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Unraveling the Molecular Requirements for Macroscopic Silk Supercontraction Tristan Giesa,† Roman Schuetz,‡ Peter Fratzl,‡ Markus J. Buehler,*,† and Admir Masic*,†,‡ †

Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States ‡ Max Planck Institute of Colloids and Interfaces, Science Park Golm, 14424 Potsdam, Germany S Supporting Information *

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 Spider photo courtesy of Charles J. Sharp. testing and Raman spectroscopy 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

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pider 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 nm (Figure 1b).7 The alanine-rich region makes up the hydrophobic β-sheet crystals which are the key for remarkable mechanical performance of dragline silk.8−11 The semiamorphous phase contains predominantly GGX motives and features a significantly poorer strand orientation.12 The order in the crystal and the disorder in the semiamorphous 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.12,14−16 Similar 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 © 2017 American Chemical Society

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 1 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 β-sheet 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 semiamorphous 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 Received: March 3, 2017 Accepted: August 28, 2017 Published: August 28, 2017 9750

DOI: 10.1021/acsnano.7b01532 ACS Nano 2017, 11, 9750−9758

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Figure 1. N. clavipes dragline silk nanostructure and supercontraction mechanism: bridging from experiments to modeling. Supercontraction is the shrinking along the main axis of the silk fiber in water in comparison to its dry state (by up to 50% depending on the silk, around 15% in N. clavipes silk). (A) In the full-atomistic molecular dynamics simulation, silk is represented by a unit of silk, with a stable β-sheet crystal and two amorphous phases. The amorphous phase is responsible for the supercontraction mechanism. (B−D) Silk assembles in nanofibrils of size 20−150 nm. Hundreds of fibrils form dragline fibers of micrometer size. The spider spins the strong dragline silk as structural support for its webs and as lifeline for escape. (E) Measurement of the supercontraction process on dragline silk fibers in a humidity chamber using tensile testing and in situ Raman spectroscopy. (F) In this multiscale approach we are able to link the macroscale supercontraction effect to nanoscale changes in the structure and propose mutations to the core sequence of the silk to suppress the supercontraction effect. Panel (D) courtesy of Charles J. Sharp.

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). Another 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 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, as a way to connect simulation results to experiments at multiple scales. Specifically, we explore the molecular origin of dragline silk supercontraction using a full-atomistic 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.

RESULTS AND DISCUSSION Figure 1e shows the experimental setup used to measure the in situ supercontraction process of natural Nephila clavipes dragline silk. After the humidity is increased, the strain in the fiber decreases under isostress conditions; see Figures S1 and S2 in the Supporting Information. The molecular dynamics study analyzes 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 endto-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-to-end 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%). 9751

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Tyr’s phenol side chain. The relative intensity ratio of the 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 ≪ 1).41 Figure 3b (left) shows values for I860/I830 peak ratios determined from the deconvoluted Raman spectra. Note that the line at peak ratio 1.5 only serves to illustrate a comparison to the simulation. On average, the Tyr residues tend to be both donor and acceptor of hydrogen bonds in dry conditions and turn into acceptors in supercontracted wet state. This suggests a specific involvement of Tyr and specifically of the OH group in the folding and supercontraction of silk. While this phenomenon has been observed,38 to our knowledge, the precise implications for the supercontraction process have not yet been investigated. The A/D ratio can be directly determined from the hydrogen bonding in the molecular dynamics trajectory in fully hydrated and dehydrated conditions, Figure 3b (right). In agreement with the Raman experimental results, Tyr changes its A/D ratio when passing form dry to wet conditions. This change is mainly associated with the OH group, as seen in the subplot of Figure 3b (right). Interestingly, we find a similar change to be prominent in other polar and/or charged side chains, such as arginine (Arg) and serine (Ser), suggesting a contribution from these residues to the macroscopic contraction of silk. Note that all these residues are located in the amorphous part of the silk sequence; see highlights in Figure 1f. To gain a more fundamental understanding of the supercontraction process, we investigate the energetics of supercontraction and how a stress can be generated from it. Figure 4a shows the supercontraction strain versus stress of silk dragline silk fibers from simulation and experiment. The supercontraction strain εSC = ΔL/L0 is the strain generated in an unconstrained fiber when immersed in a humid environment. The supercontraction stress σSC is the stress needed to retain a contracting fiber at its original uncontracted length. It can be determined from tensile tests (experiment and simulation) or a free energy balance (simulation). The simulation data points in Figure 4a are determined in two ways. The data point at zero

Figure 2. Supercontraction measured from simulation and experiment. The strand silk model is equilibrated with and without water, and supercontraction is measured by average end-to-end length of the molecule (“Simulation Length”) as well as radius of gyration of dehydrated versus hydrated molecule (“Simulation Radius”). The radius of gyration tensor yields the shape change along the three axes of the molecule, whereas the end-to-end length method cannot reveal the overall shape change. Results of the simulation in three axis directions are compared with experimental results, and agreement is found for the contraction in the axial direction (ΔL/L0).

