Segmented Nanofibrils of Spiral Silk in - American Chemical Society

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Segmented Nanofibrils of Spiral Silk in Uloborus walckenaerius Spider Zhongbing Huang, Xiaoming Liao, Guangfu Yin,* Yunqing Kang, and Yadong Yao College of Material Sciences and Engineering, Sichuan UniVersity, Chengdu, 610065, People’s Republic of China ReceiVed: NoVember 17, 2008; ReVised Manuscript ReceiVed: February 13, 2009

The structure of the adhesive silk of the Uloborus walckenaerius spider has remained unsolved due to its unremarkable material properties. In this work, we investigate the microstructure of Uloborus spider cribellar silk using probe microscopy techniques. Nanofibrils in a section of cribellar silk from the pyriform gland was observed by atomic force microscopy, and the results showed a segmented substructure with R-helices for individual protein molecules. The height and width of each nanofibril segment suggests a webing pattern of R-helix molecules. Force spectra of the cribellar nanofibrils indicate that a modular substructure within single protein molecules is demonstrated through unfolding this fibril molecule. The substructure features of this Uloborus spider cribellar silk is obviously different from the structures of other spider silks analyzed by single-molecule force spectroscopy. 1. Introduction During recent decades, research interest in spider silk has significantly increased due to its remarkable mechanical properties, which has excited us to synthesize it by biomimetic techniques, promoting the rapid development of a high property material field. Spider capture silks have outstanding viscoelastic properties, which combine both a high tensile strength comparable to Kevlar fiber (∼1 GPa)1 and extremely stretchy elasticity (as much as 500-1000%).2-9 Obviously, spider capture silk is also a natural block copolymer with its repeating structural motifs.10 The stretch of this silk protein especially gives our design inspiration in many elastic materials, such as texture, fabrics, coatings, munition ropes, and engineering structure materials.11 As a good tool for capturing insects, the structure and force properties of two-dimensional (2D) webs from orb spiders (Araneidae) is stronger than that of a 3D web-networks of spiders. In these woven-orb webs, the energy dissipation needs to stop an insect from breaking silk strands like 3D webs but by stretching the capture spiral with high elasticity. Thus, the capture spirals are required to be strong in order to not break and release the insect.3,12 When the spiral silk is even blown about by gentle breezes, it can also contract rapidly from stretching so as not to sag and stick to things. While sticking insects, the spiral silk from ecribellate orb webs needs to maintain its elasticity by remaining wet, through the action of its hydroscopic gluey coating on two supporting silks (the elasticity of dry spiral silk is much smaller than that of wet silks).4 In cribellar spiders, the stickiness of the spiral silks (produced by the pyriform gland), which are supported by a pair of axial silks (produced by the pseudoflagelliform glands),13 is not dependent on the air humidity. The extensibility of cribellar silks results in part from the separation of the fine fibrils that form its surface and in part from the extensibility of its supporting axial silk.3 A detailed study from Ko¨hler and Vollrath revealed that the extensibility of spiral silk produced by immature Uloborids was 3.8× greater than that of ecribellar * To whom correspondence should be addressed. Tel/Fax: 86-2885413003. E-mail: [email protected].

