Minor Ampullate Silks from Nephila and Argiope Spiders: Tensile

Article pubs.acs.org/Biomac

Minor Ampullate Silks from Nephila and Argiope Spiders: Tensile Properties and Microstructural Characterization G. V. Guinea,†,‡ M. Elices,†,‡ G. R. Plaza,†,‡ G. B. Perea,†,‡ R. Daza,†,‡ C. Riekel,§ F. Agulló-Rueda,∥ C. Hayashi,⊥ Y. Zhao,⊥ and J. Pérez-Rigueiro*,†,‡ †

Centro de Tecnología Biomédica, Universidad Politécnica de Madrid, 28223 Pozuelo de Alarcón, Madrid, Spain Departamento de Ciencia de Materiales, ETSI Caminos, Canales y Puertos, Universidad Politécnica de Madrid, 28040 Madrid, Spain § European Synchroton Radiation Facility, B.P. 220, F-38043, Grenoble Cedex, France ∥ Instituto de Ciencia de Materiales de Madrid (ICMM-CSIC), Campus de Cantoblanco, 28049 Madrid, Spain ⊥ Department of Biology, University of California, Riverside, California 92521, United States ‡

ABSTRACT: The mechanical behavior and microstructure of minor ampullate gland silk (miS) of two orb-web spinning species, Argiope trifasciata and Nephila inaurata, were extensively characterized, enabling detailed comparison with other silks. The similarities and differences exhibited by miS when compared with the intensively studied major ampullate gland silk (MAS) and silkworm (Bombyx mori) silk offer a genuine opportunity for testing some of the hypotheses proposed to correlate microstructure and tensile properties in silk. In this work, we show that miSs of different species show similar properties, even when fibers spun by spiders that diverged over 100 million years are compared. The tensile properties of miS are comparable to those of MAS when tested in air, significantly in terms of work to fracture, but differ considerably when tested in water. In particular, miS does not show a supercontraction effect and an associated ground state. In this regard, the behavior of miS in water is similar to that of B. mori silk, and it is shown that the initial elastic modulus of both fibers can be explained using a common model. Intriguingly, the microstructural parameters measured in miS are comparable to those of MAS and considerably different from those found in B. mori. This fact suggests that some critical microstructural information is still missing in our description of silks, and our results suggest that the hydrophilicity of the lateral groups or the large scale organization of the sequences might be routes worth exploring.

1. INTRODUCTION Spider silks are well known for their outstanding mechanical properties. Researchers have been inspired to determine the basis for these properties and for the attempts to replicate − or even to improve − silk performance through biotechnology techniques. Major ampullate silk (MAS) is the most studied silk type, for its ease of collection and, in many cases, because of its superior mechanical behavior. In contrast, minor ampullate silk (miS) from the minor ampullate gland, a thread usually spun along with MAS and also used as the temporary spiral during orb-web construction, has been little studied owing to the fact that samples are not as easy to obtain, and its biological role in spider activities is less well understood. However, miS offers an excellent opportunity to deepen our knowledge of the structure-properties relation in silks because its protein sequence shares some motifs with MAS but also shows significant differences. This study is intended to provide additional information on the tensile properties and microstructure of miS fibers from two orbicular spiders, whose MAS fibers have been extensively characterized: Argiope trifasciata and Nephila inaurata. Regarding the chemical composition of miS, Work and Young, in their pioneering 1987 paper,1 published the amino © 2012 American Chemical Society

acid composition of minor ampullate silks from various orbweb-building spiders. They noticed the lack of proline in the miS of Araneus diadematus2 and in the near absence in Argiope aurantia (∼0.9%3). With respect to the primary structure of the miS spidroin (spidroin is a contraction of “spider fibroin”), not many papers have been published. Specifically, miS spidroin cDNA transcripts from N. clavipes were sequenced by Colgin and Lewis in 19964,5 and from A. diadematus by Guerette et al. also in 1996.6 More recently, the amino acid sequence motifs of a few more species have been added including among others Uloborus diversus and Deinopis spinosa,7 Nephilengys cruentata,8 Latrodectus hesperus,9 and Parawixia bistriatra.10 To the authors’ knowledge, no miS sequences from any Argiope species have been reported. The published miS sequences show similarities to those of MAS spidroin-1 but also show marked differences. The GGX and short poly-Ala (An) motifs of MAS spidroin-1 sequence are present in the miS sequences, but miS also has extensive amounts of (GA)n repeats. Incidentally, the (GA)n Received: March 23, 2012 Revised: May 22, 2012 Published: June 5, 2012 2087

dx.doi.org/10.1021/bm3004644 | Biomacromolecules 2012, 13, 2087−2098

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and Nephila inaurata (Walckenaer, 1842). The samples were obtained from two mature females of each species. Spiders were fed on a diet of crickets. The fibers were retrieved with the monitored forced silking process described elsewhere.25 This procedure is a variation of the conventional silking process26 and allows measuring the force during silking. It has been found that silk fibers spun with the same silking stress show the same tensile behavior.27 The threads obtained by forced silking contain MAS fibers and, occasionally, miS fibers. Figure 1 shows a micrograph of a

