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Unraveling the Molecular Requirements for Macroscopic Silk Supercontraction Tristan Giesa, Roman Schuetz, Peter Fratzl, Markus J. Buehler, and Admir Masic ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b01532 • Publication Date (Web): 28 Aug 2017 Downloaded from http://pubs.acs.org on August 30, 2017
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Unraveling the Molecular Requirements for Macroscopic Silk Supercontraction
Tristan Giesa1, Roman Schuetz2, Peter Fratzl2, Markus J. Buehler1*, and Admir Masic1,2*
1
Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, USA
2
Max Planck Institute of Colloids and Interfaces, Science Park Golm, 14424, Potsdam, Germany
ABSTRACT
Spider dragline silk is a protein material that has evolved over millions of years to achieve finely tuned mechanical properties. A less known feature of some dragline silk fibers is that they shrink along the main axis by up to 50% when exposed to high humidity, a phenomenon called supercontraction. This contrasts the typical behavior of many other materials that swell when exposed to humidity. Molecular level details and mechanisms of the supercontraction effect are heavily debated. Here we report a molecular dynamics analysis of supercontraction in Nephila clavipes silk combined with in situ mechanical testing and Raman spectroscopy 1 ACS Paragon Plus Environment
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linking the reorganization of the nanostructure to the polar and charged amino acids in the sequence. We further show in our in silico approach that point mutations of these groups not only suppress the supercontraction effect, but even reverse it, while maintaining the exceptional mechanical properties of the silk material. This work has imminent impact on the design of biomimetic equivalents and recombinant silks for which supercontraction may or may not be a desirable feature. The approach applied is appropriate to explore the effect of point mutations on the overall physical properties of protein based materials.
Keywords: silk, mechanics, water, supercontraction, simulation, Raman.
Spider dragline silk is a natural protein material that has evolved over millions of years to develop finely tuned mechanical properties to serve specific functions, including the ability to tailor properties in changing environments.1-3 Dragline silk is mainly made of two different proteins and its primary structure relies on a subset of the naturally occurring amino acids as building blocks.4,5 The more abundant protein, MaSp1, contains a sequence of alanine- and glycine-rich repeats leading to a distinct hierarchical structure,6 Figure 1a-d. The silk unit cell (Figure 1a) assembles into nanofibrils of size 20-150 nanometers (Figure 1b).7 The alaninerich region makes up the hydrophobic β-sheet crystals which are the key for remarkable mechanical performance of dragline silk.8-11 The semi-amorphous phase contains predominantly GGX motives and features a significantly poorer strand orientation.12 The order in the crystal and the disorder in the semi-amorphous phase are directly linked to silk’s interaction with water.13
Water has the ability to fundamentally reorganize silk’s molecular structure and can cause dramatic changes in mechanical properties and physical characteristics.12,
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phenomena can be observed in other biological materials, such as tendon collagen17 and squid proteins.18 While most protein structures swell upon hydration, some spider dragline silk fibers will shrink along the main axis by up to 50% at high humidity, a phenomenon known as supercontraction.19,20 Nephila clavipes dragline silk reversibly shrinks by 15-20%, and if the fiber is constrained it will generate a tensile stress.6, 12-14, 21-23 Immersion in water typically results in the reduction of stiffness by up to an order of magnitude, and noticeable improvement in fracture strain.8,9, 13, 24-26
While there are numerous studies on supercontraction, the exact mechanism behind it has not yet been revealed.23, 27-30 It is believed to be an essential feature of the spinning process, since wet elastomeric silk can be processed easier.20 It has been suggested that since the βsheet crystals are hydrophobic, they do not undergo significant structural changes when hydrated, so that the origin of the supercontraction phenomenon is likely to be located in the semi-amorphous phase only.25, 31 Above a critical hydration level (~70%), water molecules intrude the H-bond network between strands in the amorphous structure and allow them to reorganize into a less ordered, more coiled, lower energy state.12, 23, 28 The response of silk to water indicates that the dry fiber is frozen into a glassy state that shows some degree of alignment. Exposure to water releases the glassy state and the wetted silk turns into an elastomer.30-33 Using nuclear magnetic resonance Yang et al. linked the supercontraction process to the highly conserved YGGLGSQGAGR block in the silk sequence.16 They identified Leucine (Leu, L) as potential key residue of the supercontraction effect, while noting the proximity of Tyrosine (Tyr, Y) and Arginine (Arg, R). Other study indicated the proline-related motif, GPGXX, as an essential player in spider silk supercontraction.34 However, nearly proline-free regenerated B. mori silks shows supercontraction (up to 5%),35