silk produced by mature Araneids of the same mass.3 However, the special microstructure of cribellar spiral silks from the Uloborids spider are not understood fully. With using atomic force microscopy (AFM) in the protein structure field based on force spectroscopy, people have revealed that the elastic behavior in molecules of many proteins, such as the muscle protein,14,15 polysccharides,16-18 DNA,19,20 silkworm silk,21 dragline silk, and flagelliform silk from ecribellar spiders.22,23 Furthermore, the surface and internal structure of dragline silk22,24 and orb web silk have also been provided by AFM. Hansma et al.23 investigated the sequence and structure of ecribellar spider capture silks through analyzing AFM force spectroscopy and their results revealed that the flagelliform protein from ecribellar spider capture silk had a nanospring molecular microstructure. However, we have not understood why the outstanding extensibility of spiral silk from the cribellate Uloborude spider exists. Capture silk of the cribellar spider is harder to investigate than that of the ecribellar spider and the less-elastic dragline silk, because capture silk is found only in orb webs of cribellar spiders and is thus difficult to isolate or collect from orb webs due to its superfine nanosize and high viscidity. Therefore, force spectroscopy is especially valuable because it can be used to investigate capture silk in situ from orb webs. With a force spectroscopy technique from AFM and extensive analysis of the pulls, we are able to investigate the adhesive cribellar silk using this technique and propose models for both its molecular and multimolecular structures. 2. Materials and Methods Sample Preparation and Observation. Genomic DNA was isolated from ethanol-preserved specimens. Tissue samples from the opisthosoma were homogenized in extraction buffer (50 mM Tris-HCl, pH 8, 300 mM EDTA, pH 8, 1 mM NaCl, 0.5% SDS, 8 mg mL-1 DTT and 0.4 mg mL-1 proteinase K), incubated for 2 h at 56 °C and centrifuged at low speed. The supernatant was phenol extracted twice and washed with chloroform. DNA was precipitated with 100% ethanol and 0.3 M sodium acetate, pH 6. Pellets were resuspended in TE buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA).

10.1021/jp810103s CCC: $40.75  2009 American Chemical Society Published on Web 03/24/2009

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Figure 1. Sketch of experimental setup, showing section of a cribellar web of Uloborus spider collected on a glass slide, and a section of cribellar fibril that is being stretched. Enlargement square shows detail of cantilever stretching a fibril from a spiral silk.

Aliquots of the DNA extractions were subjected to PCR reactions to amplify three DNA fragments corresponding to the following regions of the Drosophila melanogaster 28S rRNA sequence:25 3338 ( 3650 (primers: 5′-CCCSCTGAAYTTAAGCATAT-3′, 5′-ACTCTCTATTCARAGTTCTTTKC- 3′). Amplifications were done in 25 µL Tris (67 mM, pH 8.8) containing 2 mM MgCl2, 50 µg bovine serum albumin, 1 mM of each dNTP, 1 µM of each primer, template DNA (500-800 ng) and Ampli Taq DNA polymerase (1 unit, Perkin/Elmer-Cetus). Forty cycles of PCR (92 °C for 40 s, 50 °C for 60 s, 72 °C for 120 s) were performed. Amplified products were examined on 1.5% agarose gels. DNA fragments were excised from the agarose gel, purified using glassmilk (Gene Clean, Dianova), and redissolved in TE buffer. About 100 ng of template DNA was used per sequence reaction, which was carried out according to Casanova et al.26 DNA fragments were manually sequenced in both directions with the amplification primers. In order to observe spiral silk by AFM, spiral silks were allowed to bind on a freshly cleaved glass surface by incubating for 5-8 min and excess protein was removed. The shape and structures of pyriform protein fibrils were studied by scanning electron microscopy (SEM, JEOL-5900LV, 20 kV). AFM Image and Force Spectroscopy. AFM images in air were performed by standard procedures in tapping mode on a Scanning Probe Microscope Station of SPA400-SPI4000 (SEIKO NanoTechnology Inc., Chiba, Japan). Cantilevers for AFM imaging were also obtained from Budgetsensors Instruments. The AFM images were obtained, processed, and analyzed with accessory software with AFM unit. Force spectroscopy was performed on a Molecular Force Probe MFP-SA (Asylum Research, Santa Barbara, CA). Silicon nitride cantilevers were obtained from Park Scientific (Stanford, CA). The MFP was used to obtain force-piezo-extension curves, which were calibrated and partially analyzed by a manufacturersupplied software. In order to obtain worm-like-chain (WLC) curve-fit analyses, this software was correspondingly modified by using the built-in macro language. Stretching Fibril Molecules of Spiral Silk. A section of spider web from Uloborus walckenaerius was deposited onto a clean glass microscope slide, carefully breaking off silk strands at the edges of the slide. Webs on slides were stored in a dustfree container at 22 °C amd 25% relative humidity until used. In the previous report,23 a liquid drop needed to be placed on a part of unstretched orb web to keep its natural wet state. However, the adhesive spiral silks of cribellar will shrink in the liquid drop and change the nature of their molecular structure. Our pulling silk experiment was performed in air to maintain the natural structure state of cribellar spiral silk, and Figure 1 shows the sketch of experimental setup. For pulling, the glass slide was placed in the MFP (MFPSA, Asylum Research, Santa Barbara), and a strand of capture web attached to the slide. The tip of the end of a cantilever was slowly pressed into a strand of spiral silk laid on the glass slide, and stopped several seconds for conglutination between the tip and one of the nanofibrils. When the tip was raised, it could

