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Insight into the Structure of Single Antheraea pernyi Silkworm Fibers Using Synchrotron FTIR Microspectroscopy Shengjie Ling,† Zeming Qi,‡ David P. Knight,§ Yufang Huang,∥ Lei Huang,† Huan Zhou,† Zhengzhong Shao,† and Xin Chen*,† †

State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Laboratory of Advanced Materials, Fudan University, Shanghai, 200433, People’s Republic of China ‡ National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, 230029, People’s Republic of China § Oxford Biomaterials Ltd., Magdalen Centre, Oxford, OX4 4GA, United Kingdom ∥ Department of Material Science, National Microanalysis Center, Fudan University, Shanghai, 200433, People’s Republic of China S Supporting Information *

ABSTRACT: Synchrotron FTIR (S-FTIR) microspectroscopy was used to monitor both protein secondary structures (conformations) and their orientations in single cocoon silk fibers of the Chinese Tussah silk moth (Antheraea pernyi). In addition, to understand further the relationship between structure and properties of single silk fibers, we studied the changes of orientation and content of different secondary structures in single A. pernyi silk fibers when subjected to different strains. The results showed that the content and orientation of β-sheet was almost unchanged for strains from 0 to 0.3. However, the orientation of αhelix and random coil improved progressively with increasing strain, with a parallel decrease in α-helix content and an increase in random coil. This clearly indicates that most of the deformation upon stretching of the single fiber is due to the change of orientation in the amorphous regions coupled with a conversion of some of the α-helix to random coil. These observations provide an explanation for the supercontraction behavior of certain animal silks and are likely to facilitate understanding and optimization of postdrawing used in the conjunction with the wet-spinning of silk fibers from regenerated silk solutions. Thus, our work demonstrates the power of S-FTIR microspectroscopy for studying biopolymers.

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INTRODUCTION The complete amino acid sequence of silk fibroin of the Chinese (Oak) Tussah silkworm Antheraea pernyi (A. pernyi) cocoon silk is remarkably similar to that of the partial sequence of the major ampullate silk spidroin I (MaSp I) of the Golden Orb Web spider (Nephila clavipes) but with longer poly(alanine) repeat regions.1,2 Both proteins contain very large central repetitive regions consisting of poly(alanine) domains (poly(Ala)12 in Antheraea fibroin and mostly poly(Ala)6−7 in Nephila spidroin I) alternating with glycine-rich regions. The latter regions in both species contain similar motifs, for example GGYG, GGAG and GSGA.2,3 The shorter poly(alanine) length in the Nephila silk is thought to contribute to its lower crystallinity compared with Antheraea silk while the latter silk has a less crystalline structure than that of the silk of the domestic silkworm Bombyx mori (B. mori). Thus the β-sheet content of A. pernyi cocoon silk is intermediate between that of B. mori silk and Nephila spider major ampullate silk while the mechanical properties of the Antheraea silk are also intermediate between those of the other two silks.4 For this reason, studies on this intermediate type of silk may help to © 2013 American Chemical Society

account for the mechanical properties of the whole family of animal silks.1,5,6 The earliest studies on the secondary structure of A. pernyi silk fibroin were on regenerated silk fibroin films.7−10 Those studies show that the α-helix is the predominant conformation in these films as cast, but is converted to a β-sheet structure by heat or alcohol treatment.4,7,8 However, studies on the secondary and higher order structure of native A. pernyi silk and the relationship between the structure and mechanical properties are still fairly limited1 though work on the not dissimilar Samia cynthia ricini (S. c. ricini) silk has uncovered much useful information on the detailed secondary structure of both poly(alanine) and glycine-rich regions before and after the conformation transition.11−13 Although the mechanical properties of commercial A. pernyi silk fibers are inferior to the benchmark Nephila spider major ampullate silk, our recent research showed that silk fibers Received: February 19, 2013 Revised: March 28, 2013 Published: April 22, 2013 1885

