Liquid Crystalline Granules Align in a Hierarchical Structure To

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Liquid crystalline granules align in a hierarchical structure to produce spider dragline microfibrils Ting-Yu Lin, Hiroyasu Masunaga, Ryota Sato, Ali D Malay, Kiminori Toyooka, Takaaki Hikima, and Keiji Numata Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00086 • Publication Date (Web): 02 Mar 2017 Downloaded from http://pubs.acs.org on March 5, 2017

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Liquid crystalline granules align in a hierarchical structure to produce spider dragline microfibrils Ting-Yu Lin,1 Hiroyasu Masunaga,2 Ryota Sato,1 Ali D. Malay,1 Kiminori Toyooka,3 Takaaki Hikima,4 Keiji Numata1* 1

Enzyme Research Team, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa,

Wako-shi, Saitama 351-0198, Japan. 2

Japan Synchrotron Radiation Research Institute, 1-1-1, Kouto, Sayo-cho, Sayo-gun, Hyogo

679-5198, Japan. 3

Mass Spectrometry and Microscopy Unit, Technology Platform Division, RIKEN Center for

Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 2300045 Japan. 4

RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5198, Japan.

*Correspondence to: Keiji Numata ([email protected])

ACS Paragon Plus Environment

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ABSTRACT

The spider silk spinning process converts spidroins from an aqueous form to a tough fiber. This spinning process has been investigated by numerous researchers, and micelles or liquid crystals of spidroins have been reported to form silk fibers, which are bundles of silk microfibrils. However, the formation process of silk microfibrils has not been clarified previously. Here, we report that silk microfibrils are generated through the formation, homogenization and linkage of liquid crystalline granules without micelle-like structures. Heterogeneous granules on the submicron to micron scale were observed in the storage sac, whereas homogeneous granules with diameters of approximately 100 nm were aligned along the tapering duct. In the spun fibers, the homogeneous granules were connected along the fiber axis. This is the first clear description of the formation of granule-based microfibrils in the spinning process, which is the key conversion process leading to the unique hierarchical structure of spider dragline.


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INTRODUCTION Spider dragline silk has outstanding mechanical properties, owing to its hierarchical structures of aligned crystals embedded in an amorphous phase.1,

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The crystalline and

amorphous regions are constituted by tandem repeats of polyalanine and glycine-rich sequences that contribute to strength and ductility of silk fibers, respectively, resulting in their excellent toughness. During the spinning process, the N- and C- terminal domains of major ampullate spidroins orchestrate the formation of insoluble silk fibers in response to pH and ionic gradients.3, 4

The self-assembled spidroins form predominantly antiparallel β-sheet crystals, which are

embedded in an amorphous and mobile phase of silk microfibrils.5 The liquid-to-fiber spinning mechanism of spidroins has been probed and partially elucidated via microscopic and birefringence analyses.6-10 Another important factor for silk fiber formation is shear: as silk protein solution flows from the storage gland through a funnel-like constriction and subsequently through a gradually narrowing duct toward the spigot, the silk precursors experience progressively increasing shear forces, which have profound effects on fiber formation.11 Based on the previous results, two hypotheses have been presented to account for the hierarchal structures involved in the transformation of silk protein solutions to solid filaments at the micron and submicron scales: the liquid crystalline model9,

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and the micelle model.7 The former

suggests that fiber formation starts with fibroins in a liquid crystalline state that are subjected to shearing forces and pH changes while being drawn down through the major ampullate gland. Hexagonal columnar liquid crystals were observed in the spider dragline silk by transmission electron microscopy, suggesting that silk molecules could be folded into short rods rather than supramolecular liquid crystals within the gland.12 By contrast, the latter suggests that the spidroins form micelle structures that control water and protein interactions during spinning.7


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The formation of micelle-like structures was observed at relatively high concentrations of silk solution by atomic force microscopy (AFM).13, 14 Molecular characterization of silk molecules by solution NMR reasonably explains that the C-terminal non-repetitive domain assembles into oligomeric micelles.15 The important difference between the two models is that a building block is either liquid crystalline state or micelles.16 Accelerating elongational flow and shear forces are considered to induce a conversion from liquid crystals to rod-like structures in the tapering duct.8,9 Although the contributions of these building blocks to physical properties of silk fibers have not yet been investigated, the micelle-like building block could be observed as a globular structure in the silk fibers, as reported previously.7 Because of this characteristic spinning process, silk fibers have a unique hierarchical structure that ranges from the angstrom to the macro scale. A silk fiber is a bundle of microfibrils with a skin layer;17 however, the formation process of microfibrils with such hierarchical structures in the nano- to micrometer range remains to be elucidated. In this study, we provide direct morphological and structural information on the role of silk fibroin assembly in microfibril formation from the storage sac to the tapering duct and, finally, to the dragline fiber. Based on our structural and morphological analyses, we determined that the microfibrils are composed of homogeneous granules, which are the basic units of the submicron to micrometer hierarchical structures in silk fibers. Furthermore, the granule-based spinning indicates that liquid crystalline granules, rather than micelle of silk molecules, play an important role to realize the unique hierarchical structure of spider dragline.

