in the glycine rich domain (related to all Ala residues) that is predicted from the A. pernyi fibroin amino acid sequence.25 This further proves that the Ala residues in the poly(alanine) segments in native A. pernyi silk fibres from silkworm a take simultaneously three conformations, while those in contracted silk fibres just take both β-sheet and α-helix conformations.


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Table 4. The fractions of different conformations in Ala Cβ peak before and after water contraction Ala Fraction (%) Conformation Native

Contracted

β-sheet

41

74

Random coil

23

14

α-helix

36

12

3.4. Structural Model during Water Contraction. The three different analytical methods, i.e., S-FTIR, Raman, and

13

C CP/MAS NMR, clearly show that the structure transition occurs

remarkably for the silk fibres from silkworm a during water treatment. However, no observable structure change occurs for silk fibres from silkworm b. Obviously, the different structure variation of these two silk fibres when interacted with water arise from their intrinsic differences of structures, and S-FTIR results indeed show that the content and orientation of secondary structures (especially α-helix and β-sheet) is different for these two samples (Table 2 and 3). Besides, the different capacity of shrinkage of these two silks (Table 1) also suggests the different orientation of oriented amorphous regions, and it is much higher for silk fibre from silkworm b as it shows higher shrinkage than silk fibre from silkworm a.8 Furthermore, our previous study on hydrated A. pernyi silk suggests that water primarily penetrates into relatively disordered regions (amorphous regions) and breaks the hydrogen bonds consequently and improves the mobility of chains within these regions, while the well-defined crystalline regions remain rigid and intact.21 Based on these facts, we can now make a description of the molecular mechanism of the structure transition induced by water. When the silk fibre from silkworm a is submerged in


20

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water, largely agminated α-helix bundles in the silk fibre are easily attacked by water molecules, and they become more relaxed due to the breakage of the intra-chain hydrogen bonds within single α-helix structure. This is confirmed by the fact that α-helix structures almost become invisible (their peaks decrease significantly) in the

13

C CP/MAS NMR (Figure S6) and even

Raman (Figure S7) spectra when the silk fibres are immersion in water. At the moment, the amorphous regions in the silk fibre behavior like a rubber,21 and the stress−strain curve tested in water is nearly a straight line, with the modulus almost equivalent to the post-yield dry modulus of water contracted A. pernyi silk tested in air (Figure S8a and b). Then, in the following drying process, the adjacent unconfined α-helix structures would be closer to each other, and thus gives rise to the structure transition from α-helix to β-sheet through the conversion of hydrogen bonding pattern from intra-chain to inter-chain. The random coil conformation taken by a few part of poly(alanine) segments can also be converted to β-sheet with the aid of water molecules. Meanwhile, this structure transition would lead to the change of orientation of these secondary structural components (Table 3). For the silk fibre from silkworm b, it contains a small number of α-helix structures and they are isolated to each other. Although their intra-chain hydrogen bonds can also be destroyed by water molecules, there is no new hydrogen bonding to be formed between different α-helix structures in the drying process, hence, no structure transition occurs. In addition to the α-helix domains, water can also insinuate into the oriented amorphous regions of both silks and consequently cause the rearrangement of hydrogen bonding structure (disorientation of molecular chains), although these changes cannot be definitely determined by our methods used here.8,42 The detailed structure transition models are summarized in Figure 4. Next, we can interpret the mechanical response of these two kinds of silk fibres to water treatment. After water submersion, the chain segments that are penetrated by water molecules


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become more loosely packed, and then less strain energy is needed to unfold these chain segments when the silk fibre is subjected to tension.46 Therefore, the contracted silks display lower initial modulus, yield stress and resilience than those of native silk fibres (Figure 1 and Table 1). For silk fibre from silkworm a, the increase of content of β-sheet conformation and the increase of orientation of both β-sheet and α-helix conformations after water treatment should be the origin of the increased post-yield modulus and decreased breaking stain of contracted silk compared to the native silk (Figure 1a and Table 1). However, for silk fibre from silkworm b, the increase of breaking strain should be attributed to the reduction of orientation of molecular chains in the amorphous regions.

Figure 4. Structure transition models of as-reeled A.pernyi silk fibres from two different silkworms during water contraction.

4. CONLUSIONS


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In summary, we have investigated the water induced properties and structure transition information of as-reeled A. pernyi silk fibres from two different individuals. Detailed mechanical properties data indicate that initial modulus, yield stress, breaking stress and resilience all decrease after water contraction for these two kinds of silk fibres. However, post-yield modulus increase, and breaking strain decrease for silk from silkworm a, and they are just the reverse for silk from silkworm b. Both S-FTIR and Raman spectra show that silk fibre from silkworm a contains more α-helix conformation than silk from silkworm b, and these α-helices are partially converted to β-sheet structures after water treatment for silk fibre from silkworm a, but there is nearly no conformation transition for silk fibre from silkworm b. In addition, the orientations of both α-helix and β-sheet structures increase along with the conformation transition generated in silk fibre from silkworm a.

13

C CP/MAS NMR results further suggest that structure transition

from α-helix and random coil to β-sheet is induced by water for Ala residues for silk fibre from silkworm a and it mainly occurs in poly(alanine) regions. Besides, similar structure transition from α-helix to β-sheet is also observed for Ser residues. Finally, the structure transition models of these two as-reeled A. pernyi silk fibres are described and we propose that α-helix to β-sheet transition is driven by the conversion from intra-chain hydrogen bonds to inter-chain hydrogen bonds. In combination with the previously published work, our results present here further shed light on the effect of water on the mechanical properties and structure of animal silk, and will provide valuable information for the interaction between water and other materials with hydrophilic groups.

ASSOCIATED CONTENT Supporting Information


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The Supporting Information is available free of charge on the ACS Publications website at DOI: . FTIR, XRD, Raman spectra, 13C CP/MAS NMR chemical shifts and spectra, and stress–strain curves (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions §These authors contributed equally to this work.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (NSFC 21574024 and 21574023) and Ministry of Science and Technology of China (2016YFA0203301). The authors also thank Prof. Tetsuo Asakura at Tokyo University of Agriculture and Technology, and Dr. Jinrong Yao at Fudan University for their valuable suggestions and discussions.

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Graphic Abstract


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Understanding the Mechanical Properties and Structure Transition of

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