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directly reeled from A. pernyi caterpillars were similar in breaking stress and toughness to both B. mori silk prepared in the same way and to the benchmark spider silk.1 The other mechanical properties, such as elasticity, supercontraction, and the effect of water on the modulus of A. pernyi silk were between those of spider major ampullate silk and directly reeled B. mori silk. This suggested that the relatively poor mechanical properties of commercial, degummed A. pernyi cocoon silk may result from structural defects introduced by the “Figure of Eight” natural spinning process and by damage produced by degumming. We proposed a general model relating the mechanical properties of the silks to the primary structures of their silk proteins,1 while our previous work on B. mori silk showed that silk protein secondary structure is important in defining the mechanical of the fibers.14 A variety of characterization methods have been used to monitor the secondary structure of silk proteins in silk fiber, including X-ray diffraction (XRD),15−21 neutron spectroscopy,18 solid-state NMR,11,12,22−24 FTIR spectroscopy,25−30 and Raman spectroscopy.31−41 These complementary methods have provided a wealth of structural information about silk. However, the vast majority of these studies were carried out on silk fiber bundles rather than single fibers. The former are not optimal for studying the relationship between structure and mechanical properties because chemical composition, secondary structures, fiber morphology, and mechanical properties can vary from single silk fiber to fiber.42−44 Synchrotron XRD19−21 and Raman spectroscopy31−41 have been applied to single silk fibers, giving some useful information. XRD is useful in determining the sizes, structures, and orientations of the crystallites and for roughly quantifying the crystalline fraction but is less useful for studying the amorphous components and cannot be used for accurately determining the content of the different secondary structures. Based on its scattering nature, Raman spectroscopy is more suitable for qualitative measurement than quantitative analysis, as the later needs rather restrict conditions45,46 compared to the absorption infrared spectroscopy that obey Beer−Lambert Law. Although conventional FTIR spectroscopy is one of the oldest and well-established experimental techniques for conformational analysis of polypeptides and proteins,47 there appear to be no reports to date of its use to characterize single silk fibers except the one from Boulet-Audet et al.48 The main difficulty is that the diameter of the light beam is three magnitudes larger than the 5−20 μm width of a normal single silk fiber. Thus, a single silk fiber is only exposed to a small part of the infrared beam, creating a serious signal-to-noise problem which results in very poor quality of the spectra rendering them almost useless. Although Boulet-Audet et al. successfully applied ATR infrared spectroscopy to analyze single silk fiber, they used their own smart-designed custom-built sample holder and the diameter of focused infrared beam (about 750 μm) was still much larger than the sample size. In addition, it is well-accepted that quantitative analysis from ATR spectrum is always affected by the possible spectral distortion arisen from the principle of this mode. This problem has recently been solved by our use of synchrotron FTIR (S-FTIR) microspectroscopy.4 In our previous published Note, we briefly demonstrated that such a method can be used to analyze the tiny single silk fibers qualitatively and quantitatively because of the ultrabrightness of high spatial resolution of synchrotron infrared beam. S-FTIR can be performed as easy as the conventional FTIR, without any further needs for accessories or custom-built apparatus. In

the present communication we report the use of this technique to study in detail both the content of the secondary structures and their orientation in single A. pernyi silk fibers and how both of these are by tensile deformation.

