Drawing-Induced Changes in Morphology and Mechanical Properties

Biomacromolecules 2010, 11, 1009–1018

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Drawing-Induced Changes in Morphology and Mechanical Properties of Hornet Silk Gel Films Tsunenori Kameda,*,† Katsura Kojima,† Eiji Togawa,‡ Hideki Sezutsu,† Qiang Zhang,† Hidetoshi Teramoto,† and Yasushi Tamada† National Institute of Agrobiological Sciences, Tsukuba, 305-8634, Japan, and Forestry and Forest Products Research Institute, Tsukuba, 305-8687, Japan Received December 26, 2009; Revised Manuscript Received February 5, 2010

Complete amino acid sequences of the four major proteins (Vssilk 1-4) of silk (hornet silk) obtained from yellow hornet (Vespa simillima, Vespinae, Vespidae) cocoons have been determined. The native structure of the hornet silk (HS), in which Vssilk 1-4 have an R-helix domain with coiled-coil R-helices and a β-sheet domain, is restored when hornet silk gel films (HSGFs) are formed by pressing and drying HS hydrogel. Necking occurs when dry HSGFs are drawn; however, wet HSGFs can be uniaxially drawn with a draw ratio (DR) of 2. Drawing helps obtain high-performance films with a maximum tensile strength and tensile modulus of 170 MPa and 5.5 GPa, respectively. Drawing-induced changes in the orientation and conformation of the coiled-coil structure are investigated.

Introduction Silk has gained considerable importance because of the growing demand for materials with good renewability, sustainability, and biodegradability. Almost all the silk used in textiles and other industrial materials is obtained from lepidopterans, such as Bombyx mori. The silk of the lepidopterans consists of core protein with highly repetitive sequence that forms predominately β-sheet conformation.1 However, lepidopterans are not the only insects that produce silk. Hymenopterans such as honeybees, wasps, hornets, and ants also produce silk cocoons in the late larval stage. Recently, Sutherland et al.2 showed that silk genes/proteins obtained from Hymenopterans can be classified into two types: (1) protein in the silk produced by the parasitic wasp Cotesia glomerata (Braconidae); this protein has a highly repetitive amino acid sequence,3 and (2) proteins in the silk produced by honeybees, bumblebees, bulldog ants, weaver ants, hornets, and so on; these proteins have nonrepetitive amino acid sequences with coiled-coil structure (in the coiled-coil structure, multiple R-helices are mutually entwined4).5,6 Silk fibers made of these novel coiled-coil proteins are expected to have a unique structure; further, their properties may differ considerably from those of silkworm and spider silks.7,8 Hence, such silk fibers are considered to be promising candidates for the development and design of new-generation biomaterials for a variety of practical applications. We have carried out a detailed investigation of the silk proteins (hornet silk (HS)) obtained from hornets (Vespinae, Vespidae) and their use in the development of new materials.5,9–13 The yellow hornet (Vespa simillima) builds large nests that can have a maximum diameter of approximately 1 m. Each of these nests houses numerous hornet silk caps, which are actually cocoons (Figure 1A). We have found that HS comprises four major proteins, Vssilk 1-4, whose molecular size increases in the order Vssilk 1 < Vssilk 2 < Vssilk 3 < Vssilk 4.5,9 The proteins in HS exist in the form of R-helices and β-sheets, and some of the R-helices form coiled-coil structures.5,9,13 * To whom correspondence should be addressed. Tel.: +81-29-838-6213. Fax: +81-29-838-6028. E-mail: [email protected]. † National Institute of Agrobiological Sciences. ‡ Forestry and Forest Products Research Institute.

Figure 1. Photographs of silk cocoon (A) and silk gel film (B) obtained from Vespa simillima xanthoptera.

HS is considered to be more suitable for investigating the applications of protein materials having a coiled-coil structure than are the silks produced by other hymenopterans; this is because of the availability of a well-established method for HS purification, by which the hornet nests and wax adhering to the silk cocoons can be effectively removed.9,13 The following two issues must be resolved before carrying out detailed investiga-

10.1021/bm901472a  2010 American Chemical Society Published on Web 02/24/2010

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tions using HS as the model of a coiled-coil protein: (1) determination of the precise amino acid sequences in HS proteins; (2) production of HS materials having a coiled-coil structure, including films and regenerated fibers. Although the amino acid sequences in Vssilk 1-4 have been deduced by EST analysis in previous study,5 in general, the amino acid sequences and N-terminus deduced by EST analysis are not always accurate. Thus, to determine the precise amino acid sequences that have a certain N-terminal amino acid obtained from the cDNA with a complete open reading frame (ORF), in this study, we analyze the complete amino acid sequences of the four abovementioned silk proteins (Vssilk 1-4) by the following methods: (1) selection of the cDNAs encoding Vssilk 1-4 from the cDNA library established in a previous study5 and determination of their complete base sequences; and (2) determination of the 5′-terminus of mRNA encoding Vssilk 1-4 by 5′ rapid amplification of cDNA ends (5′-RACE). To study the practical applications of these silk fibers, it is necessary to produce silk materials having a coiled-coil structure. In this study, we developed a novel method that involves pressing and drying of HS hydrogel for the fabrication of HS films (named hornet silk gel films (HSGFs)), which are shown in Figure 1B. The most striking feature of our method is the formation of a coiled-coil structure during the generation of the HSGF. Moreover, our HSGF can be drawn both in the dry and wet states. We investigate drawing-induced changes in the morphology and mechanical properties of the HSGF to understand the changes in the orientation and conformation of the coiled-coil structure during the drawing process. By this investigation, we aim to understand the role of the coiled-coil structure in determining the physical properties of the HSGF. Information on the physical properties of HSGF with an oriented coiled-coil structure, such as stress/strain behavior and tensile strength and modulus, can be used to evaluate candidate materials for the development and design of new-generation biomaterials to be used as silk materials comprising coiled-coil proteins as well as to explain the physical properties of R-keratin fibers. R-Keratin fibers such as hair and wool comprise oriented R-helix structures with a coiled-coil structure. Several structural models of R-keratin have been proposed14–16 for explaining the stress/strain behavior of R-keratin fibers; however, no conclusive results have been obtained in this regard. The objectives of this study are as follows: (1) determination of the amino acid sequence in Vssilk 1-4 by 5′-RACE of the total RNA isolated from the silk glands of mature V. simillima larvae; (2) fabrication of a HSGF and comparison of the molecular structure of the film with that of the native cocoon; and (3) investigation of the drawability of the HSGF and drawing-induced changes in the morphology and mechanical properties of the film.