From the simulation, we are able to determine the secondary structure composition (see Supporting Information, Figure S3), indicating an increase in β-sheet in the dry structure 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

Figure 3. Polarized Raman and hydrogen bonding in the silk wildtype. (A) Left: Polarized Raman scattering of N. clavipes dragline silk in wet (85% RH) and dry (15% RH) conditions. Significant changes are observed in the 830−860 cm−1 region which can be associated with vibrations in the Tyr OH-group. Right: The peak ratio (I860/I833) is considered an indicator for the propensity of Tyrosene to act as a strong hydrogen acceptor or donor. (B) Molecular dynamics simulation results. The acceptor/donor (A/D) ratio determined from the hydrogen bonding analysis of the molecular dynamics trajectory is in agreement with the Raman experimental results. Tyr tends to be a donor in dry conditions and a donor/acceptor in wet conditions. In the simulation, other polar and/or charged residues in the amorphous part of silk such as Arg and Ser display similar behavior. 9752

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Figure 4. Molecular details of supercontraction. (A) Supercontraction strain versus stress for dragline silk fibers determined from experiment and simulation. The abscissa axis (εSC 0) corresponds to the constrained supercontraction state, where the fiber is not allowed to shrink. The ordinate axis (σSC = 0) corresponds to the unconstrained supercontraction state. The simulation data points are found by determining the entropy of spider silk protein in hydrated and dry conditions and deriving the stress needed to reverse supercontraction. The range of experimental data is indicated by the shaded area surrounded by black dots and explicated in the Supporting Information. (B) Dihedral sidechain angle distribution of Tyr determined from simulation. The χ2 angle describes the torsion angle of the aromatic ring that is coplanar with the hydroxyl group of Tyr. From dry to wet conditions, a peak shift from −90° to −10° as well as a shift from 90° to 170° is observed. (C) Two detailed snapshots from the molecular dynamics simulation showing the Tyr local environment. The same residues at a similar simulation time are shown in dry and wet conditions. Three (out of 15) polypeptide chains are visible (blue, red, and green), while Tyr and its hydrogen bond partner are highlighted. The peak at ∼−10° and ∼170° (1) in the dry state is associated with the intermolecular hydrogen bonding of Tyr’s OH group with the Gly of the adjacent chain. The hydrogen bond is formed through hydrogen of the Tyr and oxygen of the Gly involved also in forming protein backbone. In this case, Tyr acts as a hydrogen bonds donor. In the wet state, Tyr both accepts hydrogen bond from mobile water within the structure as well as donates H-bonds through intramolecular interactions with the hydrophobic Gly.

stress is obtained directly from the change of shape, where equilibrium simulations with and without water determine the shape of the silk molecule. The stress value in iso-strain configuration is computed by decomposing the free energy (see the Experimental Methods). The changes in both entropy and enthalpy generate the supercontraction stress. From simulation, we estimate a 12.3% increase of absolute entropy (TΔS ≈ 2.1 MJ/mol), from dry to wet at T = 300 K. The entropic part of the supercontraction stress σent needed to transition from the supercontracted to the uncontracted state is then determined by σent ≈ 4TΔS/(πΔrD2), where Δr is the change in length (∼13%) and D the diameter of the molecule (∼4 nm). This yields a simulated entropic supercontraction stress of σent = 64.5 ± 10.9 MPa (red shaded area in Figure 4a). Alternatively, the supercontraction stress is directly estimated from simulation with a tensile test, using the stress at supercontraction strain (see the Experimental Methods). The stress determined (σsim ≈ 70 MPa) is very close to the entropic stress, which is intuitive, since the computational tensile test is performed in solvent and the energy related to the desolvation is not taken into account. The change in enthalpy is estimated from molecular dynamics simulation as ΔAH ≈ 1.2 MJ/mol. The core sequence of silk is a slightly hydrophobic material;42