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EXPERIMENTAL SECTION

Preparation of Single A. pernyi Silk Fibers. Raw A. pernyi silk fibers obtained from Shandong Province, China, were degummed by boiling in two 30 min changes of 0.5% (w/w) Na2CO3 solution. The degummed fibers were then washed with distilled water and allowed to air-dry at room temperature. Single brins (silk monofilaments) were selected from those degummed silks. Preparation of Regenerated A. pernyi Silk Fibroin Films. A. pernyi silk fibroin films were prepared by dissolving the degummed silk fibers in 7.5 mol/L Ca(NO3)2 aqueous solution at 90 °C. The resulting solution was dialyzed against deionized water for 3 days at 4 °C, then cast onto a polyethylene plate with a smooth surface, and allowed to dry at approximately 25 °C and 50% relative humidity to give films approximately 5 μm thick. FTIR Spectroscopy of A. pernyi Silk Fibroin Films. FTIR spectra were recorded using a Bruker 66v/s FTIR spectrometer with a liquid nitrogen cooled MCT detector. To eliminate interference from water at 1500−1700 cm−1, the entire light path of instrument was continuously evacuated with a Nidec rotary vacuum pump. For each time of measurement, 256 interferograms were coadded and transformed employing a Genzel-Happ apodization function to yield spectra with a nominal resolution of 4 cm−1. Polarized S-FTIR Microspectroscopy of Single A. pernyi Silk Fibers. The experiments were performed at Beamline U4 in the National Synchrotron Radiation Laboratory (NSRL, Hefei, China). The instrumental details are presented in our previous paper.4 We shall emphasize here that a relatively large 20 × 20 μm square aperture was selected in this study because of the large width of flat A. pernyi silk (≥30 μm). We put this aperture in the center of the silk in order to avoid the diffraction and the scattering of the infrared light. In addition, for each silk sample (1 cm long), we chose three different positions to analyze, and if there is no variation in these three S-FTIR spectra, we took it as one effective datum. A KRS-5 IR polarizer was inserted in the infrared beam in order to study the dichroism of certain absorption bands. To study the effects of deformation, single fibers were firmly mounted on a custom-built apparatus with a modified engineer’s screw micrometer to precisely control the extension of a single fiber. Strains of 0.10, 0.20, and 0.30 were used. Single fibers were brought to the required extension over a period of about 1 min in air at 25 °C and 30% relative humidity and left to equilibrate for 1 h under these conditions before recording spectra in the synchrotron beam. During the measurement, the tension was kept though all the strain we chosen ensured the fiber was in its plastic region. S-FTIR microspectra were collected in the mid-infrared range of 800−3800 cm−1 at a resolution of 4 cm−1 with 256 coadded scans. Data Processing of FTIR Spectra. Background was collected each time before all FTIR spectra of silk fibroin films and single silk fibers were collected. Nine-point smoothing was used prior to obtaining second derivative spectra for single silk fibers with OPUS 6.5. Deconvolution of amide III band was carried out using PeakFit 4.12. The number of peaks and their positions were obtained from the second derivative spectra and fixed during the subsequent deconvolution process. As in our previous studies, a Gaussian model was selected for the band shape, and the bandwidth was automatically adjusted by the software.49 It should be noted that each spectrum shown in this paper was from a single experiment, but the data obtained from the spectra (e.g., β-sheet content, etc.) were the average of more than five separate deconvolutions from different samples. The significance of differences in β-sheet content as well as molecular order parameter (Smol, see below) of different silk fibers was analyzed by oneway ANOVA. Before one-way ANVOA analysis we used Shapiro-Wilk method to check data normality and Levene’s test to check homogeneity of variance. We also used Duncan’s multiple range test (DMRT) for multiple comparisons of the data. 1886

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Calculations of Absorbance Profile and Molecular Order Parameter. The orientation of certain moieties can be obtained from the angular dependence of the absorbance A(ν) at wavenumber ν which corresponds to a vibration of the molecular group under investigation. In the general case, the angular dependence of the absorbance can be determined using the following function.26−28 A(ν , Ω) = − log10{10−A max (ν)cos2(Ω − Ω 0) + 10−A min(ν)cos2(Ω − Ω 0)}

(1)

where A(ν, Ω) is the peak intensity of a certain band, Ω is the polarization angle, Ω0 is the angle at maximum absorption, and Amax and Amin are the maximum and minimum absorbance, respectively. The molecular order parameter (Smol) of the corresponding secondary structural component was calculated as follows.48

S

mol

A (ν) − A min (ν) = max A max (ν) + 2A min (ν)

Figure 1. S-FTIR microspectra of A. pernyi silk materials: (a) silk fibroin film as cast, (b) silk fibroin film treated with 70% ethanol aqueous solution at room temperature for 24 h and subsequent drying, (c) single silk fiber measured with the infrared beam parallel to the fiber axis, and (d) single silk fiber measured with the infrared beam perpendicular to the fiber axis.

