Synchrotron FTIR Microspectroscopy of Single Natural Silk Fibers

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Synchrotron FTIR Microspectroscopy of Single Natural Silk Fibers Shengjie Ling,† Zeming Qi,‡ David P. Knight,§ Zhengzhong Shao,† and Xin Chen*,† †

The Key Laboratory of Molecular Engineering of Polymers of MOE, Department of Macromolecular Science, Laboratory of Advanced Materials, Fudan University, Shanghai, 200433, 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

bS Supporting Information ABSTRACT: Synchrotron FTIR (S-FTIR) microspectroscopy was used to monitor the silk protein conformation in a range of single natural silk fibers (domestic and wild silkworm and spider dragline silk). With the selection of suitable aperture size, we obtained high-resolution S-FTIR spectra capable of semiquantitative analysis of protein secondary structures. For the first time, we have determined from S-FTIR the β-sheet content in a range of natural single silk fibers, 28 ( 4, 23 ( 2, and 17 ( 4% in Bombyx mori, Antheraea pernyi, and Nephila edulis silks, respectively. The trend of β-sheet content in different silk fibers from the current study accords quite well with published data determined by XRD, Raman, and 13C NMR. Our results indicate that the S-FTIR microspectroscopy method has considerable potential for the study of single natural silk fibers.

’ INTRODUCTION Animal silks have an outstanding portfolio of mechanical properties combining high modulus, strength, and extensibility.1,2 The structure of the predominant silk protein, fibroin (spidroin), has been widely studied at different levels in a range of species. In general, animal silk can be regarded as a semicrystalline biopolymer with highly organized antiparallel β-sheet nanocrystals embedded in amorphous matrix.3 However, the details of how these secondary structures in natural silks are affected by factors including spinning conditions and mechanical strain and how the secondary structure helps to determine the mechanical properties of silk fibers are not completely understood. In addition, the chemical composition, fiber morphologies, and mechanical properties of different silks show considerable complexity and variability,4 6 which can be traced to diversity in protein amino acid sequences,7 differences under spinning conditions,8 and also the internal and external environment of the animals.9 Accordingly, studies of defined microscopic regions of single natural silk fibers under different conditions would be useful to understand the relationship between protein secondary structure and mechanical properties of silk fibers. Single silk fibers have been examined by synchrotron X-ray diffraction (XRD)10,11 and Raman spectroscopy,12 15 giving some useful information. Although FTIR spectroscopy is one of the oldest and well-established experimental techniques for the conformational analysis of polypeptides and proteins,16 there appear to be no reports to date of its use to characterize single natural silk fibers. In principle, FTIR spectroscopy can provide both qualitative and quantitative information about protein conformations because it is an absorption spectroscopy.17 However, although recent developments in Raman spectroscopy r 2011 American Chemical Society

make quantitative analysis possible, it is still more difficult with Raman than with FTIR spectroscopy. Despite the advantages of FTIR spectroscopy, this technique has not yet been applied to single silk fibers because the aperture diameter (usually several millimeters) required for conventional globar light source is three magnitudes larger than the diameter of single silk fibers (5 20 μm). Thus, a single silk fiber is only exposed to a small part of the infrared beam, so the quality of the spectra is very poor and almost useless. However, FTIR microspectroscopy with a conventional globar light source provides the potential to study micrometer-sized samples, but the signal-to-noise ratio of the spectra is very low because the detector receives insufficient signals from a micrometer-sized aperture. Therefore, conventional FTIR microspectroscopy is mainly used for qualitative rather than quantitative analysis.18 In the past 20 years, synchrotron FTIR (S-FTIR) microspectroscopy has been developed as a rapid, direct, nondestructive, and noninvasive analytical technique for micrometer-sized samples.19,20 This technique combines the ultrahigh brightness of synchrotron infrared source (usually 100 1000 times brighter than the conventional globar source) with the powerful magnification of the microscope, allowing spectra with high signal-to-noise ratio to be obtained from micrometer-sized samples or sample areas.18,19,21 Here we report the use of S-FTIR microspectroscopy to study semiquantitatively the secondary structure of single natural silk fibers from both domestic and wild silkworms and spiders. We

Received: May 3, 2011 Revised: July 11, 2011 Published: July 26, 2011 3344

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report that using this technique the β-sheet content in single silk fibers can be determined from the amide III band.