therefore, the enthalpic term is endothermic and reduces the stress needed to reverse supercontraction (cyan shaded area in Figure 4a). The supercontraction stress determined from simulation that includes entropic and enthalpic contributions is σSC = 37.4 ± 12.4 MPa. The shaded area in between the zero-stress and zero-strain value connects the lower and upper values of the standard deviation and hence indicates only a possible pathway between those two states. This pathway is not necessarily linear. Figure 4a also presents a compilation of experimental data for silk fibers that were generated from tensile tests of dragline silk fibers (black dotted area). There is a large range of experimental results, with details shown in the Supporting Information. It is notable that these stresses are below the yield point of spider dragline silk (∼150 MPa). Among the different types of silks of orbweaving spiders, the relation between supercontraction stress and supercontraction strain tends to be monotonic, i.e., the larger supercontraction strain is related to larger supercontraction stress. This suggests that the free energy scales directly with the length, and the mechanism behind supercontraction is similar for different types of dragline silk. In the Supporting Information (Figure S4), we support this claim with experiments on dragline silk from C. salei. 9753

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and average end-to-end length) and compare them to the wildtype silk. While the natural wildtype silk exhibits strong contraction (∼15%) and the two in silico control experiments (replacement of Ser and Gln) do not show any changes in supercontraction magnitude, an in silico mutation of Tyr indeed leads almost to the suppression of the contraction, Figure 5. The effect of the

The results also indicate the contribution of enthalpy in the energy balance during the supercontraction and suggest the possibility to control the process by altering the chemical composition of the protein sequence. This was done by performing in silico a targeted mutation on the key residues that mostly contribute to the increase in entropy and minimally affect the enthalpic term. From the hydrogen-bonding analysis and the Raman spectra we identify polar and/or charged amino acid residues with large side chains such as Tyr and Arg as potential key players in the supercontraction process. This is reasonable considering the polar nature of water interacting with the protein. However, it has remained unclear through which type of interactions these residues are able to affect the protein structure. Conformational changes in proteins are often linked to changes in dihedral angles, the torsion angles in the residue backbone and the sidechain. We find prominent shifts in the χ2 angle of Tyr, shown in Figure 4b (for comparison, the dihedral angles for Arg, Ser, Gln, and Leu are shown in the Supporting Information, Figure S5−S8). From wet to dry state, a peak shift from −90° to −10° as well as a symmetric shift from 90° to 170° is observed. It is possible to match the angles to rotamer tables43 and find associated secondary structures from the conformation of the backbone (Supporting Information, Tables S2−S6). Arg’s and Tyr’s shift in side-chain dihedral angle is specifically associated with a secondary structure transition from sheet-like in dry state to coiled or helical structure in the wet state leading to a contraction in the wet state. Since each rotamer conformation contains a set of dihedral angles in a three-dimensional space, it is very difficult to correlate macroscopic uniaxial contraction to the local conformational change. However, the relation between the dihedral angles and the behavior of individual amino acids in different environments is possible. Figure 4c illustrates the local environment of Tyr in both dry and wet conditions associated with peaks in Figure 4b (marked with numbers) at −10° (1) and 170° (2). Three of the 15 polypeptide chains are visible (blue, red, and green), while Tyr and its hydrogen bond partners are highlighted. Peak 1 under dry conditions is associated with the intermolecular hydrogen bonding of Tyr’s OH-group with the Gly residue of an adjacent chain. The hydrogen bond is formed between hydrogen of Tyr and the CO oxygen of Gly that also forms the protein backbone. In this case, Tyr acts as a hydrogen bond donor. On the other hand, in the wet state, Tyr both accepts hydrogen bonds from mobile water within the structure as well as donates H-bonds through intramolecular interactions with the hydrophobic Gly, associated with peaks 2. Similar results are found for the peaks in the χ4-angle of Arg, where the interactions with water are replaced by intramolecular interactions (Supporting Information, Figure S8). In order to explore the possibility of controlling supercontraction, we alter the sequence of spider silk by performing residue mutations on Tyr and Arg, as illustrated in Figure 1f. Five additional sequences are equilibrated using molecular dynamics simulations with and without solvent. As a control experiment, where no change in supercontraction is expected, Ser is replaced by Ala and Gln is replaced by Leu. In another experiment, Tyr is replaced by Phe (similar molecular structure, but no hydroxyl group on the aromatic ring). In the fourth mutation experiment, Arg (positively charged amino acid with long side chain) is substituted with Leu and in the last mutation experiment, Tyr is replaced by Phe, Arg by Leu and Ser by Ala. We measure the contraction/expansion (by radius of gyration