(2)

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RESULTS AND DISCUSSION Peak Assignment of A. pernyi Silk Fibroin. FTIR spectra of A. pernyi silk fibroin films have been widely studied and absorption bands mainly in amide I and II regions assigned as follows.4,7−10 Generally, the peaks centered at 1655−1660 and 1545 cm−1 are attributed to random coil and helical conformation, and the peaks at 1620−1630 and 1525 cm−1 are attributed to β-sheet. It is well-known that the amide I and II bands are extremely sensitive to atmospheric water vapor, so it is difficult to analyze those two amide bands quantitatively unless water is completely excluded from the specimen and light path. However, when measuring single silk fiber under FTIR microscope, it is almost impossible to do so. In our previous work, we proved that the less-water sensitive amide III band can be used alternatively to analyze quantitatively the conformation composition of silk fibroin in place of amide I band most frequently used for this purpose.4 Using silk fibroin films, we assigned the adsorption peaks in amide III band as follows, 1222 cm−1 to β-sheet, 1242 cm−1 to random coil, and 1265 cm−1 to α-helix, by comparing them to the corresponding peaks in amide I band under well-controlled experimental conditions.4 In the present communication, we also paid attention to absorption peaks in the 1800−800 cm−1 region besides the amide I to amide III bands as these fingerprint peaks may help us to fully understand the secondary structure of A. pernyi silk fibroin. We still used ethanol to induce the conformation transition from random coil and α-helical to β-sheet in thin A. pernyi silk fibroin films as in our previous work4 and the corresponding conventional FTIR spectra before and after the transition are presented in Figure 1. This figure clearly shows that the peaks at 1332, 1306, 1105, and 892 cm−1 peaks were only present in the untreated films (curve a). These peaks are present in the FTIR spectra of polyalanine with α-helix conformation.50 Further strong evidence for the predominance of α-helix in untreated A. pernyi silk fibroin films comes from solid-state NMR, XRD, and Raman spectroscopy.3,7,8,51 Accordingly, these peaks in A. pernyi silk fibroin are assigned to α-helix conformation. After ethanol treatment, a new peak at 965 cm−1 appears (curve b). This peak also has been seen in FTIR spectra of spider dragline silk fiber and poly(alanine) with β-sheet conformation,26−30,52 so we are confident in assigning this peak to β-sheet. The detailed assignments of A. pernyi silk

fibroin in the 1800−800 cm−1 region are summarized in Table S1 based on both classical assignments and our observations. Inherent limitations in conventional infrared techniques (ATR-FTIR and FTIR microspectroscopy) prevent highquality spectra from being obtained from a single A. pernyi single silk fiber. Previous attempts have been made to use bundles of A. pernyi silks to overcome the size limitation of the large beam diameter,25,53 but the low signal-to-noise ratio and distortion of ATR-FTIR spectra, as well as the different thickness and packing the individual silk fibers affected the quality of the spectra obtained. Consequently, no detailed assignment of infrared absorption peaks or polarized FTIR spectra have been reported for A. pernyi silk fibers. The intense brightness and polarization of synchrotron sources, enabled us to obtain high quality polarized S-FTIR microspectra of single A. pernyi silk with the plane of polarization either parallel or perpendicular to the long axis of the fiber (Figure 1, curves c and d). This figure clearly shows that β-sheet is the dominant conformation in single A. pernyi silk fibers and that peaks assigned to α-helix are, depending on the polarization angle, either undetectable or relatively small. In addition, not like the major ampullate spider silk is round and B. mori silk is triangle, A. pernyi silk is flat with the maximum width of about 30 μm and quite even thickness of about 10 μm (Figure S1). This ensures the infrared beam can totally pass through the sample, so there was no problem either with the stray light or the possible band broadening from the nonuniform thickness of the sample. Infrared dichroism gave interesting new information. As would be predicted from the conventional model for the orientation of β-sheet in silk fibers, the β-sheet peaks at 1222 cm−1 (N−H bending) and 965 cm−1 (C−N stretching) showed significant dichroism parallel to the fiber long axis. Other βsheet peaks at 1373, 1054, and 925 cm−1 showed perpendicular dichroism, which may came from the movement of CH3 groups in alanine residues. On the other hand, the peaks at 1405 and 1168 cm−1 also showed significant parallel dichroism but were not sensitive to the conformation change, as they have similar size in spectra from A. pernyi silk fibroin films before and after ethanol treatment (curves a and b, Figure 1). The assignments and dichroism of these and other absorption peaks from 1800 1887

