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Proline and Processing of Spider Silks Yi Liu, Alexander Sponner, David Porter, and Fritz Vollrath* Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, United Kingdom Received August 7, 2007; Revised Manuscript Received September 13, 2007
Major ampullate (MAA) silks from a variety of spider species were collected by artificial silking that adjusted the samples to have similar breaking strains. Those silks are highly comparable in post-yield mechanical properties, but their supercontraction behaviors and initial moduli vary in large ranges and both correlate with the content of one amino acid, proline. These relationships, in combination with protein sequence data, support the hypothesis that the proline-related motif, that is, GPGXX, may play a key role in silk. This also explains the interspecific variability of spider dragline silk. Moreover, MAA silks from three representative species were prepared in a range of processing conditions and their mechanical properties were compared. Our results indicate how chemical compositions, coupled with processing conditions, shape the mechanical properties of the spider silk.
Introduction What determines a spider silk’s properties: primary sequence or processing conditions?1,2 This important question has been hotly debated for nearly ten years. As it appears now, spinning conditions tune a silk fiber’s properties,3,4 while the primary sequence, evolved over nearly 400 million years, plays an important role in defining the basic material.1 The proteinaceous constituent of the MAA silk of Nephila claVipes has been shown to be composed principally of two highly evolved proteins: the virtually proline-free spidroin MaSp1 and the proline-rich spidroin MaSp2.5,6 Similar and related spidroins were found in the MAA silks from a variety of other araneoid species.1,7 Glycine (G) and alanine (A) are always the most abundant amino acid residues in all MAA silks, whereas the proline (P) content varies considerably between the MAA silks of different species.8 This distinct interspecific variability in amino acid compositions of MAA silks suggests an evolutionary function. Selection, one might presume, acted primarily on the mechanical properties. This, then, would suggest a link between chemical structure and mechanical properties. However, attempts to correlate the amino compositions or the primary protein sequences with the mechanical performance of silks so far failed to produce conclusive results.9–11 Large, or super, contraction of the fiber, when placed in water or other wetting agents demonstrates the interference of water molecules with the integrity of the bulk material and, as such, is a peculiar feature of MAA silks.12,13 Supercontraction is probably induced by the water affecting the mobility of the glycine-rich motifs, but not the alanine-dominated crystalline regions, which remain fixed.14–16 Yang et al. studied the MAA silk of Nephila claVipes with 13C and 2H NMR, claiming its supercontraction is controlled by YGGLGS(N)QGAGR blocks in MaSp1.15,17 However, this study did not take into account the contribution of MaSp2, as the authors mentioned the absence of proline signals in their NMR spectra.15,17 Yet an earlier study showed Nephila claVipes MAA silk to contain over 4% of proline,18 which must be due to MaSp2 components.19 Indeed, a recent immunostaining study confirmed the existence of non* To whom correspondence should be addressed. E-mail:fritz.vollrath@ zoo.ox.ac.uk
negligible amounts of MaSp2 (with its high proline content) in Nephila MAA silks.19,20 Proline is not intrinsically “ordered”, with its side chain cycling back to the polypeptide backbone and forming a ring structure.21 This distinguishes it from other amino acids residues. With the pyrrolidine ring, proline introduces steric constraint and lacks one amide proton available for hydrogen bonding, which stabilizes such structures.22,23 Hence, proline disfavors the formation of R-helix and β-sheet structures but tends to induce β-turn or γ-turn structures in the backbone.24–27 Recently, Rauscher et al. have shown that the combination of proline and glycine could modulate backbone hydration and conformational order of peptides, thus leading to elastin-like or amyloid-like behaviors.28 In a number of natural elastomers, proline is widely present in the form of pentamer sequence, that is, GPGXX.26,29 Such proline-containing motif was suggested to disrupt the crystalline structure in spider silk27 and was hypothesized to account for the elasticity of silk.26 Recently, Vollrath and Porter suggested proline is related to a fiber’s sensitivity to water, that is, the capacity to shrink (Csh), because it brings about intrinsic disorder but with potential for order.21,30 This argument is supported by two facts: (1) silkworm silk and spider minor ampullate (MIA) silk, which both contain little proline, contract little if at all,31–34 and (2) spider MAA silks from a variety of species and with different amounts of proline supercontract to differing degrees.8,35 Thus, the ratio of proline might be linking amino acid composition and supercontraction behavior, potentially being a key parameter ignored in previous studies.9,11,17 Indeed, because there is a strong link between supercontraction and mechanical properties,3 proline might be a key to linking a silk’s amino acid composition with many, if not all, of its mechanical properties.36 Here we compare spider silks from eight species and demonstrate how proline, coupled with processing conditions, influences a spider silk’s properties.