EXPERIMENTAL SECTION


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Major ampullate glands and dragline silk. Major ampullate glands were obtained from adult female Nephila clavata spiders (n = 5, body size > 20 mm). The spiders were fed small crickets (body size < 5 mm) and kept at 25 °C and 45% relative humidity (RH). Silk samples in the major ampullate glands were extracted with tweezers and used for further experiments within a few minutes after extraction. Spider draglines were retrieved from adult female N. clavata with a reeling rate of 2 m/min. The fiber bundles were stored in the dark at 25 °C and 40% RH. Synchrotron wide-angle and small-angle X-ray scattering (WAXS and SAXS) measurements. Synchrotron WAXS and SAXS measurements were performed at the BL45XU beamline at SPring-8 (Harima, Japan) using an X-ray energy of 12.4 keV (wavelength = 0.1 nm) and a beam diameter of 45 µm. All the WAXS patterns were recorded with a flat-panel detector (FPD, C9728DK-10, Hamamatsu Photonics, Hamamatsu, Japan).18 The sample-to-detector distances during the WAXS and SAXS measurements were 50.2 and 2036 mm, respectively. The exposure time for each scattering pattern was 10 s. The obtained two-dimensional (2D) scattering patterns were converted to 1D profiles using Fit2D software.19 Corrections were made for background scattering and the detector geometry.18 Birefringence analysis. Birefringence was calculated by dividing the retardance by the object thickness. Because spider fibers are cylindrical, the fiber diameter was used as the object thickness. The retardances of the silk fiber, major ampullate gland and tapering duct were measured with a WPA-100 birefringence measurement system (Photonic Lattice Inc., Miyagi, Japan) and a light-polarized microscope (Olympus BX53, Olympus Corporation, Tokyo, Japan) with a 50× objective lens. The retardance was characterized by using WPA-VIEW (version 1.05) software. The WPA-100 system can measure samples with phase-difference values of several thousand nanometers using lasers with wavelengths of 523, 543, and 575 nm. The diameter of


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the silk fibers was calculated based on scanning electron microscopy (SEM) observation (see below for details). The thicknesses of the major ampullate glands and tapering ducts were measured using a digital micrometer caliper (IP65, Mitutoyo, Kanagawa, Japan). SEM observation. The morphologies of the silks before and after spinning were investigated by SEM. Silk extracted from the major ampullate glands and dragline fibers were mounted on an aluminum stub, sputter-coated with gold and examined using a SEM-6000 microscope (JEOL Ltd., Tokyo, Japan) at an accelerating voltage of 5 kV. AFM observation. A single fiber was either placed directly or mounted with superglue (Loctite 460, Henkel, Germany) on the sample stage, and the cross-section of the sample was prepared using a microtome (RM2265, Leica Microsystems GmbH, Wetzlar, Germany) with a diamond blade. The cross-section of the fiber sample was observed using AFM. Images were obtained in tapping mode at 40% RH and 25 °C via scanning probe microscopy (AFM5300E, Hitachi High-Technologies Corporation, Tokyo, Japan) and an RH controller (Hitachi). The major ampullate gland and tapering duct were mounted on mica substrates, and the skin of each organ was sliced and removed using a scalpel. The wet samples were observed using AFM under humid conditions (RH 80%). The cantilever had a spring constant of 20 N/m, and the scan frequency was 0.5 Hz. Images were obtained and analyzed with an AFM5000II controller (Hitachi). Under ambient conditions in air, the granules on mica exhibited a flat morphology, as reported previously for another protein.20 The microfibril width (distance between each nanofibril) was measured in at least 20 areas in one image, and five images were acquired for each sample to ensure that the data were representative. The granule width was determined by taking into consideration the deconvolution effect of the cantilever tip.20, 21