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to 800 cm−1 in A. pernyi single silk fibers are summarized in Table S1. Orientation of Molecular Chains in Native Single A. pernyi Silk Fiber. One of the most useful quantitative comparisons for the fiber orientation is the molecular order parameter (Smol) derived by analyzing the absorption bands unambiguously assigned to specific groups at different polarization angles.26−30 The theoretical boundary values of Smol are +1 and −0.5, which correspond respectively to perfectly parallel and perfectly perpendicular orientation with respect to the fiber axis. Figure 2 shows a series of S-FTIR microspectra for 1500− 800 cm−1 from single A. pernyi silk fibers obtained for different Figure 3. Polar plot of the absorbance of the characteristic peaks in SFTIR microspectra in the 1500−800 cm−1 region from single A. pernyi silk fibers. The symbols represent individual experimental data points, while the curves are fitted using eq 1.

represents β-sheet structure in A. pernyi silk. A similar polar plot for the 965 cm−1 band in the major ampullate spider silk of Nephila inaurata madagascariensis was obtained by Papadopoulos et al., using conventional FTIR spectroscopy on a bundle of orientated silk fibers.27 The results presented here from the polar plot give strong support to the previous studies using solid-state NMR, XRD, and Raman spectroscopy to show that the β-sheets are highly orientated in two Saturniid silkworm silk fibers (those of A. pernyi and S. c. ricini).5,11,36 Unlike the situation with β-sheet, for which we monitored the orientation using the nonoverlapping peak at 965 cm−1, there is not a single absorption peak in the S-FTIR spectrum of A. pernyi silk that uniquely corresponds to either the random coil or α-helix conformation. However, Figures 1 and 4 show that the amide III region showed a significant dependence on Ω, and this band can be deconvoluted into β-sheet, random coil, and α-helix components. There may be other intermediate conformations like turns and bends in the fiber; we considered them as random coil here as they had a large difference from the ordered α-helix and β-sheet structures. We focused on the amide III band to investigate the orientation of random coil or α-helix in single A. pernyi silk fibers. Typical deconvolution results of amide III band obtained from both the infrared beam parallel and perpendicular to the fiber axis are shown in Figure S2. By plotting the relative intensity of each conformation at different polarization angles, we obtained the polar plot of βsheet (1222 cm−1), random coil (1242 cm−1), and α-helix (1265 cm−1) component in amide III band shown in Figure 5. The order parameter Smol value of β-sheet, α-helix, and random coil were, respectively, 0.63 ± 0.07, 0.15 ± 0.04, and 0.07 ± 0.04 (see Table 1). We found if we simply used the absorbance of β-sheet peak at 1222 cm−1 to calculate Smol, it was only 0.48 ± 0.04; thus, it seems more dependable to calculate Smol value of overlap bands by deconvolution. From Figure 5 it can be seen that the α-helix conformation and random coil in the native A. pernyi silk fibers showed a slight preference for orientation parallel to the fiber axis. This observation agrees with the studies on spider major ampullate and S. c. ricini silks using Raman spectroscopy,36 solid-state NMR,24 and XRD.54 Our observation of slight but significant order in the α-helical and random coil conformations may be relevant to supercontraction. This behavior, first seen in spider major ampullate

Figure 2. S-FTIR microspectra of single A. pernyi silk fibers with different polarization angle from 0° to 90°.