Figure 5. Molecular dynamics simulations of point-mutations of the spider dragline sequence and its effect on supercontraction. Five additional sequences are equilibrated, where first Ser is replaced by Ala and Gln by Leu, and then polar and/or charged amino acids are substituted by their apolar/uncharged counterparts. The contraction/expansion of the structure is measured by radius of gyration and average end-to-end length. In the first two cases, the mutation does not change the supercontraction behavior. When Tyr is replaced by Phe (similar molecular structure, but no hydroxyl group on the aromatic ring), supercontraction is suppressed. When Arg (positively charged long side chain) is replaced by Leu, supercontraction is even reversed. When Tyr and Arg are substituted with additional replacement of Ser with Ala, the structure expands. From these results, it can be deduced that Tyr and Arg play a crucial role in the supercontraction mechanismthrough their polar and charged side-chain groupthat can be related to mostly entropic effects.

Arg mutation is even more significant, leading to an expansion in the wet state. Similarly, mutation of all three residues yields an expansion in the wet state (and, hence, a predicted axial expansion of the fiber). The effect of the Ser mutation is small, which can be explained with its comparatively small side chain and small changes in side-chain dihedral angles. In all of the mutated sequences, the β-sheet crystals remain intact (and the β-sheet content approximately constant, see the Supporting Information, Figure S9), suggesting that the mechanical properties of the entire structure is unaffected by the point mutations. A computational tensile experiment (Supporting Information, Figure S10) confirms this hypothesis. The evolutionary cause for supercontraction is not well understood. It is believed to be an essential feature of the 9754

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bonds. This marks the fundamental trigger for supercontraction in silk and highlights the importance of the sequence of specific amino acid residues in the amorphous phase. The in silico experiments for supercontraction involved molecular dynamics simulations of the N. clavipes MaSp1 sequence and its mutations in the appropriate thermodynamic ensembles. While the conditions in the simulation (hydrated and dehydrated) may not represent the most accurate condition for the supercontraction process, they reflect a limit situation of this process. Molecular modeling allows us to study in great detail the thermodynamic conditions of the two limit states without analyzing the transition between these states. Many silks display common themes in the amino acid sequence. Therefore, the findings and approach developed in this study do not only apply to N. clavipes silk, but to a much broader range of polymer materials that feature water induced structural changes.

spinning process, since wet elastomeric silk can be processed easier.20 It further has been discussed that the supercontraction process allows the spider to tailor the elastic behavior of the silk fiber by pulling and restraining the silk threads and adjusting the water content.44 In this context, the stiffness of the web needs to increase to maintain the web’s tightness when fog (e.g., morning dew) condensates on the spider web.45 Control of the dynamics in the presence or absence of solvent is crucial for the design of new polymer materials.16 In this context, genetic engineering and synthetic chemistry are offering pathways to design materials on demand if appropriate modifications to the sequence of amino acids can be proposed.46 In this study, we are able to demonstrate the crucial role of polar/charged amino acid residues in the supercontraction mechanism. Through simulations we can now investigate the effect of the substitution of these key residues with their apolar equivalents. To achieve this, we alter the sequence of spider silk, and perform residue mutations on Tyr, and Arg. This is especially of interest in view of the technical application of spider silk, where contraction may not be a desirable effect. We find that the natural wildtype silk exhibits strong contraction (∼15%), whereas a in silico mutation of Tyr indeed leads almost to the suppression of the contraction. The effect of a replacement of Arg with Leu is even more significant, leading to an expansion in the wet state. The suppression mechanism can be understood following the evolution of the system after the point mutation. In the wildtype, the change in enthalpy is dominated by the breaking of hydrogen bonds and Coulombic interactions especially in the charged and polar side chains. On the other hand, entropic contribution is driven by water “plasticizing” the polymer chains. Interestingly, a point mutation of the sequence can significantly affect the silk supercontraction behavior. This could be explained by the influence of the charged and polar residues on the enthalpic and particularly the entropic contributions that drive supercontraction as discussed above. In the wet state, the new residues form hydrophobic interactions that prevent water from plasticizing this part of the polymer chain. Hence, the point mutation changes the effective length and thus the amplitude of fluctuation in the polymer chains.47 The change in entropy from dry to wet state becomes smaller and the system does not undergo supercontraction. Our findings identify candidate residues that may be essential in the supercontraction process. The large mobile side chains of Arg and Tyr lead to a significant increase in entropy during the supercontraction process, thus shrinking the molecule. On the other hand, the changes in hydrogen bonding (and associated rotamer configuration indicated by the side-chain dihedrals) relate to the finding that the supercontraction process is additionally controlled by enthalpic effects. Note that the entropic term is so dominant that even at the freezing point of water, MaSp1 silk still uptakes water, although it contains many hydrophobic components. In dry conditions, the chains are stabilized through intramolecular interactions and the fiber compacts in radial direction, while the chains elongate in axial direction. The formation of short β-sheets in the noncrystalline part, especially in the GGX motifs (X = Arg, Ala, and Leu) increases this effect. In the wet state, mobile water disturbs these interactions and the chains, especially the long sidechains of Tyr and Arg fold onto themselves. We find that the abundance of hydrophobic Gly in the amorphous silk phase drives the large side-chains of Tyr and Arg to form hydrogen