Figure 2. (a) SEM image of spiral silk of spider Uloborus walckenaerius, arrows point two supporting axial silks; (b) a magnified image of some nanofibrils, arrows point a part of differentiable skin layers.

take up one or more fibril molecules from the strand of spiral silk and could pull these fibril molecules between the glass slide surface and the tip. During pulling fibrils, the maximum space between the tip and the sample surface was less than 0.6 µm to avoid having the tip pick up overfull fibril molecules in subsequent cycles of stretching and relaxation. The MFP head with the cantilever was played down to the MFP base with the glass slide. The spiral silk and MFP are readily able to image and place the space interval of ∼80 µm between them using the optics of the MFP. The cantilever’s tip was simply pressed onto the fibrils of a spiral silk to stretch molecules between the tip and the surface, as has been observed previously with other spider silks.22,23 Stretching Integrated Spiral Silk in Air. A region of web was collected onto a fine metal wire frame with 10 cm2 squares. A section of spiral silk that spanned a square was fixed with resin glue to two fine wires, which were placed in the grips of the testing machine (DCAT 21, Dataphysics Co., German). The section of spiral silk was suspended above the stage of an optic microscope. Length increases were measured from the changes in space between two fine wires by manipulation from the testing machine. Data for force against extension were calculated from these measurements. 3. Results and Discussion SEM of Adhesive Nanofibrils. The adhesive property of the cribellate spider’s viscid spiral relies on the cribellate thread, including two supporting axial silks with non-viscosity and thousands of extremely fine nanofibrils with high viscosity2,3,6,12,13 (shown in Figure 2). These nanofibrils possess segments with 25 ( 5 µm diameters and numerous bulges of 35 ( 5 µm width, composing a segmented structure like bead chain. As Figure 2b indicates by arrows, an obvious skin layer could be observed to coat the segments and bulges of nanofibrils, suggesting that

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Figure 3. AFM images of nanofibrils on a glass slide. The close-up AFM image (a) shows segmented structure (white arrows) and bulges (black arrows).

