Article pubs.acs.org/Biomac
Two Mechanisms for Supercontraction in Nephila Spider Dragline Silk Juan Guan, Fritz Vollrath, and David Porter* Department of Zoology, University of Oxford, Oxford OX1 3PS, U.K. S Supporting Information *
ABSTRACT: Supercontraction in dragline silk of Nephila edulis spider is shown to have two distinct components revealed by single fiber measurements using dynamic mechanical thermal analysis. The first component relies on a contraction of maximum 13% and seems to be associated with relaxation processed through the glass transition, Tg, as is induced by increasing temperature and/or humidity. The second component is induced by liquid water to the total contraction of 30%. The Tg-induced contraction is linearly correlated with the restraining stress on the fiber, and the mechanical properties of the partially contracted silk have mechanical profiles that differ from both native and fully supercontracted fibers. Here we present novel supercontraction data and discuss their structural origins, examining the relaxation of stretched orientation in the different primary structure sequences.
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the biofunctions of supercontraction, and first hypothesized orientation changes before and after supercontraction.20 Blackledge has looked at supercontraction in the form of cyclical changes to length and load under different environmental conditions.17,22 For example, virgin silks were restrained under constant length and the cyclic stress response was measured under cycles of humidity between 0% and 100%. Two different responses were reported but not related directly to structural changes, which we interpret as reduced modulus of virgin silk at high humidity, giving a reduction in stress under restraining load, and a classical supercontraction of stretched silk at high humidity to give a higher stress. Plaza quantified “supercontraction stress” to be ∼60 MPa by raising the humidity up to 100% under constant length.23 He then observed that supercontraction can be linked to the glass transition in silk by using a combination of high humidity (about 70%) and increased temperature (50 °C) and showing that supercontraction occurred under the same conditions as the reduction in modulus associated with the glass transition.24,25 No reference is made to any differences in supercontraction strain using this approach relative to simply exposing a fiber to liquid water. More recently, Fu has presented a quantitative structure−property model for the changes in modulus through the glass transition in silk, but this was not extended to supercontraction effects.9,26 Some researchers have attributed supercontraction to relaxation of orientation in the amorphous fraction of the silk polymers. For example, Michal’s work on controlled supercontraction to investigate structural mechanisms used 2D NMR
INTRODUCTION Spider dragline silks belong to a family of natural fibers that are best known for their low density and attractive mechanical properties combining strength, toughness, and environmentally benign processing conditions.1−3 The mechanical properties of a specific silk can easily be modified by variables such as chemical composition4 and processing (spinning) conditions.5,6 In addition, the ambient environment has considerable influence on the formed fiber, for example, humidity dramatically changes the performance of spider silks.7−9 On exposure to water, dragline silks can “supercontract” by up to about 50% of their stretched length. Supercontraction is an interesting phenomenon because of the very high and reproducible values of shrinkage that can be reversibly induced by cyclical loading and then unloading in the presence of water.10−12 Supercontraction varies between dragline silks from different spider species;5,13 however, other silk types have also been found to undergo supercontraction, albeit to a far smaller degree.14 Supercontraction provides new potential applications for spider silks, for example, as tunable shape memory polymers, which are of great technological interest. 15 Spider silk has also been found to show torsional memory effects, 16 and a recent study suggests that spider silks may give repeatable and effective performance as potential replacement for muscles.17 A number of research groups have used different approaches to understand supercontraction and the underlying mechanisms. The silks before and after supercontraction are usually called virgin silks and supercontracted silks, respectively. Supercontraction was first discussed and defined by Robert Work.18−21 His studies raised wide interest in spider silks and webs in science. More specifically, Work compared properties of virgin and supercontracted silks, made initial suggestions on © 2011 American Chemical Society
Received: July 25, 2011 Revised: September 19, 2011 Published: September 27, 2011 4030
dx.doi.org/10.1021/bm201032v | Biomacromolecules 2011, 12, 4030−4035
Biomacromolecules
Article