motif of sequence is extensively found in the Bombyx mori heavy chain fibroin11 but it is only scarcely found in the sequence of MAS spidroin-1, where it usually appears singly (i.e., n = 1; as −GA−) rather than in tandem arrays (i.e., n > 1).12 Structurally, miS spidroins are composed predominantly of repetitive regions, with small nonrepetitive amino and carboxyl termini.5,13 The highly repetitive regions mainly consist of alanine and glycine. Repetitive regions are interrupted by ∼137 amino acid long serine-rich regions called “spacers” because of their position between the strings of alanine and glycine rich motifs. These spacer regions have similar serine composition relative to the amorphous region of Bombyx mori heavy chain fibroin. Secondary structures for N. clavipes were considered by Liivak et al. in 199714 and Jelinski et al. in 199915 as well as by Hayashi et al. also in 1999,16 where the corresponding secondary structure for miS of A. diadematus is discussed. More recently, similar data for N. senegalensis by Riekel and Vollrath17 have been published. All of these results suggest a serial arrangement of crystalline and amorphous phases similar to that of MAS. Even so, analysis of the sequences indicates that miS has a much larger fraction of possible crystal-forming blocks (68% of the amino acids) and very short amorphous blocks (32% of the amino acids) compared with MAS. Studies using Fourier transform infrared spectroscopy of miS collected from N. edulis found a significant fraction of α-helical secondary structure and reduced β-sheet structure in the soluble protein,18 although the presence of other secondary structures, such as 310 helices, was predicted theoretically16 and found in Raman spectroscopy measurements.16,19 Solid-state NMR data suggest that the conformations of the alanine residues found in miS fibers are more heterogeneous than those found in MAS fibers, even with a large fraction of alanines in a non-β sheet conformation.14 As for tensile properties of miS, there is not much information in the literature: In 1977, Work20 investigated the influence of water on miS from A. diadematus and found no supercontraction. Later on, in 1994, Lewis and collaborators21 tested miS from Araneus gemmoides and N. clavipes and concluded that Nephila silks appear to be more elastic than those of Araneus. Recently, Hayashi and Blackledge22,23 published miS tensile tests from A. trifasciata and A. argentata showing that the mechanical performance of MAS and miS fibers are qualitatively more similar to one another than to other Argiope silks (tubuliform, aciniform, and capture spiral). Two species, Argiope trifasciata and Nephila inaurata, were used in this study, and their silk fibers were subjected to extensive mechanical characterization in air and in water. In particular, the possible existence of supercontraction as a ground state24 was assessed. Furthermore, the microstructure of the fibers was analyzed by X-ray diffraction (XRD), Raman spectroscopy, and atomic force microscopy (AFM). This analysis adds significant information to both the tensile behavior and the microstructure of miS fibers from orb-web weaving spiders, and allows a comparison with the microstructure and tensile properties of MAS and silkworm silk fibers.

Figure 1. SEM micrographs of thread from an Argiope trifasciata spider obtained by forced silking (FS). Both types of fibers discussed in this work, major ampullate (MAS) and minor ampullate (miS) silks, are shown.

thread from an Argiope trifasciata individual, with both types of fibers. MAS fibers were carefully removed during the silking process so that only miS was retrieved and, subsequently, tested. 2.2. Mechanical Tests. Details of the tensile testing procedure are given elsewhere.28 In brief, samples from the fibers were glued on aluminum foil frames with a base length of 20 mm. Tensile tests were carried out on a tensile testing machine (Instron 3309-662/8501) that was appended to an environmental chamber (Dycometal CCK-25/300). Forces

2. EXPERIMENTAL WORK 2.1. Collection of Silk. This work characterizes and compares the properties of miS and MAS fibers spun from the minor ampullate glands and major ampullate glands, respectively, of the species Argiope trifasciata (Forsskål, 1775) 2088


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time was typically 2 s/pattern. Patterns recorded outside the fiber during the same scan were used for background correction. About 30−40 patterns and an equivalent amount of background patterns were averaged for structural analysis. Data analysis was performed with the FIT2D program.34 2.5. Raman Spectroscopy. To measure the molecular alignment with respect to the fiber axis, we collected polarized Raman spectra with the incident and scattered light either parallel (Izz) or perpendicular (Ixx) to the fiber axis (labeled as z) in the Raman Laboratory of ICMM-CSIC (Madrid, Spain). A near-infrared (NIR) 785 nm wavelength diode laser (Toptica dfBeam 785-S with optical isolator) was used for excitation to reduce fluorescence emission from the sample. Light was focused to and collected from the fiber with a 100× microscope objective (Mitutoyo Plan NIR infinity corrected). Laser power was kept below 50 mW to avoid damaging the sample. Each sample was analyzed at several points to check for possible heterogeneities. Scattered light was analyzed with a singlegrating spectrometer (Princeton Acton Spectra Pro 2300i) and a CCD detector (Princeton Pixis 256). Laser light was blocked from entering the spectrometer with an edge filter (Semrock Razor Edge U grade). The spectral range between 500 and 1800 cm−1 was recorded because it contains the amide I and amide III bands. These bands arise from vibration modes that are approximately perpendicular and parallel, respectively, to the polypeptide backbone, and their intensity is very sensitive to the relative orientation between the atoms vibration direction and the incident laser light polarization.35 2.6. Atomic Force Microscopy. The nanostructural organization of the fibers was studied with AFM. The details of the experimental procedure have been described elsewhere.36 In brief, fibers were embedded in Spurr’s resin and allowed to cure for 72 h at 70 °C. The function of the Spurr’s resin is to serve as mechanical support to the fiber during ultramicrotomy. The longitudinal sections were obtained by ultramicrotomy with a diamond blade. The AFM images were recorded in a Bermad 2000 AFM (Nanotec Electrónica, Spain) using Olympus OMCL RC800PSA cantilevers, and the highest resolution was obtained with the stiffest tip (nominal stiffness 0.76 N/m). AFM images were recorded in the dynamic mode in the repulsive regime of the tip−sample interaction.37,38 The processing of the AFM images, consisting of simply equalizing and adjusting the contrast and the brightness of the micrographs, was performed with WSxM program (Nanotec Electrónica, Spain).38 No filter was used to improve the quality of the images or to highlight their details.