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RESULTS AND DISCUSSION To determine whether silk proteins exist in a crystalline form or are contained in micelles, the major ampullate glands and draglines of female Nephila clavata were characterized by smallangle and wide-angle X-ray scattering (SAXS and WAXS, respectively) (Figure 1A). The dragline silk WAXS profile exhibited (020), (210) and (040) reflections that were assigned to βsheet structures (Figure S1).18 The silk from both the storage sac and the tapering duct exhibited very weak peaks for the (210) and (040) reflections, implying that β-turns and disordered structures predominated over β-sheet structures.22, 23 The SAXS profiles show two broad peaks with d-spacings of 21 and 35 nm in the storage sac silks and the gland skin (Figure 1B). The peak at 21 nm originated from the skin of the gland, whereas the broad peak at 35 nm indicates the presence of multiple coexisting nanostructures with very low periodicity. Accordingly, we estimated that the average distance between the periodic structures was approximately 35 nm in the storage sac. Importantly, we did not identify any micelle-specific SAXS profiles,24 even in the dragline, suggesting that no micelle-like structures are present. However, we did detect a shorter periodic structure with an average distance of approximately 7 nm between the structures in the dragline. This periodic structure could have originated from the interspaces between microfibrils.


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Figure 1. The anatomy and SAXS analyses of the spider gland and dragline silks. (A) The anatomy of the storage sac, tapering duct and dragline and the samples used for the X-ray scattering measurements. The samples were attached immediately after they were removed from N. clavata. (B) One-dimensional (1D) SAXS profiles of the storage silk (red), tapering duct silk (blue), dragline silk (green) and gland skin (black). Each arrow indicates the size of the structure assigned to the peak.

To elucidate the morphology of the silk before spinning, the contents of the fresh, yellowish major ampullate glands were dissected and observed by atomic force microscopy (AFM) and scanning electron microscopy (SEM) (Figure 2). SEM analysis revealed granules with diameters exceeding 1 µm throughout the storage sac (Figure 2A), whereas much larger


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granules were found throughout the gland (Figure S2). Submicron and nanoscale granules were also observed by AFM in the gland and storage sac, respectively (Figures 2B, S3A and B). To investigate the alignment and liquid crystalline state of the protein molecules in the storage sac 11, 25

, we measured the birefringence of the gland (Figure 3A and B). The storage sac silk was

optically isotropic, indicating that the silk molecules in the storage sac were not aligned. This finding correlates well with a previous report,7 implying that the spidroins in the ampullate sac are heterogeneous in the liquid crystalline state. Together with the X-ray scattering results and the birefringence measurements, these results suggest that the granules in the storage sac are in a heterogeneous liquid crystalline phase, not in micellar structures (Figure 2C).

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Figure 2. Cross-sectional topography of spider silks in the major ampullate storage sac, tapering duct and dragline. The morphology of the storage sac silk was determined by (A) SEM and (B) AFM. Heterogeneous granules were observed by both methods. The dashed circles in B indicate granules. (C) Illustration of silk granules with various diameters in the storage sac. (D-F) The morphology of the tapering duct silk was determined by AFM (E and F are enlargements of the dashed boxes in D and E, respectively). The dashed circles in F indicate granules in microfibrils. (G) Illustration of granule-based microfibrils aligned along the fiber axis in the tapering duct. (H) AFM longitudinal cross-sectional topography of the dragline silk fiber. (1–18) Enlargements of the boxed regions shown in H. 1 and 11–18 presenting the border region between glue and silk fiber. (I) Illustration of granule-based microfibrils bundled into a dragline silk fiber.

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Figure 3. The brightfield (A-C) and retardance images (D-F) of the storage sac silk (A, D), the tapering duct silk (B, E) and the dragline (C, F), respectively.


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In the tapering duct, smaller granule features with diameters of approximately 100 nm (105 ± 29 nm) were axially aligned (Figures. 2D–F and S3C–G). Although the axially aligned granules appeared similar to microfibrils, individual granules could be seen in AFM phase images (Figure 2E and F). The heterogeneous distribution of the granule size became homogeneous as the spinning proceeded from the proximal to the distal part of the gland (Figure 4); simultaneously, the granule diameter also became uniform (Figure S4). The birefringence in the tapering duct increased drastically relative to that in the storage sac (Figure 3B and E), indicating that the silk granules in the tapering duct silk were aligned along the duct (as illustrated in Figure 2G). AFM observations revealed that the granules in the dragline silk were homogeneous in size and formed microfibrils with a width of 113 ± 20 nm (Figure 2H and 1–18), similar to the granule width in the tapering duct silk (Figure 4). An average density of proteins is estimated to be approximately 1.37 g/cm3.26 If the silk granule were hypothesized to be a sphere of a diameter of 100 nm, one silk granule would be composed of 1450 silk molecules. The homogeneous granules were connected and formed microfibrils along the fiber axis. These findings suggest that the shearing forces and pH changes that occur before silk spinning8, 11, 27 induce a transition in the size and spatial alignment of the granules, which promotes microfibril formation in the dragline. The birefringence increased additively in the dragline silk compared to the tapering duct (Figure 3C and F). As a result of the granule formation during the spinning process, the silk granules in the dragline silk were aligned along the fiber axis, formed microfibrils and became bundled as a dragline, resulting in higher birefringence and anisotropic properties.