techniques.27,28 Michal suggested that on hydration the mobile (amorphous) phase first disorientates; if more contraction is allowed, then the orientation distribution of both mobile and static (crystalline) components broadens.27,28 However, the attribution of orientation alone fails to explain quantitatively the variety of supercontraction effects across spider species. Savage show that there are major differences in the structural organization in the glycine-rich domains and different molecular mechanisms for elastic effects in proline-rich and proline-poor segments of hydrated spider silks.29 Boutry and Blackledge discuss the role of the two GGX and GPGG motifs in supercontraction and argue that the GPGG motif of MaSp2 evolved to enhance supercontraction effects in dragline silks, without actually differentiating between the shrinkage mechanisms in the two motifs.30 Liu’s work attributed supercontraction mainly to proline content.5,13 He proposes that the close link between proline and particular elastin-like motifs in spider silks affects the orientation and relaxation of these intrinsically disordered peptide segments both during fiber formation (spinning) and during fiber performance in environmental humidity (RH), respectively.5,13 Liu’s experimental measurements also showed relations between supercontraction and mechanical properties. Highly stretched native dragline silks have a relatively consistent stress−strain profile with high modulus and high strength. With greater supercontraction, the stress−strain profile changes to lower modulus and higher extensibility. Since the stress−strain profile can be predicted from the structural degree of order in the silk, this allows supercontraction to be linked directly to the structure and morphology of silk.13 Silks from three species of Nephila spiders have been used frequently in supercontraction studies: Nephila clavipes, edulis, and senegalensis. The silks from the same spider family share similar gene sequences and display comparable mechanical and processing behavior.4 Nephila draglines have been shown to consist of two different proteins, namely, MaSp1 (proline-free) and MaSp2 (proline-rich).31 Liu’s work strongly suggests that the proline-rich MaSp2 fraction makes a substantial contribution to supercontraction. However, it is not clear how other structural components might also participate in supercontraction or how the intrinsically disordered proline might contribute to both amorphous and crystal relaxation processes suggested by Michal.28 On the basis of all the work presented above, we formulated the hypothesis that there are at least two major contributions to supercontraction: (i) relaxation of oriented amorphous domains in the MaSp1 fraction and (ii) relaxation of prolinerich metastable ordered domains in the MaSp2 fraction. The empirical measurements presented here and their analysis have as their objective the test of the “two-contributions” hypothesis for supercontraction. For this we experimentally separated the two contributory effects in Nephila edulis dragline silk by focusing on the MaSp1 and MaSp2 protein components. Both these proteins have “hard” blocks of about 30% molar alanine segments that form β-sheet crystal domains and “soft” blocks of intrinsically disordered segments with characteristic sequence components -GGX- and -GPGXX- for MaSP1 and MaSp2, respectively. Since we know that exposure to liquid water induces full supercontraction, we assumed that both contributions are involved. We also noted that some experiments with controlled humidity levels seemed to induce only partial supercontraction
and speculated that only one of the contributions was activated. Refinement of our experiments showed that reproducible and stable fractions of the total supercontraction could be induced at specific combinations of stress, temperature, and humidity, which happened to correspond well with glass transition conditions for silk discussed above.9 This suggested that we were able to selectively induce relaxation of an oriented amorphous fraction. This work reports our investigation of the different contributions to supercontraction, based upon these initial screening observations, and suggests ways to relate these contributions to the composition and morphology of dragline silk.
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EXPERIMENTAL SECTION
Sample Preparation. A medium size Nephila edulis spider was first anesthetized by CO2 and then immobilized by pinning onto a platform. Dragline threads were then identified under a microscope. Once the spider recovered, draglines were forcibly reeled at a controlled speed of 10 mm/s and collected on our standard rotating spool with horizontal spaces and automatic advance. The lab environment was 20 °C at a relative humidity of 40%. Silks were stored under lab conditions before being tested. Virgin Silks. The silks directly from the spool are categorized as virgin silks. They were carefully transferred from the spool to paper holders. Supercontracted Silks. Virgin silks were first transferred carefully from the spool to dividers with a dividing distance of 25 mm. The restraining length on the dividers was adjusted from 25 to 19 mm at first before the silk was immersed in water at room temperature. The silk shrank and became tensioned under water. Further measurement of the shrinkage was conducted in a 1 mm stepwise manner. When the silk no longer shrank in water (observed as loss of tension under an optical microscope), the silk was marked as fully supercontracted and the shrinkage was noted down. In general, all silks have a shrinkage of 6−8 mm out of 25 mm (ratio: 24%−32%). The supercontracted silks were mounted on the paper holders without tensioning soon after the supercontraction (they were still wet). Both natural and supercontracted silks were kept restrained on paper holders in the testing room (temperature 20 °C, relative humidity