were measured by a 100 mN load cell with 0.1 mN resolution (HBM 1-Q11). The tests were carried out at a constant, fixed rate of 1 mm/min. The samples were stored at 20 °C and 35% RH. The tests in air (dry conditions) were performed at 25 °C and 35% relative humidity. A first group of samples obtained from the forced silking procedure and labeled as FS was tested in air. A second group of fibers was tested in water, with the sample submerged in a bath of water.29 In this case, the fiber was allowed to freely contract and was subsequently stretched until breaking. The contracted length of the fiber was used as initial length to calculate fiber strain. A third group of fibers was tested in air in the maximum contracted state and was labeled as MC. In this case, water was removed after contraction, and the fiber was allowed to dry overnight, being finally tensile tested in air at 25 °C and 35% RH. The maximum contracted length, defined as the maximum length at which the fiber was taut and not subjected to load, was obtained from the measured force−displacement curve. Fibers from two individuals of each species were tested, and three tensile tests were performed for each condition. To verify that individuals of one species produce MAS fibers with similar properties, we tested at least five samples of MAS fibers from each individual in the FS state, obtaining comparable results. These tests were shown to be representative of a specific silk type from a species by the reproducibility obtained with the use of the supercontraction process and by the absence of intraspecific variability that has been extensively documented in A. trifasciata MAS fibers.30 2.3. Cross-Section Measurements. To obtain the crosssection of the silk fibers, two small pieces, 5 mm long, adjacent to both sides of each tensile tested sample were retrieved, coated with gold, and placed in a scanning electron microscope (JEOL 6300). The samples were observed at V = 10 kV and I = 6 × 10−10 A. Diameters at a given section of the fiber were calculated as the average of at least two measurements on each micrograph, and the cross-sectional area was determined assuming a circular cross-section. The cross-sectional areas were used to compute true stress− true strain curves from force-displacement data. True stresses and true strains were calculated under the hypothesis of constant volume during deformation and contraction. This supposition has been validated for MAS fibers.31 2.4. X-ray Diffraction. To measure the fiber microstructure, crystallinity, and orientation distribution, we performed synchrotron radiation microdiffraction experiments at the ESRF-ID13 (European Synchroton Radiation Facility; Grenoble, France) beamline using a ≈ 2 × 2 μm 2 monochromatic beam from crossed mirrors at a wavelength of λ = 0.0996 nm.32 The low divergence of the beam allowed observing structural features of up to ∼10 nm in size.33 Single fibers were fixed by instant glue to MiTeGen MicroMounts that were attached to a Hampton magnetic support and fixed to the magnetic base of a raster-stage, which allows orthogonal x/y/z scans with submicrometer resolution. The samples were aligned normal to the beam by an Olympus microscope that had been calibrated to the focal spot. Experiments were performed at 100 K using an Oxford cryoflow system. A FreLon detector with 2K × 2K pixels (binned to 512 × 512 pixels) and 16-bit readout was used for data collection. The detector-to-sample distance was calibrated with Al2O3 powder to 67.5 mm. Raster scans were performed with a step-resolution of 3 μm across and 5 μm along about 50 μm of the fiber’s length. The data collection

3. RESULTS 3.1. Forcibly Silked Fibers Tested in Air. The observation of miS fibers retrieved from a forced silking process (forcibly silked (FS) samples) in an SEM showed that the cross-section of the miS fibers was reasonably homogeneous along the observed length. Compared with MAS fibers, miSs were significantly thinner. (See Figure 1.) The miS fibers from both species had the same mean diameter and standard deviation, 1.8 ± 0.1 μm. The MAS fibers produced from the same individuals were substantially thicker (N. inaurata, 5.9 ± 0.2 μm and A. trifasciata, 3.0 ± 0.3 μm). Tensile tests of the miS and MAS fibers obtained by forced silking from N. inaurata and A. trifasciata are displayed in Figure 2. MAS fibers from the two species produced similar results, as did miS fibers from the two species. Average mechanical values are summarized in Table 1. The comparison 2089


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mechanical data.20,23,40,41 A comparable conservation had been previously assessed from the comparison of MAS fibers spun by different members of the Araneoidea30 and is also illustrated in Figure 2 by the similarity between both FS MAS fibers. 3.2. Forcibly Silked Fibers Tested in Water. The effect of water on the mechanical properties has been studied by carrying out tensile tests with the fibers completely immersed in water. Figure 3a and Table 1 show that MAS and miS fibers are affected differently by water. The plasticizing effect of water42,43 is clearly stronger on MAS fibers, as evidenced by the almost three orders of magnitude reduction in the initial elastic modulus upon exposure to water (N. inaurata, 41 MPa; A. trifasciata, 22 MPa). In contrast, the elastic modulus of miS fibers tested in water shows a modest decrease, resulting in values of 400 MPa for N. inaurata and 1400 MPa for A. trifasciata. (See the inset in Figure 3a.) The reversibility of deformation of fibers in water was studied by loading−unloading-reloading tests. Three tests were carried out with each material, and it was found that the behavior of MAS fibers and of miS fibers was very different. The distinct behavior is illustrated in Figure 3b, where loading−unloading− reloading curves of A. trifasciata MAS and miS are compared. The recovery ability of MAS fibers is indicated by the concurrence of the reloading curve (labeled 3) with the initial loading curve (labeled 1). In contrast, stretching in water of miS fibers leads to irreversible deformations, as indicated by the concurrence of the reloading curve (labeled 3) with the unloading curve (labeled 2), which differs considerably from the initial loading curve (labeled 1). Consequently, miS fibers deform irreversibly in water and display a permanent deformation after unloading, even if the fiber is reloaded 24 h after unloading, as was the case of the reloading step shown in Figure 3b. 3.3. Contraction and Recovery of Fibers. A unique property of MAS fibers is the supercontraction phenomenon, which consists of a significant reduction of the length of unrestrained fibers when immersed in water or in highly humid environments.20,29 The biological significance of supercontraction is still under debate.8,30,43−45 The maximum supercontracted state24,46 has been identified as a ground state of the MAS fibers, as it can be always reached