Experimental Section 5′-RACE of Transcripts of Vssilk 1-4 Gene. Mature larvae of V. simillima were collected from nest combs while still alive, and the silk glands were excised. The silk glands were thoroughly rinsed with phosphate-buffered saline and then immersed in RNAlater (Ambion, Austin, TX). Total RNA was isolated from the silk glands using Isogen (Nippongene, Tokyo, Japan). On the basis of the cDNA sequences determined for Vssilk 1-4 by EST analysis in a previous study,5 the primers for 5′-RACE were designed as follows: 5′-GCCGAACCCGAAGCAGAAGAAG-3′ and 5′-CCGAAGCCAGAGATGAGGACAAG-3′ (nested primer) for Vssilk 1, 5′-GTGCTCTACCGGTAGCTGACG-3′ and 5′-CGCGCTCCAGGACGCATCTGAAG-3′ (nested primer) for Vssilk 2, 5′-CGCGGATAAGGCCTGAGCAGAAG-3′ and

Kameda et al. 5′-CCGGCTCCGGCATTAGCTGAG-3′ (nested primer) for Vssilk 3, and 5′-CCCGCGCTTGCACTGGATGAATC-3′ and 5′-CCCAAAGAGAAGGCGATTCCAC-3′ (nested primer) for Vssilk 4. The 5′ regions of Vssilk 1-4 transcripts were amplified by polymerase chain reaction (PCR) with the isolated total RNA using a GeneRacer Kit (Invitrogen, Carlsbad, CA) by following the manufacturer’s instructions. All the PCR experiments were conducted using a TPersonal thermal cycler (Biometra, Go¨ettingen, Germany) and KOD-Plus-Ver.2 highfidelity DNA polymerase (TOYOBO, Osaka, Japan). The amplified fragments were cloned into a pCR-Blunt II-TOPO vector (Invitrogen), and the sequences of the clones thus obtained were determined. Preparation of HSGF. Cocoon caps (700 mg) were collected from the nest of V. simillima and dissolved in 20 mL of 9 M LiBr aqueous solution by stirring for 15 min at 37 °C.9 The solution was filtered to remove the nest papers adhered to the cocoons. For four days, the solution was dialyzed against sterilized water using a cellulose tubing (size 18; Wako Pure Chemical Industries Ltd., Osaka). During the dialysis, the HS solution was transformed into a turbid gel. The concentration of silk in the gel was 1 wt %, as determined by weighing the residual solid after freeze-drying the silk gel. The diameter and length of the hydrogel obtained after dialysis were 19 ( 2 and 170 ( 5 mm, respectively. For pressing and drying the HS hydrogel to obtain an HSGF, the cellulose dialysis tube containing the gel was sandwiched between water-absorbing papers by applying a pressure of 35 N for 3 days. Finally, the cellulose tube was removed, and the films were further dried in vacuum for 24 h at room temperature to obtain the HSGF. The length and width of the obtained HSGF were approximately 170 and 20 mm, respectively, and the film had a uniform thickness of approximately 0.2 mm. Although the gel was turbid, the dried film was transparent and adopted the light-brown color of the nest paper. Drawing of HSGF. A strip of width 1 mm and length greater than 50 mm was cut out from the pressed and dried HSGF. To measure the draw ratio, ink marks separated by a distance of 1 cm were made on the strip. For dry drawing, the films were drawn using a tensile tester (EZ test; Shimadzu, Kyoto, Japan) with an initial strain rate of 2-10% min-1. For wet drawing, the film strips were prewetted for 1 h in a large water bath maintained at 15 °C and drawn by using a stretching device at various draw ratios. The ends of the drawn films were fixed on the stretching device to prevent relaxation, and the films obtained were dried at room temperature for more than 2 h. Mechanical Properties of HSGF. The tensile properties of the specimens (1 × 70 × 0.2 mm) were measured using a tensile tester (EZ test; Shimadzu, Kyoto, Japan) at 23-24 °C and 30-40% relative humidity. The gauge length was set at 50 mm, and an initial load cell of 50 kgf (1 kgf ) 9.80665 N) was applied. The tensile strength and the ratio of the relative elongation to the initial film length at break (%) were determined from the stress/strain curves. The stress/strain curves were observed in wet and dry environments. For investigations in the wet environment, the films were immersed in a large quantity of water for 1 h. The stress/strain curves were measured soon after the films were removed from the water bath. The tensile modulus test was carried out at a strain rate of 1% min-1, and the tensile moduli were determined from the initial slope of the stress/strain curves at a low strain (90%). This result indicated that the amino acid sequences in the proteins (“presumably silk proteins”) obtained from Vespula squamosa are similar to those in Vssilk 1, 3, and 4. However, there is a marked sequence divergence between the proteins obtained from

Figure 2. Schematic representations of the signal peptide, Ala-rich, Ser-rich, and coiled-coil domains in Vssilk 1-4. Symbols R1-5 show the repetitive region, which includes 5 repeats of 20 amino acids. Amino acid sequences for R1-5 are given below. Symbols (*), (:), and (.) indicate identical, strongly similar, and weakly similar amino acid sequences for R1-5, as confirmed by ClustalW analysis.27