’ EXPERIMENTAL SECTION Preparation of Single Silk Fibers. Bombyx mori (B. mori) silk fibers were degummed by boiling in two 30 min changes of 0.5% (w/w) NaHCO3 solution. Then, the degummed silk fibers were washed with distilled water and allowed to air-dry at room temperature. The single fibers (brins) were selected from these degummed domestic silkworm silks. Antheraea pernyi (A. pernyi) silk fibers were degummed by boiling in two 30 min changes of 0.5% (w/w) Na2CO3 solution. Then, the degummed silk fibers were washed with distilled water and allowed to air-dry at room temperature. The single brins were selected from these degummed wild silkworm silks. Nephila edulis (N. edulis) dragline silk fiber was artificially reeled with a drawing speed of 2 cm/s from spiders under ambient room conditions of 24 ( 3 °C and 25 ( 5% relative humidity. The single fibers were selected from these reeled spider fibers. Preparation of Regenerated Silk Protein Membranes. The preparation of B. mori silk fibroin membrane is as described elsewhere.22 The dry degummed B. mori silk fibers were dissolved in 9.3 mol/L LiBr aqueous solution. After dialysis against deionized water for 3 days at room temperature, the solution was cast onto a polyethylene plate and allowed to dry at ∼25 °C and 50% relative humidity to give films of an approximate thickness of 5 μm. A. pernyi silk fibroin membrane was 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 and was then cast in the same way as B. mori. The preparation of Nephila spidroin (spider fibroin) membrane was as described elsewhere.23,24 The dope of major ampullate gland was diluted to 2% (w/w) by adding deionized water and left overnight to dissolve at room temperature (∼20 °C) with occasional slight rotation of the tube. Films were cast by allowing 1 mL aliquots of the resulting clear solution to dry overnight at ∼20 °C and 50% relative humidity in 3 cm  3 cm polystyrene weighing boats. FTIR Spectroscopy of Silk Protein Membranes. Infrared spectra were recorded using a Bruker 66v/s FTIR spectrometer with a liquid-nitrogen-cooled MCT detector. It is well known that the fine structure of water absorption at 1500 1700 cm 1 interferes with amide I and II band in proteins, so to eliminate spectral interference due to atmospheric water vapor, we continuously evacuated the entire light path of the instrument 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. S-FTIR Microspectroscopy of Single Silk Fibers. The experiments were performed at Beamline U4 in the National Synchrotron Radiation Laboratory (NSRL, Hefei, China). The instrumental details are presented in the Supporting Information. The FTIR microspectra were collected in the mid-infrared (MIR) range of 800 3800 cm 1 at a resolution of 4 cm 1 with 256 coadded scans. Spectrum data collection and processing were performed using OPUS 6.5 (Bruker). Data Processing of FTIR Spectra. Background was collected each time before all FTIR spectra of silk protein membranes and single silk fibers were collected. Nine-point smoothing was used prior to obtaining second derivative spectra with OPUS 6.5. Deconvolution of amide I and amide III bands was carried out using PeakFit 4.12. The numbers and positions of peaks were defined from the results of second derivatives spectra and fixed during the 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.25 It should be noted that each spectrum shown in this Article was from a single

Figure 1. S-FTIR microspectra of single silk fibers: (a) B. mori, (b) A. pernyi, and (c) N. edulis. experiment, but the data obtained from the spectra (e.g., β-sheet content, etc.) were the mean and standard deviations taken from separate deconvolutions from at least seven separate samples. The significance of differences in β-sheet content in the different types of silk fibers was determined by one way ANOVA on the statistics package in OriginPro 8 using a fixed-effects model and Fischer’s test (F-test) to separate means.

’ RESULTS AND DISCUSSION FTIR Spectra of Single Silk Fibers with S-FTIR Microspectroscopy. The small diameter of single natural silk fibers (∼6 μm

for single N. edulis major ampullate filaments) necessitates a compromise in the size of the aperture in the FTIR microscope to obtain the best signal-to-noise ratio. We chose a 5 μm  20 μm aperture for N. edulis spider dragline silk fibers and a 10 μm  20 μm aperture for the wider B. mori and A. pernyi silkworm silk brins. The rationale for this choice is given in the Supporting Information. Figure 1 shows S-FTIR microspectra for the three different animal single silk fibers. All three spectra show good resolution in the amide I, II, and III regions, making them suitable for qualitative analysis. The absorbance increased very sharply at ∼1700 cm 1, probably because of the scattering effect in small samples seen in other samples with FTIR microspectroscopy.26 28 The amide I and amide II regions were unsuitable for semiquantitative analysis because of the problems of baseline correction, which increased sharply at 1700 cm 1, and atmospheric water vapor interference resulting from the inadequate purge of water vapor from the light path within the microscope. However, the amide III region in S-FTIR microspectra of single silk fibers looked promising for quantitative analysis. Peak Assignment of Amide I and Amide III Bands of Silk Proteins. The assignment of adsorption peaks of B. mori silk fibroin as well as other two silk proteins in amide I band has been widely studied, but for amide III band, the studies are few and more controversial.29,30 Here we used a conventional FTIR spectrometer to record the spectra of regenerated B. mori, A. pernyi, and Nephila silk protein membranes to yield high-quality spectra for peak assignment by evacuating the light path to avoid the interference of the atmospheric water vapor as far as possible. It is well known that silk proteins undergo a conformation transition from random coil, helical conformation, or both to β-sheet when treated with aqueous alcohol or salt solutions.24,25,31 In this Article, we used 70% ethanol aqueous solution to induce the conformation transition in B. mori and A. pernyi silk fibroin 3345