Materials and Methods Adult females of eight species from three families, that is, Araneidae (Cyrtophora citricola, Nuctenea sclopetaria, Argiope argentata, Argiope lobata, and Araneus diadematus), Tetragnathidae (Nephila edulis and Nephila senegalensis), and Theridiidae (Latrodectus hesperus) were reared in the laboratory, fed on flies, and watered regularly. Single
10.1021/bm700877g CCC: $40.75 2008 American Chemical Society Published on Web 12/04/2007
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Figure 1. Representative stress–strain curves of MAA silks from a range of species tested either in the native (solid line) or the supercontracted state (dash line). Breaking points of all tested samples are shown as solid squares (native silk, 103 samples) or hollow squares (supercontracted silk, 93 samples). The reeling condition was tuned to produce native silk samples with a breaking strain of 0.24 ( 0.03. Each species is illustrated in a distinct colour as follows: gray square, Cyrtophora citricola; red square, Latrodectus hesperus; black square, Nephila edulis; brown square, Nephila senegalensis; green square, Nuctenea sclopetaria; purple square, Argiope lobata; light green square, Argiope argentata; blue square, Araneus diadematus. The proline content and ordered fraction (see refs 39, 40) of supercontracted MAA silks are marked. The straight black line defines the upper limit of native MAA silk, where the ordered fraction is 1.0; the straight gray line roughly separates the stress–strain curves of native and supercontracted silks.
filaments of dragline silk were collected at eight controlled speeds (1, 2, 6, 10, 20, 27, 100, and 200 mm/s), as described elsewhere.3 The sampling procedure allowed for separate sections of one fiber to be removed individually for a range of experimental and analytical tests. For measurements, individual silk threads were carefully transferred with dividers from the spool to a custom-built microscale mechanical testing machine and tested at the straining speed of 50% per minute4,37 at ambient temperature (22–25 °C) and humidity (35–50%). Some samples were tested in the native state, while others were tested in the supercontracted state. A number of filaments were gold-coated, and their diameters were measured with a JEOL JSM-5510 SEM at the magnification of 10000. The value of each thread’s diameter was used to calculate its adjacent sample’s engineering stress (normalize). The diameter of a supercontracted sample was calculated with the tested assumption38 that a fiber’s volume remains constant after supercontraction. Adjacent sections of individual fibers tested mechanically were analyzed for amino acid content by Oxford University’s Immunochemistry Unit. For the analysis, 7–15 silk filaments, about 3 cm in length each, were put in pyrolyzed tubes. To each sample, 2 nmol of norleucine was added as internal standard. The samples were dried under vacuum. After hydrolysis in 5.7 N HCl for 22–24 h at 110 °C, they were dissolved in distilled water containing EDTA, and 20–40% of each sample was placed on a glass amino acid analyzer sample slide. Hydrolyzed amino acids were derivatized under basic conditions with phenylisothiocyanate (PITC) to produce phenylthiocarbamyl (PTC) amino acid derivatives on an Applied Biosystems Model 420A PTC derivatizer and then analyzed with an online Applied Biosystems Model 130A PTC Amino Acid Analyzer.