Figure 2. True stress−true strain curves of MAS and miS fibers obtained by forced silking (FS) from Argiope trifasciata and Nephila inaurata spiders tested in air (25 °C, 35% RH).

of miS and MAS fibers shows that the former are considerably more deformable. The breaking strain of the miS fibers is almost the same for both species. There was also striking correspondence between the tensile strengths of MAS and miS from a particular species. Specifically, tensile strength was systematically higher in the fibers from N. inaurata, averaging 1.80 (MAS) and 1.50 GPa (miS), than in the corresponding fibers spun by A. trifasciata (1.3 GPa MAS, 1.04 GPa miS). The initial elastic moduli of each of the four silks in the FS condition were close to 10 GPa, except for N. inaurata MAS, which was the stiffest (14.2 GPa) fiber. The concurrence of the tensile properties in air of FS miS fibers spun by two species that diverged at least 120 million years ago39 (Figure 2 and Table 1) suggests the extreme stability of this type of silk over evolutionary time, which is further supported by comparison with previously published Table 1. Mechanical Properties of MAS and miS Fibersa

σu (GPa)

Einitial (GPa) FS fibers tested in air Nephila inaurata Argiope trifasciata MC fibers tested in air Nephila inaurata Argiope trifasciata fibers tested in water Nephila inaurata Argiope trifasciata

εu

WF (J/m3)

MAS miS MAS miS

14.2 11.2 10.7 10.0

± ± ± ±

0.6 0.7 0.3 0.4

1.80 1.5 1.3 1.04

± ± ± ±

0.06 0.2 0.2 0.06

0.26 0.46 0.17 0.45

± ± ± ±

0.01 0.05 0.02 0.02

264 300 90 240

± ± ± ±

5 50 30 20

MAS miS MAS miS

4.0 12.0 3.5 8.2

± ± ± ±

0.5 0.8 0.1 0.9

1.46 1.22 0.91 0.7

± ± ± ±

0.09 0.07 0.06 0.1

0.70 0.46 0.80 0.35

± ± ± ±

0.02 0.02 0.02 0.07

270 245 236 150

± ± ± ±

20 25 7 40

MAS miS MAS miS

0.041 0.39 0.022 1.42

± ± ± ±

0.004 0.03 0.001 0.09

1.7 1.0 1.42 1.22

± ± ± ±

0.1 0.1 0.05 0.02

0.67 0.53 0.95 0.51

± ± ± ±

0.01 0.09 0.03 0.01

280 210 185 245

± ± ± ±

10 60 8 25

Einitial: slope of the initial linear part of the true stress-true strain curve. σu: True stress at breaking. εu: True strain at breaking. WF: Work to fracture, measured as the area under the stress−strain curve up to the breaking point. Values shown as mean value ± standard error. a

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Figure 3. (a) True stress−true strain curves of MAS and miS fibers obtained by FS from A. trifasciata and N. inaurata tested in water (25 °C). (b) True stress−true strain curves of FS fibers from A. trifasciata tested in water to check reversibility: loading (1), unloading (2), and reloading (3). miS does not show complete reversibility.

independently f rom the loading history of the material by simply subjecting the unrestrained fiber to water immersion and subsequent drying.47 Moreover, this state appears as a ground state from which the whole range of tensile properties of spider silk can be obtained in a reproducible way by simply modifying the alignment of the fibers using the wet stretching procedure.48,49 Therefore, recovery ability and adaptability are specific properties of MAS fibers. Until now, however, this phenomenon has not been investigated in miS fibers. The contraction of miS fibers in water and the possible existence of a ground state, comparable to that observed in MAS fibers, has been analyzed. The contraction from the FS

condition produced by immersion in water has been determined in two steps: (1) contraction during immersion in water and (2) contraction after subsequent drying. At least three tests were carried out for each material. The results are shown in Table 2, where contraction is quantified through the percentage of contraction, %C, which is defined as %C = 100 × (L0 − LC)/L0, where L0 is the initial length of the fiber and LC is the length after contraction. It is observed that all four silk types experience an initial contraction when immersed in water and a further contraction after subsequent drying. However, there are remarkable differences between MAS and miS fibers in the extent of contraction: The percentage of contraction for 2091


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Table 2. Percentage of Contraction from the FS Condition of MAS and miS Fibersa Nephila inaurata % contraction by immersion in water % total contraction after drying

Argiope trifasciata

MAS

miS

MAS

miS

24 ± 3

5.6 ± 0.3

51 ± 1

2.8 ± 0.7

41 ± 1

13.4 ± 0.5

60 ± 2

5.3 ± 0.8

% Contraction = 100 × (initial length − contracted length)/initial length. Values shown as mean value ± standard error.

a

miS fibers of both species, either in water or in total, yields values between 3 and 18 times lower than that for MAS ones. The tensile properties of dried fibers after complete contraction and drying are shown in Figure 4. Contracted

Figure 4. True stress−true strain curves of maximum contracted (MC) MAS and miS fibers from A. trifasciata and N. inaurata spiders tested in air (25 °C, 35% RH).

Figure 5. True stress−true strain curves of miS from A. trifasciata. The fiber was subjected to successive processes of loading in air−complete contraction in water−reloading in air (recovery tests). The initial length at the beginning or the test was used for the calculation of strain. The fiber does not show recovery.