nanofibrils possess a skin layer structure similar to dragline silk.27 We propose that the skin layers have a high concentration of hydrophilic proteins and hygroscopic low-molecular weight compounds present, and result in the static electric forces contributing to the viscosity of the cribellar silks.28,29 AFM of Adhesive Nanofibrils. Most of the adhesive cribellar silk emerged as an obvious nanofibrous structure with a tendency to form nanoscale aggregates on a clean glass slide due to their prominent viscosity, and their pictures imaged in air are shown in Figure 3. These images on cribellar nanofibrils were easily obtained on hydrophilic glass, indicating that nanofibril formation is an intrinsic property of the pyriform silk protein. In Figure 3a, each nanofibril appears as a distinct segmented structure, and an average segment length is about 32 ( 6 nm, average height of 15 ( 7 µm at the center of the segment, and obvious width of 25 ( 8 nm. Mean volumes of these segments could be estimated to be in the range of 3200-8000 nm3. The previous work has shown that volumes of biomolecules in AFM images tend to correspond to the volumes predicted by their molecular masses, assuming a molecular density of 1-1.3 g/mL.30,31 According to this relationship, these segments have a mean molecular mass of ∼ 3200-11000 kDa, which is on the order of 45-150× the molecular mass of the adhesive silk protein monomer of 66 kDa.32 In the images, we could also observe lots of bulges on these nanofibrils, their widths were nearly twice that of the 25 µm heights of nanofibril segments, and these bulges do not correspond with occasions when two nanofibers appear to overlap each other (Figure 3b). Obviously, these bulges result not from a simple pile-up of the nanofibrils by themselves, but possibly more from the component of the skin layer, such as glycoprotein. As seen in Figure 3a, these nanofibrils also show a little branching, which results from nanofibrils contacting each other or partly overlapping, like those bulges, due to their viscosity. Therefore, we believe that the cribellar nanofibril possesses a regular and defined secondary and tertiary structure with defined fiber-forming structures in each adhesive nanofibril segment. Molecular Modeling of Pyriform Protein Sequences. Becker et al. predicted that molecular springs are the basis for the elasticity of ecribellar spider capture silk.23,33,34 Their prediction comes from the spring-like β-spiral sequences of the flagelliform protein in capture silk. In our work, modeling of adhesive cribellar nanofibrils has been done by Vink et. al with Insight II (Molecular Simulations) essentially as previously described.35 The sequence from Uloborus walckenaerius was modeled as an R-helix in [KQSGPYX] repeats. This model

Huang et al. forms folds for the Uloborus sequence with many hydrogen bonds stabilizing the R-helix, and with all side chains facing toward the inside of the cribellar fibril, thus affording additional stabilization for this fold in a nature property. Other folds also gave compact model structures for the Uloborus sequence, but some had problems, such as a low number of hydrogen bonds and side chains of amino acid exposed to the outside rather than the inside of cribellar fibrils. The entire cDNA sequence for Uloborus walckenaerius, whose middle 114 amino acids were modeled in Figure 4 is IQSGPGV [QRAKVSASQGPGG]3QRADVSAYGPGGVYGKQGPGG [QRAKVSGAQRAKYQRADVGGV]2DSFQLAA[QGAA]3 LSDVSTASGKASTQRADVGGAKQSGPGLGRKQSGPYXGRADVG; the spacer sequence is in bold type. This is the sequence for one of the tandemly arrayed ensemble repeats from the cDNA library made by the pyriform gland mRNA: The amino acid composition of this sequence is 14.8% A, 13.3% G, 13.0% S, 11.7% Q, 5.0-4.3% each of L, V, I, and K; and 2.5% each of P, H, and N. The secretion from pyriform glands producing sticky materials are characterized by a high content of basic amino acids, especially lysine (K), and the lower content of proline and residues having small side chains makes the β-configuration unlikely.36 Thus, it can be assumed that the proteins occur in a randomly coiled and R-helix-rich state both before and after secretion of pyriform glands.37 The cribellar fibril proteins of Uloborus contain high percentages of glycine, alamine, and serine, often in the repetitive [KQSGPAX]n motifs characteristic of R-helixes. Model structures for cribellar fibril proteins in Figure 4 were designed with the assumptions that R-helices and random coils were present and that the unstretched proteins were tight, considering their certain elongation, which is reported with the extreme elasticity of both bulk spiral silk.2,3 In the R-helix models of cribellar fibril sequences from Uloborus walckenaerius (Figure 4), the large change in length before and after extension would occur. Single molecules of cribellar fibrils are composed of tandem repeats of the entire ensemble, consisting of repetitive R-helix sequences and nonrepetitive spacer sequences. Because R-helices acting as fibril molecular springs result in the elasticity of cribellar fibril silk, these springs are inevitably nonhookian. According to the classical theory of macromolecular or silk elasticity, the springs should function largely as entropic springs.34,36 For confirming the assumed entropic model with the R-helixes, the imitated stretching with using molecular dynamics was performed to investigate the structural origins of the special mechanical properties of the R-helix sequences in adhesive fibril proteins. Each spring with polypeptide has a backbone, which is theoretically free to rotate around its CsC and CsN single-bonds in this polypeptide chain. The structure is different from a simple and regular metal spring. The assembly of prolines and lysines in the cribellar fibril sequence with the side chains of the amino acids could obstruct or delay the progress of the free rotation of the polypeptide backbone chain. However, this structure makes sure that there is no hookian component in the backbone chain elasticity. We measured the force spectra and stretching curves of cribellar spider spiral silk and a sketch of the experimental setup is shown in Figure 1. These force spectra show some of the longest structures observed through the obtained force spectroscopy in Figure 5: the longest stretch is over 500 nm long, as compared with typical pulls of ∼200 nm of titin15 or less for a dragline and silkworm silk22,23 construct. The cribellar silk force spectra obviously show patterns of elasticity for this spider silk on a smaller force scale than yet seen, with stretching forces