Vespula squamosa and those obtained from honeybee silk. Vespa and Vespula are classified under the superfamily Vespoidea, whereas honeybees belong to the superfamily Apoidea. The significant sequence divergence between HS proteins and honeybee silk protein can be explained on the basis of the evolutionary distance between Vespoidea and Apoidea.6 Formation of HSGF. Recently, we have developed a method to prepare gel films from silk.18 The drawability and mechanical properties of these silk gel films are better than those of cast films. The major difference between gel films and cast films is in their preparation process: gel films are obtained by gelation, whereas cast films are directly prepared from solution. For the fabrication of gel films from silk sericin,18 an aqueous solution of sericin is poured into a mold before gelation, and then, the gel is dried to obtain a film. With this method, films of various shapes can be obtained by simply changing the mold used for gelation. However, because HS undergoes self-aggregation and gelation during dialysis, its aqueous solution cannot be obtained. For this reason, after dialysis, HS present in the cellulose tube is in the gel state. Taking into account this typical feature, we have developed a method to prepare gel films from HS. In this method, the HS gel, which remains in the cellulose tube after dialysis, is sandwiched between water-absorbing papers placed over the tube; then, a steel plate is placed over the cellulose tube and pressure is applied. The cellulose tube is peeled off after complete film formation to obtain the HSGF. Behavior of Dried HSGF During Drawing. The HSGF obtained from 1 wt % silk gel is shown in Figure 1B. Transparent HSGFs with thicknesses in the range 0.2-0.3 mm were drawn. Interestingly, the HSGF was stretchable even in the dry state (air humidity: 40-60%) and underwent necking deformation (SEM image in Figure 3), although conventional silk films prepared from fibroin and sericin were brittle and showed poor ductility when subjected to straight drawing in the dry state.19 The stress/strain curve obtained for the necking deformation is shown in Figure 4a. Initially, the draw stress increased linearly with the strain, then decreased sharply beyond the yield point, and finally remained almost constant in the yield

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Figure 3. SEM microphotographs of the necking deformation of the hornet silk gel film (HGSF).

Figure 5. Stress/strain curves and recovery responses (A) and plots of applied strain vs residual strain (B) for the swollen hornet silk gel film (HSGF). The numbers I, II, and III in (A) represent the three distinct regions characterized in the stress/strain curve.

Figure 4. Stress/strain curves obtained for dried hornet silk gel films (HSGFs) with draw ratios of (a) 1.00 (drawing speed: 2% min-1) and (b) 1.25, (c) 1.60, (d) 1.75, and (e) 2.00 (drawing speed of 10% min-1). Breaking points for HSGF drawn with a draw ratio of 2.00 at drawing speeds of 10% min-1 (2) and 100% min-1 (b) are plotted along with error bars.

region. Necking commenced from the start of the yield region. From this stress/strain curve, it was found that the strain at the breaking point was approximately 100%, which corresponded to a DR of 2; the draw ratio (DR) is the ratio of the lengths of the film after and before drawing. In this case, the drawing speed was set to 2% min-1. When the drawing speed was increased from 2% min-1 to 10% min-1, the yield stress increased from approximately 40 to 60 MPa, and the film ruptured at the necking point even before the neck propagated along the entire specimen (data not shown). Behavior of Wetted HSGF During Drawing. The HSGF is transparent when dry (Figure 1B) and becomes opaque upon wetting. Absorption of water results in changes in the stress/ strain curve obtained for the HSGF, as shown in Figure 5A; the swollen HSGF shows elastomeric behavior, that is, it has a low initial modulus and stretches uniformly to almost twice its original length without undergoing necking deformation. The curve obtained for the wetted HSGF is characterized by three distinct regions. Region I, where the stress increases rapidly with strain, corresponds to a strain of less than 15%. Region II, where the stress increases slowly and linearly with the strain, corresponds to a strain of 15-50%. At higher strain values (>50%; region III), the stress once again increases rapidly with the strain. These three regions are particularly well-defined in the case of the wetted silk films.18 Although the wetted HSGF was uniformly drawn at the desired DR values, the HSGF drawn under wetting conditions showed a certain degree of strain recovery when its ends were

released from the drawing apparatus. To examine the recovery behavior, the stress response was measured in the following manner: the film was drawn at constant speed of 100% min-1 up to a given strain and then brought back to the initial state at the same speed until the applied strain was released (Figure 5A). Figure 5A shows that the wetted HSGF was permanently deformed and was not restored to its original length, indicating that the film underwent elastic deformation and partial plastic deformation during drawing. Figure 5B shows the plots of applied strain versus residual strain. From these plots, it was apparent that permanent deformation occurred in the film even at a small strain, and the increase in the residual strain with the applied strain followed a polynomial curve. To prevent recovery after wet drawing, both ends of the drawn HSGF under wetting at the desired DR were fixed to the drawing apparatus until the film dried. Loss of water from the drawn HSGF during drying was thought to result in a decrease in the chain mobility, which in turn caused the film to remain in a state of preferred molecular orientation. To confirm this, HSGFs were drawn at various DR values and then dried, and the structures and tensile properties of the resulting films were studied in detail. Yielding Behavior of the Drawn HSGFs. The results of the tensile tests performed on the drawn HSGFs in the dry state show the effect of drawing on the mechanical properties of the films. Figures 4b-e show the representative stress/strain curves obtained for the dried HSGFs with different DR values. From these figures, it is apparent that the shape of the stress/strain curve differs with the DR, indicating that the mechanical properties of the films are significantly affected by the drawing process. Figure 6A shows the stress/strain curves in the lowstrain region for the dried HSGFs with various DR values when wet drawing is carried out at a drawing speed of 10% min-1. From this figure, it is seen that the yield point, which corresponds to a stress of 60 MPa in the case of the original gel film, shifts gradually to the high-stress region, and a corresponding increase is observed in the DR; this indicates

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Figure 7. Plots of tensile modulus (A) and birefringence (∆n; B) against draw ratio for the dried hornet silk gel film (HSGF). The relationship between tensile modulus and ∆n is also plotted (C). The dotted line in (B) represents the birefringence value of the neckingdeformed film during dry drawing and the draw ratio corresponding to wet drawing.