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implying that proline is most likely not the critical constituent for supercontraction capability.36
Here, we report an approach to study molecular mechanisms not only in silk, but also in other materials, a way to connect simulation results to experiments at multiple scales. Specifically, we explore the molecular origin of dragline silk supercontraction using a fullatomistic model and molecular dynamics combined with a well-established experimental approach17, 37-39 based on in situ Raman spectroscopy and mechanical testing in a humidity controlled chamber (Figure 1e). The described experimental platform can monitor the extent of supercontraction and molecular interactions simultaneously, whereas molecular dynamics simulations (Figure 1a) provide a detailed view on the thermodynamics of the material and the behavior of individual residues. We exemplify the power of this combined effort by proposing a genetic engineering strategy to alter silk’s behavior to industrial requirements (Figure 1f). We identify the most important parts of the silk amorphous structure that control supercontraction and then test in silico mutations to the core sequence of N. clavipes dragline silk (indicated in colors in Figure 1f) that reduce or even reverse the supercontraction mechanism. Our study demonstrates the importance of a combined experimental and computational approach for genetic engineering and innovative materials design.
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RESULTS/DISCUSSION Figure 1e shows the experimental setup used to measure the in situ supercontraction process of natural Nephila clavipes dragline silk. After increasing the humidity, the strain in the fiber decreases under isostress conditions, see Figure S1 and Figure S2 in the supporting information. The molecular dynamics study analyses a representative unit of MaSp1 silk (Figure1a), with a stable β-sheet crystal and two independent amorphous phases, equilibrated by Replica Exchange Molecular dynamics and explicit water and dehydrated simulation. In the simulation, supercontraction is measured by the change in the average end-to-end length of the molecule chains as well as the radius of gyration. Both measures are deduced from the molecular dynamics equilibrium trajectory of the silk dehydrated and hydrated model, shown schematically in Figure 2. The radius of gyration (weight averaged ellipsoid) reflects the shape of a 3D molecule and is indicated in Figure 2 together with an overlay of snapshots of the hydrated and the dehydrated structure. In the wildtype, we find a contraction from dry to wet state of 13.2 ± 5% (radius of gyration) and a contraction of 8.6 ± 1.7% (average end-toend length). Agreement between molecular simulation and macroscopic experiment (13.2 ± 0.2% ) of N. clavipes dragline silk fibers is found for the contraction in the axial direction. The values also reflect other literature results for dragline silk fibers.13 The contraction also leads to a change in volume (about 5%), as determined from the radius of gyration in the three axis directions of the fiber, Figure 2. Note that neither the end-to-end length method nor the experiments yield an estimate of the radial shape change of the molecule. Swelling of the fiber in radial direction has been reported during supercontraction leading to a constant volume.40 The radius of gyration measurement predicts a small decrease in volume (~5%) which is less than the contraction in axis direction (~15%). From the simulation we are able to determine the secondary structure composition (see supplementary information, Figure S3), indicating an increase in β-sheet in the dry structure
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and small increase in β-turns content. In Figure 3a the Raman spectra of N. clavipes dragline silk in wet (85% RH) and dry (15% RH) conditions are reported. While slight differences can be detected in the analyzed spectral range, the most striking change is observed in the 830 860 cm-1 region associated with vibrations in the Tyrosine (Tyr) side chain (Fermi resonance between the in-plane breathing mode of the phenol ring and an overtone of the out-of-plane deformation mode).41 The relative intensity of the two bands is sensitive to the extent of mixing of the two modes, and thus to the hydrogen bonding condition of Tyr’s phenol sidechain. The relative intensity ratio of two peaks (I860/I830) is up to 2.5 when the OH-group of Tyr serves as an acceptor (A) of a strong hydrogen bond (A/D >> 1) and is down to 0.3 when the OH-group serves as a donor (D) of a strong hydrogen bond (A/D