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Figure 4. Molecular models for relaxed and extended pyriform protein from cribellar spiral fibril. Side and end views of possible pyriform protein conformation, there is a segment and two bulges that include at least 4 R-helixs domains in a single molecule.

Figure 6. Force changes for pulling and relaxation of cribellar adhesive fibril silk molecules. Force changes are exponential over a force range from several pN to ∼300 pN. Figure 5. Force spectroscopy of cribellar spider adhesive fibrils molecules.

on the nanonewton range, corresponding to the previous force measure of ecribellate capture silk23 performed by AFM. These results from sections of webs of Uloborus were combined with cribellar fibril and supporting silk to establish multimolecular models for nanofibril of cribellar spiders. The upper curve in each pair of curves shows stretching or pulling of the fibril molecules, and the lower curve shows retraction or relaxation. The curves of force against extension were then observed in Figure 5, in which the pair of curves revealed the stretching and relaxation of the same fibril molecules. A lot of irregular saw-teeth, which could be observed in the stretch curve of the fibril molecules, were also seen previously in pulling plots of the proteins from many tissues, such as extracellular matrix,14 muscle titin,15 collagen,31 red blood cell spectrin,37 dragline silk protein,22 and ecribellar capture silk.23 In our experiment, these saw-teeth possibly result from the complexity of some fibrils and the difference in the exact molecular refolded structures. During the pulls of spiral silk, the continuous rupture peaks always appeared with increasing forces. As previously reported about pulling titin, spectrin, capture silk, dragline silk, and other proteins, there were nonlinear increases in force progress with the rupture peaks. According to the elastic theory of an ideal polymer stretched in its entropic theory, these nonlinear increases were fitted to the worm-like chain (WLC) model.36,38-40 As reported by other groups about the structure of protein molecules,15,23 it is possible that there was not only one molecule between the tip and surface in the pulls. The fibril-molecule pulls in Figure 5 show that the force slowly reduces to near zero at the relaxation, implying that the last molecule pulled between the tip and the surface has exactly ruptured. According to these results in Figures 5 and 6, it could be deduced that the pulls are from several molecules in a fibril.

WLC was a poor fit for the force increases progressing to rupture peaks, which resulted in a poor fit for the overall force changes during pulling. Obviously, the forces increased exponentially with pulling, and decreased exponentially with relaxation, over a range of forces from a few pico-newtons to as high as ∼300 pN (shown in Figure 6). These rupture peaks showed a few force maxima, followed by force drops with the exponential pull curves. The length constants calculated for the relaxation curves were 80 ( 20 µm (n ) 10). Although WLC fit has been used to characterize pulling and relaxation curves for the tenascin, spectrin, capture silk, and proteins titin,14,15,23,41 it was not much better than exponential fits for all of the relaxation curves in Figure 6 and many segments of the pulling curves. Thus, the results of WLC fit show a different behavior of cribellar fibril molecules from other spider silks, and exponential curve fits for relaxation curves had correlation coefficients of R > 0.980. We also obtained exponential force-distance curves on the ordinary scale integrated cribellar fibrils (shown in Figure 7). The distance constant of each fibril was 26 ( 7 µm (n ) 37) for pulling integrated cribellar fibrils in air. This exponential force increase is consistent with the previous studies of Vollrath3 and Becker23 about pulling integrated spiral cribellar silk. In contrast, integrated dragline silk does not show an exponential force increase on stretching42 due to its β-sheet-rich structure. How should two exponential force-distance curves of pulling on one or a few molecules and stretching integrated cribellar fibrils be explained? Because an integrated cribellar fibril is obviously composed of many molecules, we can naturally suppose that the force spectroscopy pulls of a cribellar fibril are far and away more than that of a molecule. Thus, these two exponential results are very easy to be inosculated. On the basis of the above-mentioned results and analysis, we can propose a reasonable model that many cribellar fibrils