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Storage sac Tapering duct Dragline silk Dragline silk @20% strain

Figure 4. The granule diameters of the storage sac silk, tapering duct silk and microfibril silk. The granule width distributions from 0 to 2 µm are summarized. The granules of microfibril silk were characterized before and after the stretching deformation (20% strain). (Inset) An enlargement of the granule width distribution ranging from 0 to 200 nm. The total numbers of samples of storage sac silks, tapering duct silks, microfibril silks and microfibril silks at 20% strain were 128, 116, 124 and 133, respectively. To further clarify how the granules are connected within the microfibrils, we investigated the deformation of granules during stretching of the microfibrils. At 20% strain, which is almost the strain at dragline break, the fiber exhibited a higher birefringence and a smaller diameter than those observed in the unstretched state, indicating that the silk granules were highly aligned when subjected to stretching pressure (Figures 5A–D and S5). AFM images of longitudinal cross-sections of the dragline fibers at 20% strain showed that the stretched fibers were composed of uniaxially oriented microfibrils (Figure 5E). The stretched silk microfibrils were


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approximately 48 ± 13 nm wide, whereas the unstretched microfibrils were approximately 113 ± 21 nm wide (Figure 4 inset). Even after stretching and thinning, each individual microfibril was still clearly composed of granules aligned with the fiber axis (Figures 4F and G and S6). The thinning of the granules in the microfibrils resulting from the stretching of the dragline fibers confirmed that the granules contribute to the deformation behavior of the fibers. Granuleconnected microfibrils were also found in the ultrahigh strength, high-density polyethylene (HDPE) fiber Dyneema, which is produced by gel spinning.28 During gel spinning, the HDPE forms granules and, subsequently, a shish-kebab structure. By stretching the HDPE fiber, the kebab is transformed into the shish, which results in tough microfibrils. Thus, the granule-based microfibrils in the dragline silk are potentially responsible for the toughness of the spider dragline.

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Figure 5. Dragline silks at 20% stretching deformation. (A and B) SEM images of the dragline silks before and after 20% stretching, respectively. (C and D) Retardance analyses of single dragline silk fibers at 0% and 20% strain, respectively. (E) Overview of the dragline silk fiber longitudinal cross-sections at 20% stretching. (F) Higher-magnification image of the stretched dragline silk fiber cross-sections. The arrow indicates the fiber axis. (G) Schematic illustration of the silk microfibrils. The stretched microfibrils also have granule-like morphologies. The composed granules are stretched, and their widths are decreased, as observed in F.

The drastic decrease in average microfibril width from 113 to 48 nm by axial deformation can be explained in terms of Poisson’s ratio, which is a representative value used to characterize material deformation (Figure 4 inset). Dragline silk from N. clavipes was reported to have a Poisson’s ratio of 1.52, which is remarkably high and indicates that the silk is an anisotropic and elastic material.29 By contrast, the dragline silk of N. edulis has a moderate Poisson’s ratio of approximately 0.37 (Table 2);30 thus, the Poisson’s ratios of spider dragline are an interesting subject. In the present study, the average Poisson’s ratio of N. clavata dragline was found to be 0.26 based on SEM observation of the dragline fibers at 20% strain (Figure S7), whereas the average Poisson’s ratio of the microfibrils exceeded 0.5, which is not a typical value for an isotropic material. Previous reports have indicated that spring-like zigzag structures in metamaterials and helical structures in collagen can produce atypical Poisson’s ratios.31,

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Therefore, their granular features found in this study may make the microfibrils anisotropic and cause spider dragline to exhibit atypical Poisson’s ratios. However, the relationship between the microfibrils and dragline silk in Poisson’s ratio is not clear on the basis of the current study.