CONCLUSION Here, we report an approach to study molecular mechanisms not only in silk but also in other materials as a way to connect simulation results to experiments at multiple scales. Specifically, we explore the molecular origin of dragline silk supercontraction using a full-atomistic 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. The described experimental platform can monitor the extent of supercontraction and molecular interactions simultaneously, whereas molecular dynamics simulations provide a detailed view on the thermodynamics of the material and the behavior of individual residues. We link 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. The approach applied is appropriate to explore the effect of point mutations on the overall physical properties of protein based materials. Furthermore, our study demonstrates the importance of a combined experimental and computational approach for genetic engineering and innovative materials design. EXPERIMENTAL METHODS Sample Type and Preparation. Samples of N. clavipes web threads were collected and separated during the summer 2014 and stored at −20 °C. The threads we collected were primarily dragline silk (MaSp1/MaSp2). Measurement Chamber and Sample Preparation. The samples were tested in a sealed chamber of volume of about 140 cm3. The chamber was kept at a constant temperature of 25 °C by means of cooling bath circulation thermostat (Huber). The humidity inside the chamber was controlled by means of a “Wetsys (Setaram)” humidity generator under a flow of 200 mL/min. Temperature and humidity were monitored via a SHT75 digital humidity and temperature sensor (SENSIRION) that was placed in the vicinity of the sample. Samples were clamped to two aluminum holders and fixed with adhesive. The strain was controlled by one of the holders which was connected to a PI (Physik-Instrumente) M-126.DG1 linear motor stage, while the axial tensile force was measured using a Honeywell Sensotec R-31E load cell (0.5 N max. equipped with an overload stop) attached to the other holder. Force values were read out every 100 ms from the load cell and averaged over 1 s in order to improve the signalto-noise ratio. 9755