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Figure 7. Force increases are exponential for pulling integrated cribellate capture silk in air: (a) The maximum peaks are the two rupture pseudoflagelliform silks; (b) the numerous smaller peaks are the ruptures of cribellate fibrils.

Figure 8. Schematic diagram for a hypothetical network of unique R-helices in a cribellar adhesive fibril that gives exponential force vs distance curves on pull.

are separated and rupture in succession or even in multiple steps when they are stretched due to the fibrilar molecular chain helix wrapping and coiling. When cribellar fibrils are in large stretch, their molecules are possibly unfolded, and the elasticity of the fibril molecular chain backbones would result in the slope of the extension curve. In the previous work, the elastic models of unfolded proteins are based on entropic arguments with enthalpy differences.36,38-40 Exponential curves and WLC with freely joined protein chains converge altogether at high forces and high extension.43 Although the exponential fit about the data are feasible and necessary, the fit of the data is not adequate to explain the nonentropic quality of the fibril’s elasticity. Obviously, many random coiling structures in cribellar fibril molecules would also impact their elasticity with nonentropy. In addition, the influence of air humidity in this explination should also be analyzed in future work, due to the high concentration of hygroscopic components present in the cribellar sticky fibrils.36 According to above-mentioned results and Becker’s work,23 we propose an alternate model that it is a molecular network composed of interconnected R-helices. The network, a system of essentially equal helices, can result in an exponential forceextension curve (shown in Figure 8). Each helix possesses a spring constant k and unfolds at force f; its length increases ∆L when it unfolds. When the larger pull force F is exerted to the network, its unfolding will occur, and the pull force F will be changed into f, 2f, 4f,..., 2nf with the extension of the network being L, 2L, 3L, · · · , (n + 1)L. The change relation function between the overall force and extension is exponential, because

the force constant after the rupture is k, 2k, 4k, · · · , 2nk with the network extension. When this cross-linked network is pulled, its properties may be similar to this model in Figure 8. However, cribellar fibril structure is a complicated cross-linked network with the random coils and a great deal of H-bonds, which makes the change function poorly fit the above-mentioned exponential relation. Thus, a model of helical springboarding with random coils and H-bonds need to be further designed in our experiments. Cross-links between the molecule chains in cribellar fibrils might result from spacer sequences in repetitive motifs of the cribellar fibril, as shown in Figure 4. In general, the spacer sequences in spider silk proteins are relatively rich in the acidic amino acids Asp (D) and Glu (E).23,34 Salt bridges of polypeptides would be formed between these acidic amino acids, and these salt bridges might provide attachments between adjacent pyriform protein molecules. In cribellar spider silks, the chelation of divalent inorganic cations between histidine residues44 will be in favor of forming cross-links. Thus, the crosslinks in cribellar fibrils might result from components of the adhesive glue in the skin layer, such as the ammonia and lysine or the amino-sugars of the glue glycoproteins,36 like ecribellar spider capture silk glue.45 4. Conclusions Spiral silks are an excellent natural adhesive material, and adhesive silk of the cribellar spider (such as Uloborus walckenaerius) is centuries older than that of ecribellar spider.46 After a long period of evolution, this cribellar spiral silk in the weaved orb web has come to possess remarkable material properties, which result from the unique protein molecular structure of its adhesive silk. We investigated the surface morphology and molecular structure of Uloborus spider adhesive silk using probe microscopy techniques, and their results showed a segmented substructure. The segment length and amino acid sequence are consistent with a spring-like structure for individual protein molecules. The height and width of nanofibril segments suggest a webbing pattern of R-helix molecules in each nanofibril segment. Force spectra of the cribellar pyriform fibrils demonstrate that this fibril molecule unfolds through many rupture events, indicating a modular substructure within single protein nanofibril molecule. A minimal unfolding module size is estimated to be around 40 nm, which corresponds to the extended length of a single repeated module, 114 amino acids long. The novel structure features of this Uloborus spider adhesive silk is distinctly different from the structures of other spider silks analyzed by single-molecule force spectroscopy. Acknowledgment. This work has been supported by the National Natural Science Foundation of China (Project Nos.