Figure 6. Stress/strain curves in the low-strain region for the dried hornet silk gel films (HSGFs) with various draw ratios when wet drawing is carried out at a drawing speed of 10% min-1 (A); plots of magnitude of stress drop (MPa) as a function of draw ratio (B). Numbers in (A) represent the draw ratio of the films for each stress/ strain curve.

that the yield stress increases with the DR. However, it is known that the yield stress increases as the cross-sectional area of the specimen decreases.20 Indeed, in the present study, it is observed that the cross-sectional area of the drawn HSGFs tends to decrease with an increase in the DR. Thus, for investigating the dependence of yield stress on the cross-sectional area, films with cross-sectional areas in the range of 0.1-0.5 mm2 (the widths of the films are changed gradually) are prepared. The yield stress in these films is measured as a function of the crosssectional area. The obtained results indicate that the yield stress remains almost constant (data not shown) and is independent of the cross-sectional area of the films; in other words, the effect of cross-sectional area on the yield stress is negligible. Therefore, we confirm that the increase in the yield stress with the DR is because of the drawing effect and not because of the decrease in the cross-sectional area. It is noteworthy that the magnitude of stress drop beyond the yield point decreases with an increase in the DR (Figure 6A). For a detailed investigation of this phenomenon, the magnitude of stress drop is plotted against DR (Figure 6B). From this figure, it is found that the magnitude of stress drop decreases with an increase in DR and asymptotically approaches zero at a DR of 1.5. An HSGF with a DR of 1.5 is prepared by drawing a wetted HSGF at 50% strain. Interestingly, in the stress/strain curve of the wetted HSGF (Figure 5A), 50% strain corresponds to the boundary between regions II and III. This indicates that the stress drop (Figure 6A), which is related to the necking phenomenon, is also related to the structural change corresponding to region II in the stress/strain curve of the wetted HSGF (Figure 5A). Maximum Tensile Strength and Modulus of the Drawn HSGFs. Above a DR of 1.6, the draw stress continues to increase with the strain even beyond the yield point, and the rate of increase increases with the DR; thus, the HSGF drawn at the highest DR (2.0) has the maximum tensile strength. The stress at the break (tensile strength) is affected by the drawing

speed. At a given DR, the tensile strength increases with the drawing speed. Figure 4 shows the break points for the HSGFs with a DR of 2.0 at drawing speeds of 10% min-1 and 100% min-1; the tensile strength and break elongation at a drawing speed of 10% min-1 are 155 ( 8 MPa and 16.3 ( 3.9% (mean ( SD; number of samples (n) ) 13), respectively. The tensile strength and break elongation at 100% min-1 are 168 ( 13 MPa and 18.2 ( 5.0% (n ) 23), respectively. The tensile strength and break elongation at 100% min-1 are slightly higher than those at 10% min-1. These results indicate that even though the maximum DR is only 2, the tensile strength of the dried HSGFs increases significantly from 40 to 170 MPa in the draw direction when the DR is increased from 1 to 2. The maximum tensile strength (170 MPa) of the HSGF is higher than the maximum tensile strengths of the films fabricated from B. mori silk fibroin and sericin.21,22 Moreover, the breaking elongation of the HSGF with a DR of 2.0 is relatively high, 18.2%, a value comparable to the characteristic break elongation of the native B. mori silk fibers (20%).23 The tensile modulus, which is defined as the initial slope of the stress/strain curve, also changes with the DR. Figure 7A shows the plot of tensile modulus against DR for the HSGFs. Clearly, the tensile modulus tends to increase linearly with the DR, reaching a maximum of approximately 5.5 GPa at a DR of 2.0. Moreover, optical birefringence (∆n) measurements are carried out to investigate how the overall chain orientation in the crystalline and amorphous regions in the HSGF is affected by the drawing process. Figure 7B shows ∆n as a function of DR for the HSGFs. ∆n of the undrawn HSGF is close to zero, suggesting that the specimen is isotropic. ∆n increases linearly with the DR, indicating the formation of an ordered structure in the film; the linear relationship between ∆n and the DR (Figure 7B) is similar to that between the tensile modulus and the DR (Figure 7A). The stress/strain plot of the wet drawn HSGF is not linear but a curve with three distinct inclinations (regions I, II, and III in Figure 5A); however, the tensile modulus and ∆n of this film increase linearly with the DR. From Figure 7A,B, we conclude that there exists a linear relationship between the tensile modulus and ∆n (Figure 7C), which in turn indicates that the tensile modulus is predominately governed by the preferred orientation. Solid-State NMR Analysis of the HSGF. To investigate the conformation of protein molecules in HSGF and the drawinginduced changes in the conformation, 13C CP-MAS NMR experiments were carried out. The conformations of the Ala

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Figure 9. Wide-angle X-ray diffraction spectra: undrawn (draw ratio ) 1.00) hornet silk gel film (HSGF) with random orientation (A); film with a draw ratio of 2.00 (B); undrawn HSGF after heat treatment (C); and film with a draw ratio of 2.00 after heat treatment (D). The numbers corresponding to the arrows in (B) indicate the deconvolution peaks in Figure 10 and Table 1. Figure 8. 13C CP/MAS NMR spectrum of the native hornet cocoon produced by larva of Vespa simillima xanthoptera (A) and expanded spectra for the 0-90 ppm region, along with the 13C CP/MAS spectra for the hornet silk gel films (HSGFs) with various draw ratios (B).