Results and Discussion MAA silks from eight species were reeled under one set of highly controlled conditions that had been tuned3 to achieve breaking strains of about 25%. The native silks prepared in such
a way displayed similar mechanical performance, and their stress–strain curves conformed to a common pattern (Figure 1). We use the term “native silk” to refer to forcibly reeled as well as naturally released silk in contrast to fibers artificially spun from silk dope or from reconstituted material. A statistical study indicates that the post-yield properties of these “native” MAA silks, that is, breaking stress and breaking energy, were indeed comparable (Table 1). Variability did exist between the different silks, as illustrated by the solid squares in Figure 1. However, interspecific variability did not necessarily override the intraspecific variability (Table 1). For instance, the c.v. (coefficient of variation) of single thread breaking energy across the eight species was 17%, while Nephila edulis fibers alone had a comparable c.v. of 21%. Therefore, according to the model proposed by Porter et al., our MAA silks should contain similar fractions of “order”. This model shows that the mechanical properties of any silk are determined by the fraction of “ordered” states, which are defined as having two hydrogen bonds per amide group in a peptide segment and which in turn correspond very roughly to the degree of crystallinity that is distributed on a nanometer scale.39,40 In contrast to the observed similarity in the stress–strain curves of native MAA silks, the supercontracted MAA silks of the eight species differed significantly in their mechanical behavior (Figure 1). A supercontracted MAA silk could behave like a relative “hard” material (Cyrtophora citricola and Latrodectus hesperus, breaking strain of 0.4–0.6, initial modulus of 7–10 GPa, and breaking stress of 1.2–1.6 GPa) or like extremely “soft” materials (Nuctenea sclopetaria, Argiope sp., and Araneus diadematus, breaking strain of 1.2–1.6, initial modulus of 3–5 GPa, and breaking stress of 0.6–0.9 GPa), with the silks of Nephila sp. (breaking strain of 0.8–1.1, initial modulus of 5–6 GPa, and breaking stress of 0.9–1.2 GPa) laying
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Table 1. Post-Yield Mechanical Properties of Native MAA Silks from Different Speciesa species
proline (mole%)
breaking stress (GPa)
breaking energy (mJ m-3)
N (spiders)
n (tests)
Cyrtophora citricola Latrodectus hesperus Nephila edulis Nephila senegalensis Nuctenea sclopetaria Argiope argentata Argiope lobata Araneus diadematus all species
0.6 1.9 4.2 5.7 8.8 9.3 10.4 14.3
1.65 ( 0.24 1.44 ( 0.12 1.44 ( 0.16 1.84 ( 0.17 1.66 ( 0.13 1.91 ( 0.18 1.52 ( 0.14 1.67 ( 0.18 1.65 ( 0.23
230.59 ( 31.20 243.10 ( 29.12 198.45 ( 42.08 283.98 ( 30.16 245.44 ( 35.08 224.67 ( 19.68 196.04 ( 29.20 224.94 ( 29.11 228.91 ( 39.76
3 1 4 2 3 2 4 4 24
13 10 15 13 11 14 12 15 103
a
The reeling condition was tuned to produce samples with a breaking strain of 0.24 ( 0.03.
Figure 2. Correlations between proline content (expressed in mole percent) and physical properties of native (solid squares) and supercontracted (hollow squares) MAA silks from different species: (a) capacity to shrink; (b) initial modulus; (c) breaking stress; and (d) breaking strain. Each data point represents the average in physical properties of each species, taken from n ) 1–4 spiders, with N ) 9–15 measurements and is marked in a distinct colour as stated in the legend of Figure 1. The solid lines in (a)–(d) are the fitting curves obtained by a least-squares approximation, with blue lines for supercontracted fibres and red lines for native fibres. The red dash lines each represent the average of a certain mechanical property that is similar across species and does not correlate with proline content.
in between. How it behaves seemingly depends on its proline content, as shown in Figure 1. Indeed, native MAA silks showed significant differences in Csh, and such species-specific differences correlated exponentially with the proline contents of the different silks (Figure 2a). Thus, native silks, though displaying similar mechanical performance, shrank to varying degrees in water and resulted in supercontracted counterparts with pronounced interspecific differences. MAA silk of Cyrtophora citricola contained the least amount of proline (about 0.5%) and shrank only 10% in water. MAA silk of Araneus diadematus, which together with silks of Nephila sp. is probably the most intensively studied of all spider silks, had the highest proline content (nearly 15%), and shrank more than 50% in water. Interestingly, in general, the increase rate of Csh declined with increasing proline content, especially at proline ratios above 9%, which corresponds to MAA silk of Nuctenea sclopetaria. A plateau in this Csh-proline correlation divided the otherwise rather similar silks of Argiope sp. and Araneus diadematus. The native and supercontracted MAA silks fit into the model developed with the theoretical
framework provided by the mean field theory for polymers.40 After supercontraction, MAA silks lost different degrees of “order” (Figure 1),40 therefore assuming significantly different stress–strain curves. The initial modulus of native silks, which is an excellent preyield mechanical indicator, correlates extremely well with proline content (Figure 2b). The relationship between initial modulus of native silks and proline content is described by a negative linear equation (Table 2), with Cyrtophora silk having the highest value of 18 GPa and Araneus having the lowest of 10 GPa (Figure 2b). This is consistent with the conclusions of Swanson et al. who reported that MAA silks of Kukulcania and Plectreurys (two species that both lack glycine–proline repeats) had the highest initial modulus among the seven species they studied (Nephila claVipes, Argiope argentata, Araneus gemmoides, Latrodectus hesperus, Leucauge Venusta, Kukulcania hibernalis, and Plectreurys tristis).11 After supercontraction, the initial modulus of each MAA silk in our study decreased appreciably, and its correlation with proline content can be best described by a negative exponential equation. Though those silks