Segmented Nanofibrils in U. walckenaerius Silk 60871062 and 50873066). The support of Sichuan Province through a Science Fund for Distinguished Young Scholars of Sichuan ProVince (08ZQ026-007) and Key Technologies Research and DeVelopment Program of Sichuan ProVince (2008SZ0021 and 2006Z08-001-1) are also acknowledged with gratitude. This work was also supported by the Research Fund for the Doctoral Program of Higher Education from Ministry of Education of China (No. 20070610131). We thank the Analytical & Testing Center of Sichuan University for the assistance with the microscopy work. Supporting Information Available: This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Hinman, M. B.; Jones, J. A.; Lewis, R. V. Trends Biotechnol. 2000, 18, 374–379. (2) Opell, B. D.; Bond, J. E. Biol. J. Linnean Soc. 2000, 70, 107–120. (3) Ko¨hler, T.; Vollrath, F. J. Exp. Zool. 1995, 271, 1–17. (4) Vollrath, F.; Edmonds, D. T. Nature 1989, 340, 305–307. (5) Vollrath, F.; Knight, D. P. Nature 2001, 410, 541–548. (6) Vollrath, F. ReV. Mol. Biotechnol. 2000, 74, 67–83. (7) Kitagawa, M.; Kitagama, T. J. Mater. Sci. 1997, 32, 2005–2012. (8) Blackledge, T. A.; Hayashi, C. Y. J. Exp. Biol. 2006, 209, 3131– 3140. (9) Swanson, B. O.; Blackledge, T. A.; Hayashi, C. Y. J. Exp. Zool. 2007, 307A, 654–666. (10) Hausdorf, B. J. EVol. Biol. 1999, 12, 980–985. (11) Alper, J. Science 2002, 297, 329–331. (12) Vollrath, F. Sci. Am. 1992, 266, 70–76. (13) Foelix R. F. Biology of Spiders, 2nd ed.; Oxford University Press: New York. (14) Oberhauser, A. F.; Marszalek, P. E.; Erickson, H. P.; Fernandez, J. M. Nature 1998, 393, 181–185. (15) Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E. Science 1997, 276, 1109–1112. (16) Rief, M.; Oesterhelt, F.; Heymann, B.; Gaub, H. E. Science 1997, 275, 295–1297. (17) Grandbois, M.; Beyer, M.; Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Science 1999, 283, 1727–1730. (18) Marszalek, P. E.; Oberhauser, A. F.; Pang, Y. P.; Fernandez, J. M. Nature 1998, 396, 661–664. (19) Lee, G. U.; Chrisey, L. A.; Coulton, R. J. Science 1994, 266, 771– 773. (20) Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Nat. Struct. Biol. 1999, 6, 346–349.