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Figure 2. FTIR (solid line) and second derivative (dashed line) spectra of regenerated silk protein membranes: (a) B. mori silk fibroin membrane as cast, (b) B. mori silk fibroin membrane treated with 70% ethanol aqueous solution at room temperature for 24 h, (c) A. pernyi silk fibroin membrane as cast, (d) A. pernyi silk fibroin membrane treated with 70% ethanol aqueous solution at room temperature for 24 h, (e) Nephila spidroin membrane as cast, and (f) Nephila spidroin membrane treated with 0.3 mol/L KCl aqueous solution at room temperature for 24 h.

membranes and 0.3 mol/L KCl aqueous solution to induce the conformation transition in Nephila spidroin membrane, respectively. The FTIR spectra of as cast silk protein membranes as well as those after treatment to form the β-sheet are shown in Figure 2. The assignment of adsorption peaks in amide I band is generally agreed upon: the broad peak centered at 1655 1660 cm 1 to random coil, helical conformation, or both; the peak from 1620 to 1630 cm 1 to β-sheet conformation; and the small peak from 1690 to 1700 cm 1 to β-turn conformation of the hairpin-folded antiparallel β-sheet structure.22,29,31,32 Therefore, comparison of the contents of different conformations before and after induction of the conformation transition obtained by deconvolution of both the amide I and amide III bands together with other published evidence was used as a basis for assigning the characteristic peaks in amide III region.

In the amide III band of B. mori silk fibroin, the peak at 1233 cm 1 is assigned to the random coil, helical conformation, or both, and the one at 1266 cm 1 is assigned to β-sheet conformation (Figure 2a,b) in accordance with the predominant opinion in the literature.29 For the amide III band of A. pernyi silk fibroin, there is no dispute that the 1270 cm 1 band is assigned to R-helix conformation (Figure 2c,d).29 However, the assignment of β-sheet is controversial. The adsorption band at ∼1220 cm 1 or the band from 1235 to 1241 cm 1 has been assigned to β-sheet structure,29 even in different papers from the same research group.33 36 Figure 2c,d clearly shows that the 1221 cm 1 peak appears only after the ethanol treatment, indicating its assignment to β-sheet conformation. However, the absorption peak at 1235 1241 cm 1 shows almost no variation before and after 3346

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ethanol treatment, indicating that this peak is instead due to a random coil component present in the silk fibroin films both before and after ethanol treatment and in agreement with the classical assignment in other proteins.30 Compared with B. mori and A. pernyi silk fibroin, there are few reports concerning the assignment of Nephila spidroin in amide III band. From Figure 2e,f, we find that there are two characteristic peaks (1265 and 1235 cm 1) in amide III in the as-cast spidroin membrane, but the one at 1235 cm 1 shifts to 1224 cm 1 after KCl treatment. We know the building block of β-sheet in Nephila spidroin is poly(Ala)n also found in A. pernyi silk fibroin but not in B. mori silk fibroin,15,37,38 so it is reasonable to assign these peaks using those of A. pernyi silk fibroin as reference. Therefore, we assign the 1265, 1235, and 1224 cm 1 peaks of Nephila spidroin in amide III band to R-helix, random coil, and β-sheet conformation, respectively. After assigning conformations to the characteristic absorption peaks in the amide III band, we performed a semiquantitative analysis of β-sheet content from both amide I band and amide III band in all three species. Table 1 shows that the β-sheet content calculated from amide I band and amide III band is almost the same within experimental error. This means that the amide III band in the FTIR spectra of silk protein can, in addition to the Table 1. Comparison of β-Sheet Content (%) in Different Silk Protein Membranes from Amide I Band and Amide III Band B. mori silk fibroin