MAS fibers are more deformable and compliant than contracted miS fibers. As explained above, maximum supercontracted MAS fibers represent a ground state to which the material can revert by supercontraction. To investigate the possibility of a ground state for the miS fibers, we subjected three samples from each species to successive processes of loading in air and complete contraction in water (i.e., complete contraction was allowed between two consecutive tensile tests in air). The results of one of the tests with A. trifasciata miS are shown in Figure 5 as an example. As shown in the Figure, miS fibers do not recover their initial length in the contraction stages, and the permanent deformation of the fibers increases with the maximum strain reached in the tensile tests. From these results, it may be definitively concluded that miS fibers from both species are not able to reach a ground state after contraction in water that is independent of the loading history. 3.4. X-ray Diffraction. The diagrams obtained by XRD from A. trifasciata miS are shown in Figure 6, and the related parameters are displayed in Table 3. The spots in the diagrams can be labeled with the same indices used for the reflections

found in MAS fibers (included for comparison) and in B. mori silk.50 In particular, the most intense reflections correspond to (210) and (002) planes, as indicated in the Figure. Crystallinity was estimated by the ratio between the Bragg peaks intensity and the total intensity diffracted by the fiber. The calculated values are 12% in the FS fibers and 13% in the contracted (MC) fibers. The low crystallinity of MAS fibers, in the 10−15% range, was identified as an essential microstructural characteristic linked to the ability to supercontract51 in previous studies. Correspondingly, the much higher crystallinity in Bombyx mori fibers,50 around 60%, was associated with the absence of supercontraction in this material. The initial microstructural analysis of miS spun by Eriophora f uliginea suggested a higher crystallinity in these fibers compared with MAS,52 although still far from the values yielded by B. mori, so that the importance of crystallinity in the supercontraction effect remained to be elucidated. The low values of crystallinity in miS found in this work indicate clearly 2092


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Figure 6. X-ray diffraction patterns of MAS and miS fibers from A. trifasciata in the FS (MAS, miS) and MC (miS) conditions. The fiber axis lies along the vertical direction.

Table 3. Parameters of Minor Ampullate Silk Fibers from A. trifasciata Obtained by X-ray Diffractiona particle size (nm)

orientation, fc forcibly silked (FS) contracted (MC) a

(020)

(210)

crystallinity (%)

(020)

(210)

0.93 0.92

0.94 0.94

12 13

2.6 3.2

3.3 3.4

Parameters are defined in the text.

that crystallinity on its own cannot explain the different effect of water on silk fibers. XRD data can also be used to determine the orientation between the polypeptide backbone and the macroscopic axis of the fiber. In miS fibers, the equatorial position of the 020 and 210 reflections indicates that the miS polypeptide backbone in the crystallites is oriented along the macroscopic fiber axis. The degree of orientation, fc, defined as fc = (3 − 1)/2, where is computed from the azimuthal broadening of those reflections, indicates the mean value of the angle formed by the c axis of the unit cell (aligned with the protein backbone of the β-nanocrystals) and the macroscopic axis of the fiber. The mean orientation parameter fc is close to 0.93 in both fibers, so that their alignment is slightly lower than the alignment of FS MAS fibers, 0.96, as measured in previous works.51 A lower orientation in miS was also found when comparing miS and MAS fibers spun by Eriophora fuliginea.52 Interestingly, SAXS studies17 suggested that MAS shows a longer range ordering that is absent in miS fibers, consistently with a higher orientation in the former fibers. Finally, from the equatorial profile of the reflection peaks 020/210, particle sizes perpendicular to the fiber axis were calculated as L = 0.9λ/(B cos θ), where λ is the wavelength, B is the radial width (fwhm), and θ is the Bragg angle, yielding values on the order of 3 nm, with no significant difference between as-spun and contracted samples. The sizes are again similar to the sizes calculated in MAS fibers.53,52 3.5. Raman Spectroscopy. The Raman spectra in the two polarizations, Ixx and Izz, are shown in Figure 7 and compared with the Raman spectra of MAS fibers in the FS condition. The amide I vibrational mode, corresponding to the stretching vibration of the carbonyl group of the protein is observed in both polarizations at 1668 cm−1. The amide III vibrational mode corresponding to the vibration of the C−N group of the peptide bond is clearly identifiable in the zz polarization at

Figure 7. Representative polarized Raman spectra for the zz and xx polarizations of MAS and miS fibers from A. trifasciata under the FS condition. The fiber axis lies along the Z direction. The spectra are normalized relative to the peak height of the amide I bands to allow an easier comparison.

1228 cm−1, but it does not appear as a resolved individual peak in the xx polarization. The differences between the spectra recorded in both perpendicular polarizations reflect the relative orientation between the average direction of the protein backbone of the silk molecules and the macroscopic axis of the fiber (z axis). In this context, a simple and direct measurement of the overall orientation of the protein chains is provided by the parameter p 2 defined as p2 = I(xx)III(zz)/I(zz)III(xx). 54 The p2 parameter presents the advantage of allowing the comparison of both spectra independently from possible variations in the absolute intensities resulting from the measuring process. In the definition of p2, the intensities of the amide I peak at 1668 cm−1 in the xx and zz polarizations are indicated by I(xx) and I(zz), respectively, and those of the amide III peaks at 1228 cm−1 by III(xx) and III(zz). The calculation of the parameter p2 from the spectra presented in Figure 7 yields a value of p2 = 4.1 for miS fibers. 2093


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4.1. Tensile Behavior of miS Fibers. True stress−true strain curves for minor ampullate silks from A. trifasciata and N. inaurata, tested either in air (FS and contracted) or in water, are plotted in Figure 9a for comparison purposes. miS curves

3.6. Atomic Force Microscopy. A representative micrograph of the microstructure of miS fibers determined by AFM is shown in Figure 8, where the macroscopic axis of the fiber lies

Figure 8. AFM micrograph of miS from A. trifasciata under the FS condition. The histogram shows the distribution of the size of the nanoglobules, which are approximated by ellipses.

along the horizontal direction. It is observed that miS shows a nanoglobular structure that is characteristic of silk fibers spun from both silkworms and spiders,36 and artificial fibers.54,55 The nanoglobules were approximated by ellipses, and the distribution of sizes of the major and minor axes is shown in the inset. The average values are 10 ± 3 nm for the major axis and for the 7 ± 2 nm minor axis.