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Table 2. Correlations between Mechanical Properties and Proline Content (P) of Supercontracted and Native MAA Silks physical property
fitting equation/mean
state
R2a
capacity to shrink, Csh initial modulus, Min (GPa)
Csh ) 0.560 × (1 – exp(-0.217 × P)) Min ) 3.604 + 6.895 × exp(-0.289 × P) Min ) 18.561 - 0.625 × P b ) 0.705 - 1.510 × exp(-0.154 × P) b ) 0.24 ( 0.03 σb ) 0.665 + 0.980 × exp(-0.204 × P) σb ) 1.65 ( 0.23
native supercontracted native supercontracted native supercontracted native
0.982 0.985 0.936 0.963
breaking strain, b breaking stress, σb (GPa) a
0.926
R2 is the square of the correlation coefficient.
contracted to different degrees, the loss of initial modulus in each silk seems fairly comparable across species. Post-yield mechanical properties of native MAA silks were comparable and uncorrelated with proline content, because those silks had been adjusted to similar breaking strains by our tuning of the reeling conditions (Table 1). However, after MAA silks supercontracted, those mechanical properties showed significant interspecific differences and were closely correlated with proline content (Table 2). The two intensively studied mechanical properties (breaking stress and breaking strain) of both native and supercontracted silks were plotted against proline content in Figure 2c,d. Our data show that, depending on the proline content, a supercontracted MAA silk can become “rubberlike”,41 for example, in Araneus and Argiope, or fails to change significantly mechanical behavior, for example, in Cyrtophora. Because the corresponding silks had comparable properties to one another before supercontraction, these differences in response to the action of water (demonstrated by different shrinkages) suggest differences in lost degrees of order that in turn correlate with differences in proline ratios. How does proline act to result in the observed interspecific differences in native MAA silks? If we assume (as is likely6,7) that proline (as measured by amino acid analysis of entire fibers) exists principally in the bulk material of one (or several) constituent silk protein, such as, for example, MaSp2,1,6,7,42 then the link is relatively easily explained. First, proline is known as a strong β-sheet breaker due to its unique pyrrolidine ring structure and the lack of one potential H-bond donor.24 This particular residue side group will not be able to hydrogen-bond fully with adjacent amino acid residues to form a β-sheet structure. Such partial bonding naturally leads to the weakening in the initial modulus, which is closely related to the hydrogen bonding network.39,43 Thus greater proline content would result in smaller initial modulus. Second, as a β-sheet breaker, proline might even prevent neighboring, more ordered motifs from crystallizing, opening them up to attack by water molecules. Such neighboring motifs would most probably be glycine because 70% of all glycine present in silk was found (by NMR) to be mobile during supercontraction.14,17 Moreover, a higher proline content suggests a larger fraction of MaSp25,6 and a lower ratio of “skin”.19,44 This might naturally lead to a more effective plasticization of water and influence the different supercontraction behavior patterns observed. The exponential, rather than linear, relationship between Csh and proline content (Figure 2a and Table 2) might suggest a limit to the contraction of molecular backbones. For example, the MAA silks of Araneus diadematus, Argiope lobata, and Nuctenea sclopetaria might contain so much proline that their supercontraction approaches this limit. Alternatively, this relationship might be attributed to a dense concentration of proline: in proline-rich MAA silks, some proline residues locate side by side, thus reducing the overall effect on glycine-rich domains. A previous study showed that, within one species (Nephila edulis), supercontraction and mechanical properties can be
adjusted with reeling conditions.3 Taking into account the effect not only of chemical provenance (i.e., genetic differences in the silk feedstock) but also of processing conditions, we examined the effect of reeling speed on the mechanical properties of the other two MAA silks produced for this study (Cyrtophora citricola and Araneus diadematus) and previously recorded (Nephila edulis3). These three species are typical in that they represent the low (Cyrtophora citricola) and high (Araneus diadematus) extremes of proline content, as well as the middle ground (Nephila edulis). All the three silks display a series of mechanical behaviors from “soft” to “hard” as the reeling speed increases, and their varying ranges are very similar (Figure 3). However, their Csh vary in very different ranges: 0.05–0.10 for Cyrtophora citricola, 0.17–0.42 for Nephila edulis, and 0.37–0.56 for Araneus diadematus, as marked on the typical stress–strain curves. Not surprisingly, these differences were linked with differences in the proline content of the three silks. Thus, our simple experimental test seems to strongly support the “proline hypothesis”. Taken together, a spider silk’s properties are affected by the interplay of processing conditions and chemical composition. Processing conditions can adjust a spider silk’s Csh in a certain range, while the proline content appears to determine in which range a silk’s Csh can be adjusted. Mechanical properties are tuned by processing conditions, which mainly change the “order” of molecular chains, while an important parameter, that is, initial modulus, is linked with proline content if the effect of “order” is taken out.