polarization angles (Ω, 0−90° in 10° steps). It clearly shows the coupled absorption β-sheet peaks at 1222 and 965 cm−1 became progressively smaller from Ω 0° (beam parallel to the fiber axis) to Ω 90° (perpendicular to the fiber axis), indicating parallel dichroism, while β-sheet peaks at 1373, 1054, and 925 cm−1 progressively increased, indicating perpendicular dichroism. In addition, we found that although the band attributed to the CH3 in-plane bending vibration shifted progressively from 1455 to 1445 cm−1 when Ω changed from 0° to 90° the intensity remained constant. Figure 3 is a polar plot of the S-FTIR absorbance for different peaks at 1500−800 cm−1 obtained from single A. pernyi silk fibers, in which the solid lines are fitted with eq 1 (see section 2.6). The figure shows that many absorption bands have dichroism, indicating polarized S-FTIR microspectroscopy is a suitable tool to study the orientation and order parameter of vibration modes and conformations in single silk fibers. All of the β-sheet peaks as mentioned above showed considerable order, for instance, the absorbance of the peak at 965, 1222, and 1405 cm−1 showed considerable order parallel to the fiber axis, while on the other hand, the absorbance of the peak at 1054 and 1373 cm−1 showed significant order perpendicular to fiber axis. Among these β-sheet peaks, that at 965 cm−1 showed the most obvious dichroism (Smol = 0.93 ± 0.04, almost no absorption appearing at Ω = 90° or 270°), indicating the βsheet in the single A. pernyi silk is almost perfectly orientated along the fiber axis. We know that silk fibers are composed of different secondary structures, so the change of certain specific absorption peaks may not totally be attributed to β-sheet, and that is why the Smol of 965, 1222, and 1405 cm−1 band are different. However, the adsorption peak at 965 cm−1 is −CH3 rocking, which mainly comes from alanine residues, so it mainly 1888

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Figure 4. Amide III band of S-FTIR microspectra of single A. pernyi silk fibers subject to difference strain: (a) the infrared beam is parallel to the fiber axis; (b) the infrared beam is perpendicular to the fiber axis.

occur in the spinning of spider silks in nature.59 However, little is known about what happens at the molecular level when silk fibers are strained or drawn.21 Therefore, for this article, we used S-FTIR microspectroscopy to study structural changes of single A. pernyi silks that were maintained at known tensile deformations. We used the same methods described above to deconvolute the amide III bands in spectra obtained at four different strains and to determine order parameters (Smol) for each conformation at different strain. Tables 1 and 2 (also see Table S2 and S3 for statistical details) summarize the results from this study. It can be seen in Table 1 that the order parameter of the βsheet component determined from the amide III band showed no significant increase over the range of strains from 0 to 0.3. This lack of change in order with strain was confirmed for individual β-sheet peaks at 1373, 1222, 1054, and 965 cm−1 (see Table 2). By referencing Table 3 (also see Table S4 for statistical details) it can be seen that the content of the β-sheet component determined by deconvolution of the amide III band was within the experimental error maintained at about 38% at all strains. These results strongly indicate that stretching has no detectable effect on the β-sheet content and orientation in single A. pernyi silk fibers. However, this is not to say that an increase in strain from 0 to 0.3 strain had no effect on the βsheet component; indeed, the position of the corresponding absorption peaks changed, the peak at 1222 cm−1 shifting continuously to 1217 cm−1, while that at 965 cm−1 shifts to 963 cm−1 (see Figures 4 and S3). Band shifts to lower wavenumbers have also been observed in strained B. mori silk using FTIR28 and Raman microspectroscopy.37,39−41 This behavior has been attributed to alterations in bond lengths, bond angles, and internal rotation angles of polypeptide chains,37 which shows the β-sheet crystals could be elastically deformed under external strain. Interestingly different types of silks with different

Figure 5. Polar plot of the relative intensity of different component from the deconvolution of amide III band in S-FTIR microspectra of single A. pernyi silk fiber. Letters α, β, and c represents α-helix, β-sheet, and random coil conformation, respectively.