4. DISCUSSION The influence of composition and processing on the microstructure and properties of silk fibers is a central question for the production of bioinspired fibers.56 Spinning of bioinspired fibers relies on designing the sequence of the recombinant proteins57,58 and on the processing conditions.59 However, decisions regarding these aspects must be based on a limited knowledge of the natural system. Despite the fact that significant advances have been made,54 the properties of bioinspired silks still fall far from those exhibited by naturally spun fibers. In this context, an analysis and comparison of the microstructure and properties of natural silks of known sequences appears as a promising approach to the problem. However, its practical implementation is hampered by the intrinsic difficulties of the microstructural and mechanical characterization of silks, so that only partial results are usually available. In this regard, the access to a comprehensive characterization of different types of silk fibers provides very valuable information because it can be used to render support or to reject some of the hypothesis that have been proposed to relate composition, processing, microstructure and properties in these materials.

Figure 9. Comparison of the true stress−true strain of miS (a) and MAS fibers (b) from A. trifasciata and N. inaurata.

for both species and irrespective of their environment − dry or wet − and condition − FS or MC − show remarkable concurrence beyond the experimental scatter, a result that is further confirmed by data in Table 1. It is worth noting that a similar effect is not shown by MAS fibers: tensile curves in the three conditions considered here (FS, MC and tested in water) are clearly divergent, as shown in Figure 9b. The effect of water on miS fibers is more evident in the initial part of the stress−strain curve, specifically in the initial elastic modulus and the existence of a yield plateau (Figure 9a). The plasticizing effect of water reduces the value of the initial elastic modulus with respect to the fibers tested in air, as expected from the unavailability of hydrogen bonding reinforcement present in dry samples. Collapse of hydrogen bonding is considered to be the key mechanism behind the greater flexibility and loss of yield plateau of MAS fibers submerged in water.60 Nevertheless, miS fibers retain an initial modulus in water more than an order of magnitude larger than MAS 2094


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samples (Figure 3), suggesting the presence of stable, waterinsensitive interactions within their protein chains. Besides the basic interest of this result, the lower sensitive of miS to water environments is relevant for applications of bioinspired fibers to biomedical uses because most of these applications take place under hydrated conditions. Strikingly, tensile properties of miS fibers seem to be shared by a number of species, as illustrated in Figure 9 with A. trifasciata and N. inaurata, and further corroborated by Figures 10 and 11, where our results from A. trifasciata are compared with previously published data from other authors. Blackledge and Hayashi23 performed tensile tests in air with Argiope argentata silks, in particular, with MAS and miS fibers. These results are shown in Figure 10a and when compared with

Figure 11. Comparison of the tensile behavior of MAS and miS fibers tested in water. The data by Work20 are represented.

our present measurements from A. trifasciata, it is clear that there is striking agreement between the species. In a previous study, Work20 in 1977 performed similar tensile tests using Araneus diadematus MAS and miS. These tests are summarized in Figure 10b, alongside our measurements from A. trifasciata. We have computed stresses on miS fibers from Work’s force measurements using an average value of 1.80 μm. Work used an average value of 1.3 μm for his calculations but recognized that this value was affected by a significant uncertainty, as fibers were very thin and diameters were measured using optical procedures. Under this assumption, both test results concur, as it was found with the MAS and miS from the other species. Regarding fibers tested in water, Figure 11 shows the results reported by Work20 for minor ampullate silks from A. diadematus. Work provided only values for miS under “water restrained conditions” − not unrestrained as for MAS fibers. Nevertheless, it is expected that unrestrained results would be similar as miS fibers do not supercontract. MAS curves are also shown in the Figure for comparison. It is evident that minor ampullate silks from both A. diadematus and A. trifasciata behave similarly, with their initial elastic modulus being much higher than the corresponding MAS modulus. The above results complement previous findings by the authors30 showing that Argiope argentata, Argiope trifasciata, and Araneus diadematus, which are members of the same family (Araneidae), belong to the same group regarding the tensile behavior of their MASs. Nephila inaurata, which is in a different family (Nephilidae), has stiffer MAS. Contrary to MAS, the tensile behavior of minor ampullate silk seems to be largely insensitive to both the species and the condition of the fibers. 4.2. Microstructural Comparison of miS and MAS Fibers. Minor ampullate gland silk (miS) offers an excellent opportunity to establish correlations among composition, microstructure, and mechanical properties by comparing them with those of major ampullate gland silk (MAS). The analysis of the sequence of miS fibers from different species5,8,12 revealed that miS and MAS share three amino acid sequence motifs, An, GGX, and GA, but miS is deficient in proline and thus lacks the proline containing motif, GPG, which is a characteristic feature of MAS Spidroin 2 protein.