Conclusions Interspecific differences in the mechanical37,45 and supercontraction35,37 properties are a well established “window” into silk structure–function relationships. Here we attempted to add a deeper level of understanding on the compositional level. Thus our experiments, for the first time, demonstrate the quantitative relationships between the physical properties of spider major ampullate silks and their contents of a key amino acid: proline. These relationships, in combination with protein sequence data,1,6 support the hypothesis that the proline-related motif, that is, GPGXX, may be a key motif in silk. This motif possibly prevents tight packing and thus allowing water molecules to access and disorient ordered glycine-rich motifs, which in turn leads to softening and to supercontraction.21 Furthermore, the initial modulus was found to decrease with increasing proline content. We attribute this to a decreasing fraction of hydrogen bonds owing to proline’s lack of one H-bond donor. Csh appears constant for proline-rich MA silks probably due to a corresponding dense packing of proline and a limit of contraction in the molecular backbone. Because of their different capacities to shrink, supercontracted MAA silks of different species display a variety of mechanical behaviors. Such findings, alongside a previous experimental study on the supercontraction phenomenon,3 help us to better understand
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link density of silks can vary depending on the larger scale sequence architecture of the dragline fibroins. It is possible that proline, alongside details in protein sequence (which cannot be identified by our comparative study), tune the mechanical behaviors by changing the crystal cross-link density and hydrogen bonding network. Acknowledgment. For funding we thank the European Commission and US-AFOSR for general support and NERC/ Hutchison Whampoa for a Dorothy Hodgkin doctoral studentship to Y.L. We thank C. Dicko, C. Holland, Z. Shao, and Y. M. Liu for comments and suggestions on the research and manuscript.
References and Notes
Figure 3. Representative stress–strain curves of MAA silks from three species: (a) Cyrtophora citricola (proline % ) 0.6), (b) Nephila edulis (proline % ) 4.2), and (c) Araneus diadematus (proline % ) 14.3). The silks were reeled at various conditions to lock in different degrees of order. Cyrtophora MAA silk was reeled at 1, 2, 6, 20, 100, and 200 mm/s; Nephila MAA silk was reeled at 2, 10, 20, 100, and 200 mm/s; and Araneus MAA silk was reeled at 1, 6, 20, and 200 mm/s. Typical stress–strain curves are represented by the red line (highly ordered), black line (mildly ordered), and green line (low ordered). Breaking points of all test results are shown by blue squares. Csh and ordered fraction (see refs 39,40) of typical samples are indicated.
the link between mechanical properties and the structure of a spider MAA silk. We conclude that, for a given proline content (e.g., a species-specific MAA composition), a fiber’s physical properties would be determined largely by the degree of order locked into the final product by the processing conditions.3 Between silks of different species, on the other hand, both the chemical composition (the proportion of proline as proposed in this study) and the degree of order in the noncrystalline region (as determined by processing) would together determine the physical properties of the fiber. This conclusion must be true at least for those eight species of spiders we studied. Apparently, not all MaSp2 proteins of the MAA silks in this study are identical in their domain architectures, and the crystal cross-
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