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Based on the present results and discussion, we propose a silk granule-mediated spinning process (Movie S1). This granule-based assembly process provides new insight into the spinning mechanisms and structure–function relationships of spider dragline silk. This knowledge about spider silk holds great potential as a basis for the production of artificial spider silk fibers and protein-based tough materials and the development of eco-friendly material processes utilizing mild, water-based conditions without organic solvents.

CONCLUSIONS The spinning process for silk fibres has been explained according to two theories: the liquid crystalline theory and the micelle theory. In this study, we present the sequential transition of spidroins in Nephila clavata major ampullate glands and dragline to explain the mechanism underlying the spinning process using multiple microscopic and structural analyses. We determined that the spidroins form granules instead of micelles in an ampullate sac. These granules are axially connected by shear force and pH change in the tapering duct and spun into dragline silk. Together with previous reports addressing not only silk studies but also materials science, the granule-connected fiber structure described here demonstrates the outstanding toughness and Poisson’s ratio of spider dragline. This novel finding relies on the granule-based hierarchical structure in spider’s spinning mechanism and will serve as a basis for the design of tough materials. In future, other high-resolution microscopic methods will clarify nano-scale structures and properties of spider silk fibers and microfibrils.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Figure S1-S7 and Movie S1 (PDF) AUTHOR INFORMATION Corresponding Author *Keiji Numata. Email: [email protected] Author Contributions T-YL and KN performed AFM and SEM experiments. HM, TH and KN performed X-ray measurements. RS, ADM and KN prepared samples. KT contributed to SEM experiments. KN organized the experiments and research strategy. All authors wrote the paper. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the Japan Science and Technology Agency (JST) Impulsing Paradigm Change through Disruptive Technologies Program (ImPACT) (to K.N.). REFERENCES (1) Keten, S.; Buehler, M. J. J. Roy. Soc. 2010, 7, 1709-1721. (2) Van Beek, J. D.; Hess, S.; Vollrath, F.; Meier, B. H. Proc. Natl. Acad. Sci. USA 2002, 99, 10266-10271. (3) Askarieh, G.; Hedhammar, M.; Nordling, K.; Saenz, A.; Casals, C.; Rising, A.; Johansson, J.; Knight, S. D. Nature 2010, 465, 236-U125. (4) Rising, A.; Johansson, J. Nat. Chem. Biol. 2015, 11, 309-315.