Figure 10. Tensile tests in air of A. trifasciata MAS and miS forcibly silked fibers. (a) Comparison with Argiope argentata23 fibers. (b) Comparison with Araneus diadematus20 fibers. Similar results are obtained with N. inaurata fibers, not represented for simplicity. 2095


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The large differences that silk fibers can exhibit when tested in water were first assessed when comparing the tensile properties of spider major ampullate and silkworm silk.63 It was assumed that in both silks immersion in water provokes the collapse of the intra- and interchain hydrogen bonds.60 This collapse leads to large conformational changes in spider silk, which are reflected in the supercontraction process.42 In contrast, it is assumed that the conformation of the protein chains in silkworm silk is not significantly affected by immersion in water, so that the tensile properties in the elastic regime of fibers tested in water are dominated by van der Waals forces. This hypothesis is supported by the agreement between a shear lag model of the elastic modulus of silkworm silk tested in air and in water under the previous assumption.64 The shear lag model of silkworm silk assumes that the material can be described as a composite of an amorphous phase reinforced with nanocrystals. The basic equation of the elastic modulus in this model is

The microstructural analysis of both materials reveals strong similarities between the basic microstructural parameters, as shown in Table 4. Therefore, the FS miS values of crystallinity, Table 4. Comparison of the Microstructural Parameters Measured from miS, MAS, and Silkworm (Bombyx mori) Silk method

parameter

X-ray diffraction

orientation: βsheet, fc crystallinity (%) particle size, avg. (nm) orientation: protein chains, p2 nanoglobular size (nm)

Raman spectroscopy atomic force microscopy a

miS

MAS

B. mori

0.94a

0.96b

0.96b

12a 3.0a

10−15c 3.3b

60e 4.2b

4.1a

4.0f

6.8b

10 × 7a

13 × 13d

23 × 16d

This work. bRef 51 cRefs 53 and 66. dRef 36. eRef 50. fRef 61.

⎛ tanh(ns) ⎞ Esilk = fEf ⎜1 − ⎟ + (1 − f )Em ⎝ ns ⎠

fc, orientation of the nanocrystals and nanocrystal size closely match those of FS MAS (12%, 0.94, and 3 nm for miS vs 10− 15%, 0.96, and 3.3. nm for MAS). The comparison of the p2 parameter as obtained from the polarized Raman spectra conveys information of the overall orientation of the protein chains with respect to the macroscopic axis of the fiber. In this case, miS fibers yield a value of p2 = 4.1, that matches the average value of 4.0 obtained from MAS fibers in their most aligned (forcibly silked or FS condition).61 Although previous works19,35 show a comparable trend, precise comparison with the present data is made difficult by the absence of information on the alignment of the analyzed MAS fibers. Finally, the microstructural similarity between both materials extends to the size of the nanoglobules observed by AFM, with the possible exception of its eccentricity (miS: 10 × 7 nm; MAS: 13 × 13 nm). The analysis of composition and microstructure of FS miS and FS MAS fibers might suggest that both materials are very similar and should essentially show comparable tensile behaviors. Nevertheless, the results in Figure 2 show that although both types of silk exhibit similar initial elastic modulus and tensile strength miS fibers are more compliant and have larger strain at breaking, leading to an overall different tensile behavior clearly distinguishable beyond the experimental scatter. In addition, the extreme divergence of the properties exhibited by both types of silks when exposed to water gives additional corroboration of their mechanical differences. The comparison of the percentage of contraction by immersion in water of miS and MAS fibers (Table 2) indicates that FS miS fibers do not exhibit the large values of contraction that are characteristic of MAS fibers.20 The different influence of water on both types of fibers is even more evident when the tensile properties of fibers tested in water (Figure 3 and Table 1) are considered: the initial elastic modulus of miS fibers decreases to 10% of its initial value in air, in contrast with the decrease to 0.1% of its initial value in air shown by MAS fibers. Not surprisingly, miS fibers do not show recovery or the existence of a ground state, as demonstrated by Figures 3 and 5 because these properties in MAS fibers are ordinarily attached to higher percentages of contraction. Whereas it could be argued that the lack of proline and hence the absence of the − GPG − motif in miS is at the root of this behavior, previous works by the authors have shown that proline is not essential for supercontraction.51,62

(1)

where the parameter “n” is defined as n=

2Em Ef (1 + νm)ln(1/f )

(2)

and the following symbols are used: f, volume fraction of the nanocrytalline phase; Ef, elastic modulus of the nanocrystalline phase; Em, elastic modulus of the amorphous phase; s, the aspect ratio of the nanocrystals, and νm, the Poisson coefficient of the amorphous phase. The use of this expression with the assumption that the elastic modulus of the amorphous phase was dominated by hydrogen bonds (Em = 8 GPa) when tested in air and by van der Waals forces (Em = 1 GPa) when tested in water led to a very good agreement between the calculated and experimental values of the elastic modulus of silkworm silk. Therefore, it was found that by taking the accepted values of the microstructural parameters of silkworm silk, the model yields a value of Eair = 14 GPa (experimental value Eair = 16 GPa) and Ewater = 4 GPa (experimental value Ewater = 5 GPa).64 The availability of the microstructural parameters obtained from the different characterization techniques presented in this work allows studying the robustness of the previous analysis when applied to Argiope trifasciata miS. In this regard, the required microstructural parameters were taken as (Table 4): nanocrystalline fraction, f = 0.12, and aspect ratio, s = 3.33 (calculated from the quotient between the major axis of the nanoglobules, 10 nm, taken as an upper limit of the longitudinal dimension, and the transverse size of the nanocrystals, 3 nm, as presented in Table 4). Using the same values of the elastic modulus for the amorphous matrix in air (Em = 8 GPa) and in water (Em = 1 GPa), the Poisson’s ratio of the amorphous matrix νm = 0.3, and the elastic modulus of the nanocrystalline phase Ef = 25 GPa, as were used for silkworm silk,64 the calculated values for the elastic modulus of miS are Eair = 8.3 GPa and Ewater = 1.2 GPa. These values compare well with the experimental values measured for Argiope trifasciata miS and presented in Table 1: Eair,exp = 10.0 GPa and Ewater,exp = 1.4 GPa. This agreement gives further support to the hypothesis that the mechanical behavior of miS is more similar to silkworm silk than to MAS, despite miS and MAS being closely related to each other (members of the same gene family) and silkworm silk having an independent evolution. 2096