silk but now known to be quite common though not seen in the highly crystalline B. mori silk, is thought to be driven by the recoverable disorientation of the molecular chains in oriented amorphous regions of the fibroin.1,55 Interestingly, we found supercontraction in forcibly reeled A. pernyi silk, thus, this may arise from reversible loss of the small but detectable orientation we have demonstrated by polarized S-FTIR microspectroscopy in α-helical and random coil conformations both of which are known to be located in noncrystalline regions of the silk fibroin. Effect of Tensile Deformation on the Orientation of Secondary Structure in Single A. pernyi Silk. The mechanical properties of silk fibers formed artificially by wetspinning can be improved by post drawing to increase the molecular chain orientation.56−58 Post drawing is also known to

Table 1. Molecular Order Parameter (Smol) of Different Conformations in Single A. pernyi Silk Fibers Subjected to Different Strainsa Smol conformation

strain = 0

strain = 0.1

strain = 0.2

strain = 0.3

p value

β-sheet random coil α-helix

0.63 ± 0.07a 0.07 ± 0.04b 0.15 ± 0.04c

0.65 ± 0.09a 0.10 ± 0.04b 0.20 ± 0.06b

0.66 ± 0.09a 0.15 ± 0.07a 0.26 ± 0.10ab

0.65 ± 0.08a 0.18 ± 0.06a 0.28 ± 0.09a

0.648 4.21 × 10−7 5.91 × 10−5

All values measured were expressed as mean ± standard deviations (SD). Statistical analysis was performed by one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test (DMRT) for multiple comparisons. Different letters indicate significant difference (p < 0.05).

a

1889

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Table 2. Molecular Order Parameters (Smol) of Individual Absorption Peaks in Single A. pernyi Silk Fibers Subjected to Different Strainsa Smol −1

−1

wave number (cm )

band shift (cm )

strain = 0

strain = 0.1

strain = 0.2

strain = 0.3

p value

1405 1373 1242 1222 1054 965

−2 0 −2 −5 0 −2

0.34 ± 0.03b −0.28 ± 0.02a 0.05 ± 0.02c 0.48 ± 0.04a −0.31 ± 0.02a 0.93 ± 0.02a

0.38 ± 0.04a −0.28 ± 0.03a 0.07 ± 0.03b 0.47 ± 0.04a −0.31 ± 0.04a 0.94 ± 0.03a

0.42 ± 0.04a −0.28 ± 0.03a 0.11 ± 0.05a 0.48 ± 0.05a −0.30 ± 0.04a 0.94 ± 0.02a

0.42 ± 0.05a −0.26 ± 0.03a 0.12 ± 0.02a 0.46 ± 0.06a −0.32 ± 0.03a 0.94 ± 0.03a

5.14 × 10−7 0.208 2.20 × 10−8 0.601 0.601 0.823

All values measured were expressed as mean ± standard deviations (SD). Statistical analysis was performed by one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test (DMRT) for multiple comparisons. Different letters indicate significant difference (p < 0.05).

a

Table 3. Comparison of Secondary Structures of Single A. pernyi Silk Fibers from the Amide III Band Subjected to Different Strainsa content (%) conformation

strain = 0

strain = 0.1

strain = 0.2

strain = 0.3

p value

β-sheet random coil α-helix

38 ± 3a 44 ± 1c 18 ± 3c

38 ± 2a 46 ± 2b 17 ± 3abc

38 ± 4a 46 ± 2ab 15 ± 4ab

37 ± 3a 48 ± 3a 15 ± 3ab

0.603 4.92 × 10−5 1.62 × 10−2

All values measured were expressed as mean ± standard deviations (SD). Statistical analysis was performed by one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test (DMRT) for multiple comparisons. Different letters indicate significant difference (p < 0.05).