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(5) Nova, A.; Keten, S.; Pugno, N. M.; Redaelli, A.; Buehler, M. J. Nano. Let. 2010, 10, 2626-2634. (6) Davies, G. J. G.; Knight, D. P.; Vollrath, F. Tissue Cell 2013, 45, 306-311. (7) Jin, H. J.; Kaplan, D. L. Nature 2003, 424, 1057-1061. (8) Vollrath, F.; Knight, D. P. Int. J. Biol. Macromol. 1999, 24, 243-249. (9) Vollrath, F.; Knight, D. P. Nature 2001, 410, 541-548. (10) Kerkam, K.; Viney, C.; Kaplan, D.; Lombardi, S. Nature 1991, 349, 596-598. (11) Knight, D. P.; Vollrath, F. P. Roy. Soc. B 1999, 266, 519-523. (12) Knight, D.; Vollrath, F. Tissue & cell 1999, 31, 617-620. (13) Zhong, J.; Ma, M.; Li, W.; Zhou, J.; Yan, Z.; He, D. Biopolymers 2014, 101, 1181-1192. (14) Zhong, J.; Liu, X.; Wei, D.; Yan, J.; Wang, P.; Sun, G.; He, D. Int. J. Biol. Macromol. 2015, 76, 195-202. (15) Hagn, F.; Eisoldt, L.; Hardy, J. G.; Vendrely, C.; Coles, M.; Scheibel, T.; Kessler, H. Nature 2010, 465, 239-U131. (16) Heim, M.; Keerl, D.; Scheibel, T. Angew. Chem. Int. Ed. 2009, 48, 3584-3596. (17) Augsten, K.; Muhlig, P.; Herrmann, C. Scanning 2000, 22, 12-15. (18) Numata, K.; Masunaga, H.; Hikima, T.; Sasaki, S.; Sekiyama, K.; Takata, M. Soft Mat. 2015, 11, 6335-6342. (19) Hammersley, A. P. . European Synchrotron Radiation Facility Internal Report 1997, ESRF97HA02T. (20) Numata, K; Kikkawa, Y; Tsuge, T; Iwata, T; Doi, Y; Abe, H. Macromol. Biosci. 2006, 6, 41-50. (21) Numata, K; Kikkawa, Y; Tsuge, T; Iwata, T; Doi, Y; Abe, H. Biomacromolecules 2005, 6, 2008-2016. (22) Asakura, T.; Ashida, J.; Yamane, T.; Kameda, T.; Nakazawa, Y.; Ohgo, K.; Komatsu, K. J. Mol. Biol. 2001, 306, 291-305. (23) Hronska, M.; Van Beek, J. D.; Williamson, P. T.; Vollrath, F.; Meier, B. H. Biomacromolecules 2004, 5, 834-839. (24) Manet, S.; Lecchi, A.; Imperor-Clerc, M.; Zholobenko, V.; Durand, D.; Oliveira, C. L. P.; Pedersen, J. S.; Grillo, I.; Meneau, F.; Rochas, C. J. Phys. Chem. B 2011, 115, 11318-11329. (25) Holland, C.; O'neil, K.; Vollrath, F.; Dicko, C. Biopolymers 2012, 97, 368-373. (26) Erickson, H. P. Biol. Proced. Online 2009, 11, 32-51. (27) Vollrath, F.; Madsen, B.; Shao, Z. Z. P. Roy. Soc. B 2001, 268, 2339-2346. (28) Ohta, Y.; Murase, H.; Hashimoto, T. J. Polym. Sci. Polm. Phys. 2010, 48, 1861-1872. (29) Koski, K. J.; Akhenblit, P.; Mckiernan, K.; Yarger, J. L. Nat. Mater. 2013, 12, 262-267. (30) Vollrath, F.; Madsen, B.; Shao, Z. Proc. Biol. Sci. 2001, 268, 2339-2346. (31) Eidini, M.; Paulino, G. H. Sci. Adv. 2015, 1, e1500224. (32) Wells, H. C.; Sizeland, K. H.; Kayed, H. R.; Kirby, N.; Hawley, A.; Mudie, S. T.; Haverkamp, R. G. J. Appl. Phys. 2015, 117. (33) Kiviranta, P.; Rieppo, J.; Korhonen, R. K.; Julkunen, P.; Toyras, J.; Jurvelin, J. S. J. Orthop. Res. 2006, 24, 690-699. (34) Anderson, M. L.; Mott, P. H.; Roland, C. M. Rubber Chem. Technol. 2004, 77, 293-302.


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Table 1. The granule sizes and birefringence of the storage sac, tapering duct and dragline silks. The total sample numbers of storage sac silks, tapering duct silks, dragline silks and dragline silk at 20% strain are 128, 116, 124 and 133, respectively. Storage sac silk

Tapering duct

Dragline silk

silk Granule width

Dragline silk at 20% strain

923.2 ± 286.2

105.0 ± 29.3

113.1 ± 20.7

48.0 ± 13.1

33.1 ± 22.3

2.5 ± 1.7

4.6 ± 0.7

0.3 ± 0.1

8x10-6 ± 1x10-6

3.8x10-4 ±

4.1x10-2 ±

6.2x10-2 ±

0.3x10-4

0.2x10-2

0.5x10-2

(nm) Granule height (nm) Birefringence


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Biomacromolecules

Table 2. Poisson’s ratios of silks and other materials, with references. Poisson’s ratios were calculated based on the change in the average dragline silk diameter (determined by SEM) and that of the microfibrils (determined by AFM). Material

Poisson’s ratio

Spider dragline

0.2–0.3

(N. clavata)

(average: 0.26)

Nanofibrils in spider dragline (N. clavata)

1.9–3.7 (average: 2.9)

Ref.

This study

This study Calculated from the results in

Spider dragline

0.37–0.38

Vollrath et al. Proc. R. Soc. Lond. B 2001 30

0–1.52

Koski et al. Nat. Mater. 2013 29

0–1.50

Koski et al. Nat. Mater. 2013 29

Collagen fibril

2.1 ± 0.7

Wells et al. J. Appl. Phys. 2015 32

Cartilage: matrix of primarily collagen (type II) and proteoglycans

0.1–0.4

Kiviranta et al. J. Orthopaedic Res. 2006 33

Natural rubber

0.4999

Anderson et al. Rubber Chem. Technol. 2004 34

(N. edulis) Spider major ampullate silk (Nephila clavipes) Spider minor ampullate silk (N. clavipes)

Metamaterials

Approximately 4.5 (the max. value)

Eidini et al. Sci. Adv. 2015 31


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TOC


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Liquid Crystalline Granules Align in a Hierarchical Structure To

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