and Ser residues in HSGF can be separately determined from the 13C NMR chemical shift. The 13C NMR peaks of Ala residues of HSGF are considered to indicate the structure of the Ala-rich region mainly concentrated at the center of the Vssilk 1-4 chains (Figure 2), whereas the peaks of Ser mainly correspond to the structure of the Ser-rich region concentrated at the ends of the Vssilk 1-4 chains. Thus, the focus will be on the CR and Cβ peaks of the Ala and Ser residues; these peaks are sensitive to conformational changes and are relatively well resolved. The 13C CP-MAS NMR spectrum of the native hornet cocoon (HS in the native state) is shown in Figure 8A. Figure 8B shows the expanded spectra for the 0-90 ppm region, together with the 13C CP/MAS spectra for the HSGFs with DR values of 1.00, 1.25, 1.50, 1.75, and 2.00. Peak assignments for CR and Cβ in the Ala and Ser residues were made by comparing the chemical shifts in these spectra with those observed in the spectra of peptides and other silk fibers (obtained from silkworms, spiders, etc.) in solution and solid states.9 Figure 8B reveals that the NMR spectrum of the undrawn HSGF (DR ) 1.00) is very similar to that of the native cocoon; this evidence the fact that during the generation of the HSGF, a structure similar to that of the native cocoon is restored. The spectrum shows peaks corresponding to CR and Cβ in the Ala and Ser residues in the R-helix and β-sheet conformations, indicating the coexistence of the R-helix and β-sheet in the HSGF. The peaks due to CR and Cβ in the R-helix conformation resonate more strongly than those due to CR and Cβ in the β-sheet conformation; this indicates that the Ala residue in the HSGF is predominantly in the R-helix form. Because Ala is mainly concentrated at the center of the Vssilk 1-4 chains, it can be said that this Ala-rich region is predominantly in the R-helix form.

The intensities of the Ala peaks in the 13C NMR spectrum are affected by changes in the DR values (Figure 8B). With an increase in the DR, the intensity of the peaks corresponding to CR and Cβ in Ala in the R-helix decreases, while that of the peaks corresponding to CR and Cβ in the β-sheet increases; this change indicates a conformational transition from the R-helix to the β-sheet upon drawing. From this result, we infer that the structure of the Ala-rich region changes during drawing. In contrast, the NMR peaks due to CR and Cβ in Ser are almost unchanged. Since Ser is mainly concentrated at the ends of the Vssilk 1-4 chains, we state that drawing does not induce any significant change in the structure of the Ser-rich region. This result is in sharp contrast to that obtained for the Ala-rich region. Therefore, it is possible that the drawing-induced structural changes are different in the Ala-rich and Ser-rich regions. The difference between the structural changes in the Ala- and Serrich regions is discussed later in this paper. The 13C NMR peaks corresponding to Ala residues indicate a conformational transition from the R-helix to the β-sheet; however, because the degree of R-β transition is not very high, a significant amount of the R-helix remains in the gel film whose DR is 2.00. This finding will be also discussed later. WAXD Analysis of the Undrawn HSGF. The WAXD photograph of the undrawn HSGF shown in Figure 9A reveals the presence of several isotropic rings. In addition to this, the WAXD profiles obtained for the undrawn film in two mutually perpendicular directions chosen arbitrarily in the film plane are almost identical, as shown in Figure 10a. These results indicate that there are no apparent orientations in the undrawn HSGF. We carried out deconvolution of the WAXD profiles to perform detailed analyses of the crystalline structure and orientation behavior of the undrawn HSGF. The lowermost trace shown in Figure 10 represents the deconvolution of the WAXD profile of the undrawn HSGF. We can observe nine sharp peaks and a broad peak at around 2θ ) 20°; all these peaks are fitted using a Gaussian function. The NMR spectra reveal the



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Figure 10. Equatorial (black line) and meridional (blue line) WAXD scans for hornet silk gel film (HSGF) with draw ratios of 1.00 (a), 1.25 (b), 1.50 (c), 1.75 (d), and 2.00 (e) and WAXD scan for the hornet cocoon (f). The lowermost trace represents the Gaussian deconvolution of the WAXD profile of the undrawn HSGF (a). Table 1. d-Spacings and Assigned Conformations and Indexation for Each Reflection in the X-ray Diffraction Patterns of the Hornet Silk Gel Film (HSGF)a peak

2θ (°)

d-spacing (nm)

conformation and indices

1 2 3 4 5 6 7 8 9

9.2 11.0 18.2 19.9 20.3 21.9 24.0 26.0 30.0

0.96 0.80 0.49 0.45 0.44 0.41 0.37 0.34 0.30

R R R R β(020) R β(021) β(002) β(030)

a The number for each peak in the table corresponds to the respective deconvolution peak in Figure 10.

coexistence of R-helix and β-sheet structures in the HSGF, and hence, these nine peaks can be attributed to the R-helix or the β-sheet. To determine whether the nine peaks can be assigned to R-helix or β-sheet diffractions, we carry out heat-treatment of the HSGF in an autoclave (121 °C, 20 min). The 13C CPMAS NMR spectra of this heat-treated film (Figure 11A) reveal a marked decrease in the intensity of the peaks attributable to the R-helix form in the spectrum of the untreated film; however, the intensity of the peaks attributable to the β-sheet increases after the heat treatment. This is indicative of a morphological transformation from the R-helix to the β-sheet during heat treatment. By drawing a comparison between the WAXD profiles of the HSGF before and after the heat treatment, the diffraction peaks attributable to the R-helix can be distinguished from those attributable to the β-sheet. The WAXD profiles of the HSGF with a DR of 1.00 (undrawn) and 2.00 before and after heat treatment are shown in Figure 11B. From this figure, it is found that diffraction peaks 1, 2, 3, 4, and 6 disappear after the heat treatment, indicating that they correspond to the R-helix. On the other hand, peaks 5, 7, 8, and 9 remain after the heat treatment, indicating that they are attributable to the β-sheet. The d-spacings corresponding to peaks 5, 7, 8, and 9 are essentially consistent with those observed for silkworm silk