J. Phys. Chem. B, Vol. 113, No. 15, 2009 5097 (21) Zhang, W. K.; Xu, Q. B; Zou, S.; Li, H. B.; Xu, W. Q.; Zhang, X.; Shao, Z. Z.; Kudera, M.; Gaub, H. E. Langmuir 2000, 16, 4305–4308. (22) Oroudjev, E.; Soares, J.; Arcidiacono, S.; Thompson, J. B.; Fossey, S. A.; Hansma, H. G. Proc. Natl. Acad. Sci. 2002, 99, 6460–6465. (23) Becker, N.; Oroudjev, E.; Mutz, S.; Cleveland, J. P.; Hansma, P. K.; Hayashi, C. Y.; Makarov, D. E.; Hansma, H. G. Nat. Mater. 2003, 2, 278– 283. (24) Li, S. F. Y.; McGhie, A. J.; Tang, S. L. Biophys. J. 1994, 66, 1209– 1212. (25) Tautz, D.; Hancock, J. M.; Webb, D. A.; Tautz, C.; Dover, G. A. Mol. Biol. EVol. 1988, 5, 366–376. (26) Casanova, J. L.; Pannetier, C.; Jaulin, C.; Kourilsky, P. Nucl. Acids Res. 1990, 18, 4028. (27) Eles, P. T.; Michal, C. A. Macromol. 2004, 37, 1342–1345. (28) Opell, B. D. J. Exp. Zool. 1995, 273, 186–189. (29) Vollarth, F. General Properties of Some Spider Silks. In Silk Polymers. Materials Science and Biotechnology; Kaplan, D., Wade, W. W. , Eds.; American Chemical Society: Washington DC, 1994, 17-28. (30) Gould, S. A. C.; Tran, K. T.; Spagna, J. C.; Moore, A. M. F.; Shulman, J. B. Int. J. Biol. Macromol. 1999, 24, 151–157. (31) Thompson, J. B.; Kindt, J. H.; Drake, B.; Hansma, H. G.; Morse, D. E.; Hansma, P. K. Nature 2001, 414, 773–776. (32) Huang, Z. B.; Yan, D. H.; Yang, M.; Liao, X. M.; Kang, Y. Q.; Yin, G. F.; Yao, Y. D.; Hao, B. Q. J. Colloid Interface Sci. 2008, 325, 356–362. (33) Hayashi, C. Y.; Lewis, R. V. Science 2000, 287, 1477–1479. (34) Hayashi, C. Y.; Lewis, R. V. J. Mol. Biol. 1998, 275, 773–784. (35) Vink, C. J.; Hedin, M; Bodner, M. R.; Maddison, W. P.; Hayashi, C. Y.; Garb, J. E. Mol. Phylogenet. EVol. 2008, 48, 377–382. (36) Szent-Gyorgyi, A. G.; Cohen, C. Science 1957, 126, 679–698. (37) Andersen, S. O. Comp. Biochem. Physiol. 1970, 35, 705–711. (38) Everaers, R.; Kremer, K. J. Mol. Modell. 1996, 2, 293–299. (39) Rief, M.; Pascual, J.; Saraste, M.; Gaub, H. E. J. Mol. Biol. 1999, 286, 553–561. (40) Dessinges, M. N.; Maier, B.; Zhang, Y.; Peliti, M.; Bensimon, D.; Croquette, V. Phys. ReV. Lett. 2002, 89, 248102. (41) Bustamante, C.; Marko, J. F.; Siggia, E. D.; Smith, S. Science 1994, 265, 1599–1600. (42) Marko, J. F.; Siggia, E. D. Macromolecules 1995, 28, 8759–8770. (43) Rief, M.; Pascual, J.; Saraste, M.; Gaub, H. E. J. Mol. Biol. 1999, 286, 553–61. (44) Vollrath, F.; Knight, D. P. Nature 2001, 410, 541–548. (45) Bustamante, C.; Smith, S. B.; Liphardt, J.; Smith, D. Curr. Opin. Struct. Biol. 2000, 10, 279–285. (46) Waite, J. H.; Vaccaro, E.; Sun, C.; Lucas, J. M. Philos. Trans. R. Soc. London, B 2002, 357, 143–153. (47) Vollrath, F.; Fairbrother, W. J.; Williams, R. J. P.; Tillinghast, E. K.; Bernstein, D. T.; Gallagher, K. S.; Townley, M. A. Nature 1990, 345, 526–528.

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