A. pernyi silk fibroin

Nephila spidroin

amide I

25 ( 3

21 ( 3

17 ( 2

amide III

25 ( 1

25 ( 1

18 ( 2

amide I band, is used for the quantitative analysis of conformation content.39,40 Estimation of β-sheet Content in Single Natural Silk Fibers using Amide III Band from S-FTIR Microspectra. Figure 3 shows the results of the method of peak recognition and deconvolution described above. This gave estimates of the β-sheet content of single silk fibers as follows: B. mori, 28 ( 4% (n = 9); A. pernyi, 23 ( 2% (n = 7); and N. edulis, 17 ( 4% (n = 8). One-way ANOVA analysis showed that these differences in estimated β-sheet content were highly significant (p < 0.001). Although we cannot totally exclude the possibility that the standard degumming process may affect the protein secondary structure in both the crystalline and amorphous components of silkworm fibroin, wide-angle X-ray scattering shows that the structure of degummed silk fibers is identical to those of the fibers before degumming.41 This provides strong evidence that the secondary structure of the crystalline regions is not significantly affected by the standard degumming procedure we have used. Raman spectroscopy,15 13C CP/MAS NMR,38,42 and XRD43,44 have previously been used to measure, respectively, the β-sheet content and the degree of crystallinity of silk fibers (which is closely correlated with the β-sheet content in silks15). Table 2 compares the β-sheet content (or the degree of crystallinity) of domestic silkworm, wild silkworm, and spider dragline silk fibers obtained with different methods. The Raman and XRD data of wild silkworm silks are from Samia cynthia ricini (S. c. ricini), which has an amino acid sequence similar to that of A. pernyi.45 It is not surprising that the absolute values for the β-sheet contents obtained from various method are different;46 49 however, the trend in β-sheet content B. mori > A. pernyi > N. edulis from our S-FTIR data is the same as those from Raman,

Figure 3. Deconvolution results of amide III band of single natural silk fibers: (a) B. mori, (b) A. pernyi, and (c) N. edulis (circles, original spectrum; dashed curve, deconvoluted peaks; solid curve, simulated spectrum from summed peaks). 3347

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Table 2. Comparison of β-Sheet Content (%) of Domestic Silkworm, Wild Silkworm, and Spider Dragline Silk Fibers Obtained by Different Methods domestic silkworm a

FTIR spectroscopy (current study)

wild silkworm

28 ( 4

23 ( 2

b

d

spider dragline 17 ( 4e

Raman spectroscopy 13 C CP/MAS

50 (ref 15)

45 (ref 15)

36 37e,f(ref 15)

NMR

62b(refs 41, 51, and 52)

50d(ref 48)

34f(ref 38)

b

37 56 (refs 43, 53, and 54)

XRD ordered fraction

b

c

50

d

11 15f (ref 44)

c

29 31f

25 (ref 43)

b

77

57

Standard error of mean (SEM) for the domestic silkworm, wild silkworm, and spider dragline silk fibers was, respectively, 1.5, 0.8, and 1.3%. The p value obtained from one-way ANOVA analysis was 5.9  10 5, indicating highly significant differences in the β-sheet content in the different silk fibers. b B. mori. c A. pernyi. d S. c. ricini. e N. edulis. f N. clavipes a

NMR, and XRD analysis. This accords with the ordered fraction of these silk fibroins and spidroins, predicted from their amino acid sequences to form β-sheet structure.50

’ CONCLUSIONS FTIR spectroscopy is useful for studying different conformations in proteins or polypeptides and how these change in time with treatment. However, the narrowness of natural silk fibers does not allow single fibers to be investigated by conventional FTIR spectroscopy or microspectroscopy. In this Article, we report our use of S-FTIR microspectroscopy to investigate single animal silk fibers. Our results show that rather good-quality FTIR microspectra can be obtained for a range of different silk fibers. From these microspectra, we obtained the β-sheet content in silk fibers, important for their structure and mechanical properties. S-FTIR microspectroscopy, as in other applications of synchrotron technique in biomedical, food science, and analytical chemistry fields,19,55,56 is likely to provide an advanced and powerful technique for studying silk, for example, in the realtime analysis of structure changes in single silk fibers during stretching, supercontraction, and heating. Also, it provides the possibility of studying the effect of figure-eight spinning of silkworm on changes in conformation along the length of the silk thread, which is thought to be the main reason for the inferior mechanical properties of silkworm cocoon silks compared with spider dragline silks.57 ’ ASSOCIATED CONTENT

bS

Supporting Information. Details of the infrared beamline U4 at NSRL and the choice of aperture in S-FTIR microspectroscopy. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Tel: +86-21-6564-2866. Fax: +86-21-5163-0300. E-mail: chenx@ fudan.edu.cn.

’ ACKNOWLEDGMENT 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. 2011CB605700). We thank Dr. Jinrong Yao and Dr. Lei Huang

at Fudan University and Dr. Chengxiang Li at NSRL for their valuable suggestions and discussions.

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