Figure 11. 13C CP-MAS NMR spectra of undrawn hornet silk gel film (HSGF) before (untreated) and after (treated) heat treatment (A); WAXD scans for undrawn (×1.00) film and equatorial and meridional WAXD scans for drawn (×2.00) HSGF before (untreated) and after (treated) heat treatment (121 °C, 20 min; B). The symbols R and β in (A) represent the peaks attributable to the R-helix and β-sheet, respectively. The numbers in (B) correspond to the deconvolution peaks in Figure 10.

fibroin having a β-sheet structure, as reported by Shen et al.24 With reference to this literature, the indices for each β-sheet peak are listed in Table 1. WAXD Analysis of Formation of the Coiled-Coil Structure in HSGF. Although the 13C solid-state NMR results reveal that the conformation similar to that of the native cocoon is restored during the generation of the HSGF, it is still unclear whether a coiled-coil structure similar to that of the native cocoon is restored. Therefore, WAXD analysis is carried out to investigate the formation of the coiled-coil structure in HSGF. The 2θ angles corresponding to peaks 1, 2, and 3 are essentially consistent with those for honeybee silk fibroin that has a coiledcoil structure.4 We confirm that the above-mentioned three reflections indicate the formation of a coiled-coil structure in the HSGF only if peaks 1 and 2 appear on the equator and peak 3 appears on the meridian.4 Thus, to ascertain whether peaks 1-3 are equatorial or meridional, we investigate drawinginduced changes in the WAXD patterns obtained for the HSGF. The X-ray photograph of the HSGF with a DR of 2.00 (Figure 9B) shows arcs against a diffused background; this indicates that the silk molecules in the HSGF are oriented (aligned parallel to the drawing direction). The equatorial and meridional WAXD scans for HSGFs with DR values of 1.25, 1.50, 1.75, and 2.00 are shown in Figures 10b, c, d, and e, respectively. Considerable differences in the scans can be recognized on the basis of the DR values. The intensities of the equatorial diffraction peaks 1 and 2 at 2θ values of 9.2° (0.96 nm) and 11.0° (0.80 nm) (black curves in Figure 10) are stronger than those of the meridional

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Figure 12. Curves with azimuthally integrated intensity of diffraction at 2θ ) 10° (a), 18° (b), and 20° (c) for the hornet silk gel film (HSGF) with a draw ratio of 2.0; diffraction peak at 2θ ) 20° for the heattreated HSGF with a draw ratio of 2.0 (d).

diffraction peaks (blue curves in Figure 10), and this difference is more pronounced at a high DR. For the HSGF with a DR of 2.00, a curve with an azimuthally integrated intensity is observed at 2θ ) 10° (Figure 12a), and a maxima at an azimuthal angle (φ) of 90° corresponding to equatorial diffraction (in this case, the drawing direction corresponds to φ ) 0°) is observed. Therefore, it is confirmed that peaks 1 and 2 are attributable to equatorial diffractions. In contrast, from Figure 10, it is seen that the intensity of meridional diffraction peak (peak 3 at 2θ ) 18.2° (0.49 nm)) for the drawn HSGF is stronger than that of equatorial diffraction peak. The two maxima at 2θ ) 18° for the curve with the azimuthally integrated intensity appear at φ ) 0° and φ ) 180° (Figure 12b), thereby indicating meridional diffraction. Thus, peak 3 can be confirmed to be due to meridional diffraction. The small peak at φ ) 90° in Figure 12b corresponds to peak 5 at 2θ ) 20.3°. From these results, it is inferred that the diffraction peaks at 2θ ) 9.2° (0.96 nm) and 11.0° (0.80 nm) are on the equator, while the diffraction peak at 2θ ) 18.2° (0.49 nm) is on the meridian. This indicates that these three diffraction peaks are in good agreement with those seen in the spectrum of honeybee silk that has a coiled-coil structure, which was studied by Atkins.4 Thus, we confirm that in the HSGF, coiled-coil structures are present in the R-helix domain. Similar to a native hornet cocoon, the HSGF comprises an R-helix domain and a β-sheet domain; in the R-helix domain, some of the R-helices form coiled-coil structures. This indicates that a structure similar to that of a native hornet cocoon is restored during the generation of the HSGF. This inference is further supported by the fact that the nine diffraction peaks in the WAXD profile of the HSGF with a DR of 1.00 (Figure 10a) also appear in the WAXD profile of a native cocoon (Figure 10f). WAXD Analysis of Drawing-Induced Changes in the Orientation of the r-Helix and β-Sheet. The width of the curve with the azimuthally integrated intensity is related to the orientational order of the anisotropic crystalline domains along the drawing axis. The degrees of molecular orientation (order parameters) of the chains in the crystalline domain can be

Kameda et al.