a

mechanical properties show variation in the relationship between peak position and strain.40 In contrast to the behavior of β-sheet, the orientation of random coil and α-helix both improved significantly with increasing strain, the Smol value for random coil increasing progressively from 0.07 to 0.18 with strains from 0 to 0.3, while that for α-helix increased from 0.15 to 0.28 (Table 1). One-way ANOVA analysis showed that the differences in orientation of random coil and α-helix produced by stretching were highly significant (p < 0.0001). These changes in orientation with strain were accompanied by significant changes in content of random coil, which increased from 44 to 48% with a parallel decrease in α-helix from 18 to 15% (Table 3), suggesting that this silk strain converts some α-helices to random coil. Our hypothesis that deformation upon extension depends on changes in the amorphous region of the silk fiber based on SFTIR microspectroscopy of single A. pernyi silk fibers is supported by evidence from S-XRD, neutron spectroscopy, and conventional FTIR spectroscopy on spider major ampullate silk.18,26 It is well-accepted that β-sheet crystallites lying in matrix formed by amorphous regions acts as multifunctional cross-links, which contribute stiffness and strength of the silk.60 Accordingly, much of the tensile deformation is thought to occur within the amorphous region and to result from chain reorientation and some conversion of the α-helix to random coil. Thus the remarkable combination of β-sheet crystallites and amorphous matrix is thought to endow silk with an outstanding portfolio of mechanical properties. Our results presented here strongly suggest that in A. pernyi silk the contribution of different conformations to the mechanical properties is very similar to that of spider major ampullate silk. The results obtained from spider major ampullate silk and our results for A. pernyi silk reported here both agree well with Maxwell model proposed by Krasnov et al., that is, the high extensibility of silk principally results from the disordered phase, but the relatively stiff crystals are also elastically deformed.61

However, one of the limitations of S-FTIR microspectroscopy is how small the spot size can be reduced to as this is restricted by diffraction. Fortunately, such a limitation would be overcome by the breakthrough of use multibeam synchrotron imaging system, which combine a multibeam synchrotron source with focal plane array (FPA) detectors, to greatly improve spatial resolution by reducing the diffraction limited spot size an order of magnitude to 0.54 × 0.54 μm2 pixels.62 Such a new technique not only has incomparable advantages in applications in the biomedical and cultural heritage fields,63,64 but also for single silk fiber research and other applications in materials science. This new technique should make it possible to collect high signal-to-noise spectra of smaller diameter single silk fibers (down to about 2 μm), enabling spider minor ampullate silk, flagelliform silk, and other silks to be studied.

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CONCLUSIONS Despite the fact that FTIR spectroscopy is the most widely used technique for the characterization of polymers, providing a rich source of information on diverse aspects from the composition of polymeric materials to the molecular configuration, conformation, orientation of polymer chains, very few studies have been performed using S-FTIR microspectroscopy.65 In this article, we report our use of S-FTIR microspectroscopy to investigate the conformation and order of molecular chains in single A. pernyi silk fiber, taking advantages of the high spatial resolution and polarization afforded by synchrotron radiation. The polar plots and order parameter values showed that the highly oriented β-sheet, slightly oriented α-helix, and slightly oriented random coil structures coexist in native A. pernyi silk. After stretching single fibers of this silk, it was found that both the content and orientation of β-sheet were almost unchanged, both the content and the orientation of random coil increased significantly, while the order of α-helix increased but its content declined. All these results may further understand the structure−property relationships of silks and optimize the postdraw treatment in the production of high-performance silk fibers by wet-spinning. 1890

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ASSOCIATED CONTENT

S Supporting Information *

Details of the FTIR band assignments of A. pernyi silk fibroin, the descriptives of one-way ANOVA analysis, the optical microscopy image of A. pernyi silk fiber, the deconvolution results of amide III band of single A. pernyi silk fibers when the infrared beam is parallel or perpendicular to the fiber axis, and the derivative spectra of amide III band of single A. pernyi silk fibers subjected to different strain when the infrared beam is parallel or perpendicular to the fiber axis. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86 21 51630300. Tel.: +86 21 65642866. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (Nos. 10979022, 20974025, and 21034003), 973 Project of Ministry of Science and Technology of China (No. 2009CB930000). We thank Dr. Jinrong Yao for his valuable suggestions and discussions.

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