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regarding the correlation between composition, microstructure, and mechanical behavior in these materials. Under their FS condition and tested in air, miS fibers display a tensile strength and work to fracture comparable to MAS while being more compliant. However, miS exhibits a tensile behavior well-differentiated from MAS fibers when the effect of water on the fibers is considered. Thus, if immersed in water, miS fibers do not show supercontraction and retain an initial elastic modulus in water more than an order of magnitude larger than that of MAS samples. Contrary to MAS, miS fibers do not show recovery or the existence of a ground state to which the fiber can return independently from its previous loading history. The lower sensibility to water of miS fibers resembles the behavior of silkworm silk fibers, and it has been shown that the variation of the initial elastic modulus in the presence of water can be justified with the same model used for Bombyx mori silk. Despite the large mechanical differences between miS and MAS, their microstructures reveal strong similarities in all of the features considered in this work; measurement of the crystalline fraction, orientation of the nanocrystals, size of the nanocrystals, protein chain alignment, and size of the nanoglobules result in concurrent values for the two silks. Additionally, miS microstructural parameters differ considerably from those of B. mori silk, which calls into question the identity of the actual parameters governing the macroscopic behavior of silks. A hint could be given by the degree of hydrophilicity of the protein chains or by the large scale organization of the sequence, hypotheses that imply possible and simple explanations for the extreme divergence of mechanical properties and plasticizing effect of water in miS, MAS, and silkworm silk. Finally, miS tensile properties seem to be traits extremely conserved during evolution, as demonstrated by the agreement on mechanical properties between Araneidae and Nephilidae, families estimated to have diverged at least 120 million years ago. Results from Argiope trifasciata, Argiope argentata, Araneus diadematus, and Nephila inaurata display a striking correspondence that suggests that miS properties were locked very early in the evolutionary history of orb-web weaving spiders. Why miS behaves the way it does and what the specific biological role of miS is that led to the conservation of such mechanical properties for millions of years are questions that remain unanswered.

Intriguingly, the microstructure of miS differs more from that of the nonsupercontracting B. mori silk than from that of the supercontracting MAS, as shown in Table 4, where the basic microstructural features of all three types of silk are compared. In particular, silkworm silk fibers show extreme values of the crystalline fraction (60 vs 12% for miS), alignment of the protein chains (p2 = 7.5 vs 4.1 for miS), and size of the nanoglobules (23 × 16 nm vs 10 × 7 nm for miS). Microstructural parameters of miS fall in the range of values exhibited by supercontracting fibers such as MAS (Table 4), regenerated silkworm silk,51 and bioinspired spider silk fibers.54 Besides the negligible amount of proline in both sequences, a fact which, as mentioned above, does not necessarily imply the absence of supercontraction,62 the comparative analysis of miS and Bombyx mori silk sequences identifies two additional major features in which both silks differ from MAS. One of them is the content of small, hydrophobic, amino acids, a feature that is likely to be related to the degree of affinity of these materials for water. Polar glutamine residues present in the − GGQ − motif of sequence have been proposed to explain the tendency of MAS to absorb water and supercontract.65 The analysis of the sequences of miS from Nephila clavipes (GenBank Accession numbers O17434 and O17436) and Argiope argentata (Accession numbers JQ713003 and JQ713004), species that are closely related to those studied in this work, MAS from Argiope trifasciata (Accession numbers Q9BIU6 and Q9BIU7), and Bombyx mori silk (Accession numbers P05790 and P21828) leads to an estimation of the mean hydrophilicity of these materials: 0.8 to 0.9 for N. clavipes and A. argentata miS, 0.7 for B. mori silk, and 1.2 for A. trifasciata MAS. In this context, mean hydrophilicity is defined as the average extended to all of the amino acids in the sequence considering the maximum number of hydrogen bonds that can be established by the side chains of the amino acids. In addition, analysis of the organization of the motifs within each silk protein shows that miS spidroin is characterized by large crystalline-forming regions in which the motifs − GA − and An are repeated many times. The longest crystalline forming regions of the sequenced fragments include 22 and 24 residues for A. argentata and N. clavipes, respectively. These lengths were calculated as the longest fragment that does not include any single amino acid residue, except those found in both aforementioned motifs of sequence. Assuming a β-conformation for these regions, the length spanned by the crystalline forming regions yields a value of 8 nm, which is very close to the size of the nanoglobules as measured by AFM. When a similar analysis is performed on silkworm silk, the maximum length of the crystalline-forming regions assuming β-conformation reaches a value of 37 nm, with an average value of 17 nm. These lengths are in the range of the sizes determined by AFM for the major axis of the nanoglobules, 23 ± 6 nm.36 Although further refinement and validation are necessary, the possible difference between hydrophilicity and the long-scale organization of the sequences might underlie the large differences in the tensile properties observed between major and minor ampullate gland silks.

■ ■

AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS Spiders were reared in Reptilmadrid S.L. by Oscar Campos. Ultramicrotomy was performed by E. Baldonedo (Centro de Microscopiá Electrónica, Universidad Complutense de Madrid). Dr. A. Gil and L. Colchero (Nanotec Electrónica, S.L., Spain) offered support for AFM observations. We are grateful ́ to José Miguel Martinez for his help with the artwork. The work was funded by Ministerio de Educación y Ciencia (Spain) through project MAT 2009-10258, by the Comunidad de Madrid (Spain) (Grants CCG10-UPM/MAT-5698 and S2011/ ́ BMD-2460), and by Fundación Marcelino Botin.

5. CONCLUSIONS The extensive microstructural and mechanical characterization of minor ampullate silk from two species, Argiope trifasciata and Nephila inaurata, performed in this work adds significant information to both the tensile behavior and the microstructure of miS fibers from orb-web spiders and has made possible the comparison with other silks to check some hypotheses

■

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