estimated from the curves with azimuthally integrated intensity for the drawn HSGF with a DR of 2.00 (Figure 12a-c). The order parameters for the R-helix chain are calculated to be 62 and 75% from Figure 12a and b, respectively. The order parameter for the β-sheet is estimated from the diffraction at 2θ ) 20° in Figure 12c. We can observe three peaks at φ ) 0, 90, and 180° in Figure 12c. Among these peaks, the peaks at φ ) 0 and 180° disappear after the heat treatment, as shown in Figure 12d. Therefore, the meridional diffraction peaks at 0 and 180° in Figure 12c are attributable to the R-helix structure (peak 4), whereas the equatorial peaks at 90° are attributable to the crystalline β-sheet (peak 5). From the width of the peak at φ ) 90° (Figure 12c), the order parameter of the crystalline β-sheet is calculated to be 85%. This order parameter indicates that the crystalline β-sheet is highly oriented at a DR of 2.00. However, a detailed examination of the WAXD photograph of the HSGF with a DR of 2.00 (Figure 9B) reveals the presence of ring diffraction patterns with isotropic intensity distributions, indicating a small degree of orientation; these ring diffraction patterns are observed at 2θ ) 20°, and they overlap with the arcs. These ring diffractions remain after the heat treatment (Figure 9D), thereby indicating that they are attributable to the crystalline β-sheet. These results reveal the existence of two components with different orientations, a highly ordered component and a partially ordered component, in the β-sheet region. The highly ordered component is thought to be attributable to the β-sheet in the Ala-rich domain, whereas the partially ordered component is due to the β-sheets in the Ser-rich domain. The reason for this assumption is described in the next section. Deformation Mechanism During Drawing. The results of birefringence measurements (Figure 7B) demonstrate that wet drawing results in a marked improvement in the degree of molecular chain orientation in the HSGF but no significant R-β transition, as shown by the solid-state NMR spectra (Figure 8) and WAXD profiles (Figure 10). A large amount of the R-helices remains unaffected when the HSGF is drawn with a maximum DR of 2.0. Thus, we consider that during wet drawing, molecular chain orientation occurs preferentially instead of the R-β transformation. We predict that wet drawing of the HSGF first induces molecular orientation of the coiled-coil R-helix chains in the draw direction, subsequent to which R-β transformation occurs. As described in Figure 12, the order parameter of the highly ordered β-sheet components is 85%, which is higher than that of the R-helix (62-75%). This result supports the abovementioned prediction that alignment of the R-helix chains occurs first, after which the ordered R-helix chains are drawn into highly ordered β-sheets. Thus, it can be inferred that a high DR is required for inducing a drastic R-β transformation. When a cast film fabricated using the silk fibroin obtained from Samia cynthia ricini is drawn (in this fibroin, the R-helix structure is randomly oriented before drawing), a remarkable structural transition from the R-helix to the β-sheet structure occurs when the DR is between 4 and 6.25 As described above, the ordered R-helix is considered to be transformed into the highly ordered β-sheet. However, along with the highly ordered β-sheet, partially ordered β-sheets also exist in the drawn HSGF, as shown in the WAXD photographs (Figure 9B,D). The partially ordered β-sheet is considered to exist even before drawing and is almost unaffected by the drawing process. The results of solid-state NMR experiment (Figure 8) indicate that the Ser residues, which mostly exist in the Ser-rich domain, are almost unchanged even after drawing, while the Ala residues, which mostly exist in the Ala-rich domain, transform from the R-helix to the β-sheet upon drawing.



The Ser-rich domain in HS preferentially adopts a β-sheet conformation.5,13 Therefore, we consider that the highly ordered component is attributable to the β-sheet in the Ala-rich domain and not to that in the Ser-rich domain. Next, the effect of water absorption on the drawing behavior of the HSGF is discussed. During wet drawing, water molecules are expected to act as lubricants and contribute to the smooth alignment of the molecules. To confirm this expectation, the efficiency of chain orientation between dry and wet drawing is compared. As described above, necking deformation occurs when dry drawing is carried out with a corresponding DR of approximately 2, as shown in Figure 4a. Birefringence measurements are carried out to characterize the effect of necking deformation on the molecular order. The average birefringence of the portion of the HSGF deformed by necking is approximately 5.2 × 10-3. From the dotted line in Figure 7B, it is found that the DR corresponding to wet drawing is approximately 1.4 for a birefringence value of 5.2 × 10-3. This result indicates that the orientation efficiency in dry drawing is considerably lower than that in wet drawing. This difference can be attributed to the presence of the water molecules in the HSGF. Furthermore, water molecules are considered concentrate predominantly in the Ala-rich domain rather than in the Serrich domain (the Ala-rich domain mainly comprises R-helices, some of which form coiled-coil structures, while the Ser-rich domain mainly comprises β-sheets). This is because the exterior of the coiled-coil structure is generally occupied by hydrophilic residues that cover the hydrophobic strands.26 For this reason, molecular orientation occurs predominantly in the domain containing coiled-coil R-helix structures rather than in the domain containing β-sheets, as has been described earlier. Finally, we discuss the role of the coiled-coil structure in determining the physical properties of HSFG. From the above discussion, it is clear that the marked improvement in the degree of molecular orientation during wet drawing (Figure 7B) is mainly due to the orientation of coiled-coil R-helices; this indicates that the improvement in tensile modulus by wet drawing (Figure 7A) is governed by the orientation of the coiledcoil R-helices. Therefore, we conclude that the presence of a coiled-coil structure in the protein brings about rapid improvement of the physical properties of the protein materials by wet drawing.

Conclusion The conclusions of the present study are summarized: (1) We determined the complete amino acid sequences of the four major silk proteinssVssilk 1-4sobtained from the cocoons of the yellow hornet (Vespa simillima); the amino acid sequences were identical to those deduced previously by EST analysis. (2) The native structure of the HS cocoon is restored when HSGFs are fabricated by pressing and subsequent drying of the HS hydrogel. (3) The HSGF can be stretched in the wet as well as dry state. Because the HSGF stretches uniformly in the wet state, we can obtain drawn samples with accurate DR control. This makes it possible to carry out a detailed analysis of the changes in the structure and physical properties of the HSGF during wet drawing. Wet drawing induces orientation of the molecular chains with coiled-coil R-helix structures. The maximum DR achieved during wet drawing is only 2, and the molecular weight of HS is low; despite this, the maximum tensile strength and tensile modulus of the HSGF fabricated in this study are 170 MPa and 5.5 GPa, respectively; these values are higher than those of the films prepared from silkworm silk.

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Acknowledgment. This study was supported in part by JSPS Grant-in-Aid for Young Scientists (B; 18780041) and for Scientific Research (C; 21580072).

References and Notes (1) Zhou, C. Z.; Confalonieri, F.; Medina, N.; Zivanovic, Y.; Esnault, C.; Yang, T.; Jacquet, M.; Janin, J.; Duguet, M.; Perasso, R.; Li, Z. G. Fine organization of Bombyx mori fibroin heavy chain gene. Nucleic Acids Res. 2000, 28, 2413–2419. (2) Sutherland, T. D.; Young, J. H.; Sriskantha, A.; Weisman, S.; Okada, S.; Haritos, V. S. An independently evolved Dipteran silk with features common to Lepidopteran silks. Insect Biochem. Mol. Biol. 2007, 37, 1036–1043. (3) Yamada, H.; Shigesada, K.; Igarashi, Y.; Takasu, Y.; Tsubouchi, K.; Kato, Y. A novel asparagine-rich fibrous protein (Xenofibron) from the cocoons of the parastic wasp Cotesia ()Apanteles) glomerata. Int. J. Wild Silkmoth Silk 2004, 9, 61–66. (4) Atkins, E. D. T. A four-strand coiled-coil model for some insect fibrous proteins. J. Mol. Biol. 1967, 24, 139–141. (5) Sezutsu, H.; Kajiwara, H.; Kojima, K.; Mita, K.; Tamura, T.; Tamada, Y.; Kameda, T. Identification of four major hornet silk genes with a complex of alanine-rich and serine-rich sequences in Vespa simillima xanthoptera Cameron. Biosci. Biotechnol. Biochem. 2007, 71, 2725– 2734. (6) Sutherland, T. D. S.; Weisman, S.; Trueman, H. E.; Sriskantha, A.; Trueman, J. W. H.; Haritos, V. S. Conservation of essential design features in coiled-coil silks. Mol. Biol. EVol. 2007, 24, 2424–2432. (7) Rudall, K. M.; Kenchington, W. Arthropod silks: The problem of fibrous proteins in animal tissues. Annu. ReV. Entomol. 1971, 16, 73– 96. (8) Shi, J.; Lua, S.; Du, N.; Liu, X.; Song, J. Identification, recombinant production and structural characterization of four silk proteins from the Asiatic honeybee Apis cerana. Biomaterials 2008, 29, 2820–2828. (9) Kameda, T.; Kojima, K.; Miyazawa, M.; Fujiwara, S. Film formation and structural characterization of silk of the hornet Vespa simillima xanthoptera Cameron. Z. Naturforsch., C: J. Biosci. 2005, 60, 906– 914. (10) Kameda, T.; Kojima, K.; Zhang, Q.; Sezutsu, H.; Teramoto, H.; Kuwana, Y.; Tamada, Y. Hornet silk proteins in the cocoons produced by different Vespa species inhabiting Japan. Comp. Biochem. Physiol., Part B: Biochem. Mol. Biol. 2008, 151, 221–224. (11) Kameda, T.; Niizeki, T.; Sonoyama, M. In situ observation of tryptophan in larval cocoon silk of the hornet Vespa simillima xanthoptera Cameron by ultraviolet resonance raman spectroscopy. Biosci. Biotechnol. Biochem. 2007, 71, 1353–1355. (12) Kameda, T.; Akino, T.; Kojima, K. Wax components of larval cocoon silk of the hornet Vespa analis Fabricius. Anal. Bioanal. Chem. 2007, 387, 2895–2902. (13) Kameda, T.; Tamada, Y. Variable-temperature 13C solid-state NMR study of the molecular structure of honeybee wax and silk. Int. J. Biol. Macromol., 2009, 44, 64–69. (14) Worthmann, F. J.; Zahn, H. The stress/strain curve of R-keratin fibers and the structure of the intermediate filament. Text. Res. J. 1994, 64, 737–743. (15) Hearle, J. W. A critical review of the structural mechanics of wool and hair fibres. Int. J. Biol. Macromol. 2000, 27, 123–138. (16) Cao, J. N. Is the R-β transition of keratin a transition of R-helices to β-pleated sheets. II. Synchrotron investigation for stretched single specimens. J. Mol. Struct. 2002, 607, 69–75. (17) Hoffman, E. A.; Goodisman, M. A. Gene expression and the evolution of phenotypic diversity in social wasps. BMC Biol. 2007, 5, 23. (18) Teramoto, H.; Kameda, T.; Tamada, Y. Preparation of gel film from Bombyx mori silk sericin and its characterization as a wound dressing. Biosci. Biotechnol. Biochem. 2008, 72, 3189–3196. (19) Lv, Q.; Hu, K.; Feng, Q.; Cui, F. Preparation of insoluble fibroin/ collagen films without methanol treatment and the increase of its flexibility and cytocompatibility. J. Appl. Polym. Sci. 2008, 109, 1577– 1584. (20) Shen, Z. L.; Dodge, M. R.; Kahn, H.; Ballarini, R.; Eppell, S. J. Stressstrain experiments on individual collagen fibrils. Biophys. J. 2008, 95, 3956–3963. (21) Jin, H. J.; Park, J.;V., K.; Kim, U. J.; Valluzzi, R.; Cobe, P.; Kaplan, D. L. Water-stable silk films with reduced β-sheet content. AdV. Funct. Mater. 2005, 15, 1241–1247. (22) Teramoto, H.; Miyazawa, M. Molecular orientation behavior of silk sericin film as revealed by ATR infrared spectroscopy. Biomacromolecules 2005, 6, 2049–2057.

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(23) Altman, G. H.; Diaz, F.; Jakuba, C.; Calabro, T.; Horan, R. L.; Chen, J.; Lu, H.; Richmond, J.; Kaplan, D. L. Silk-based biomaterials. Biomaterials 2003, 24, 401–416. (24) Shen, Y.; Johnson, M. A.; Martin, D. C. Microstructural charactrization of Bombyx mori silk fibers. Macromolecules 1998, 31, 8857–8864. (25) Yang, M.; Yao, J.; Sonoyama, M.; Asakura, T. Spectroscopic characterization of heterogeneous structure of Samia cynthia ricini silk fibroin induced by stretching and molecular dynamics simulation. Macromolecules 2004, 37, 3497–3504.

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Drawing-Induced Changes in Morphology and Mechanical Properties

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