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ACS Nano Confocal Polarized Raman Microscope. For confocal Raman microspectroscopy, a continuous laser beam was focused down to a micrometer sized spot on the sample through a confocal Raman microscope (CRM200, WITec, Ulm, Germany) equipped with a piezo-scanner (P-500, Physik Instrumente, Karlsruhe, Germany). The diode-pumped 785 nm near-infrared (NIR) laser excitation (Toptica Photonics AG, Graefelfing, Germany) was used. For in situ mechanical tests, a 20× (Nikon, NA = 0.4) objective was used. The spectra were acquired using a CCD (PI-MAX, Princeton Instruments Inc., Trenton, NJ) behind a grating (1200 g mm−1) spectrograph (Acton, Princeton Instruments Inc., Trenton, NJ). 60 accumulations with integration time of 1 s were used for single spot analyses. In Situ Mechanical Testing and Raman Spectroscopy. Each fiber, typically 10 mm long and 6.0 ± 0.2 μm thick, was characterized at three different humidity conditions. Starting at room temperature and 25%RH, humidity was gradually raised to 50%RH, which is known to be slightly below the supercontraction conditions. Eventually, the RH was raised to 85%RH at which supercontraction takes place. At each humidity level the examination included a stretching and releasing of the fiber within the elastic range of the stress−strain curve. This procedure was cycled several times and was found completely reversible in terms of force development and the fiber elongation. In order to guarantee sufficient time for relaxation of the fiber, the stretching was performed with a speed of 5 μm/s (which corresponds to less than 0.05%/s of the whole length). Such a rate allowed fiber to relax and stresses measured can be considered as static stresses. To confirm this assumption, the force was also measured at different strains after long relaxation periods (up to 30 min at each RH value, see Supporting Information). For stress calculations the fiber diameter in the dry state was used. The diameter of each fiber was measured by means of the optical mode of the Raman microscope and values found for few samples were also confirmed using environmental scanning electron microscope (ESEM). For the calculation of the contraction ratios (L − L)/L0 of the individual static stresses the related length pairs L0 and L were obtained from the stress−strain curves of 25% RH and 85% RH, respectively. Raman spectra were collected in situ at different RH conditions in the contraction region after relaxation. The polarization of the laser was always parallel to the main axis of the silk fiber. Molecular Dynamics Simulations. Molecular dynamics simulations were performed using a model of N. clavipes MaSp1 dragline silk, predicted from Replica Exchange Molecular Dynamics Simulations and equilibration in explicit solvent.3,11,48 The 15-strand sample of MaSp1 including a crystal and two amorphous phases was further equilibrated in an explicit water box for 30 ns without holonomic constraints and a 0.5 fs time step. The GROMACS software package with CHARMM27 force field and the Tip3P water model was used for the explicit water simulations of this complex biological molecule. This force field is able to capture electrostatic interactions without chemical reactions.49 We modeled isobaric−isothermal conditions (1 bar, 300 K) with charge-neutralizing solvent and 150 mmol sodium chloride. Equilibration was performed with Particle Mesh Ewald (PME) electrostatics, a velocity-rescale thermostat, and a Nose− Hoover barostat. The dehydrated model was also simulated for 30 ns in a canonical ensemble with tabulated charge interactions (to prevent the collapse of the simulation box). To prevent image interactions, the periodic box wrapped the protein by at least 10 Å distance. Visual Molecular Dynamics (VMD) including the STRIDE secondary structure algorithm was used for visualization and analysis of protein molecules and their equilibration trajectories.50 Hydrogen bonding (H-bonds) and hydrogen bond energies were determined by geometric proximity of hydrogen donor and acceptor, using DSSP.51 For the H-bonds, a 3.0 Å cutoff distance and a 30° cutoff angle were used. Shape Calculation. A common way to measure the shape of a molecule is the radius of gyration. This second-order tensor is calculated as the (mass or charge weighted) root-mean-square distance of all the atoms from the center of mass of a molecule. It is used here to quantify the size of the ensemble of atoms in the molecular structure. The volume of the equivalent ellipsoid described by the gyration tensor is V = (4/3)πλxλyλz, where λi is the eigenvalue of the gyration

tensor associated with direction i. Usually, the tensor is calculated with respect to the principal components of the system. One of the principal components coincides with the axis direction and the associated eigenvalue is the molecule size in that direction. By comparing the change of radius for the same structure in two different environmental conditions one can calculate the shape and volume change of the structure. Alternatively, the average end-to-end distance of the chains gives an estimate of the molecule size in axial direction. Free Energy, Entropy, and Supercontraction Stress. The supercontraction stress, i.e., the stress needed to pull a contracted fiber back to its original uncontracted length, is given by σSC ≈ − 4ΔA/ (πΔrD2), with ΔA is the change in free energy, Δr is the change in molecule length during the supercontraction process, and D is the molecule diameter. We decompose ΔA = Awet − Adry ≈ ΔAH − TΔS, where ΔAH is the change in enthalpy associated with the hydration (hydrogen bonding), ΔS is the change in entropy, and T isthe temperature. A more detailed discussion is presented in the Supporting Information. The pulling simulation was performed in explicit solvent using the steered molecular dynamics algorithm and boundary conditions similar to those reported in previous studies.3 The stress strain data shown is the pulling force divided by the effective area plotted up to the point where the strain matches the measured supercontraction strain.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b01532. Detailed description of experimental methods, modeling equations and energy parameters estimation as well as protein secondary structure evaluation (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Markus J. Buehler: 0000-0002-4173-9659 Admir Masic: 0000-0002-8791-175X Author Contributions

T.G., M.B., and A.M. conceived the idea, and all authors contributed to the writing of the manuscript. T.G., R.S., and A.M. carried out the experiments and evaluated the data. T.G. performed and analyzed the computer simulations. All authors contributed to the interpretation of the results and commented on the manuscript. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank Prof. David Kaplan and Dr. Lena Tokareva (Tufts University) for providing dragline silk samples for mechanical testing. Figure 1,, has been created with support from Dr. James Weaver (Wyss Institute, Harvard). We thank Prof. Carole Perry (Nottingham Trent University), Dr. Luca Bertinetti and Dr. Emanuel Schneck (Max Planck Institute of Colloids and Interfaces) for insightful discussions. T.G. and M.J.B. acknowledge support from ONR-PECASE (N00014-10-1-0562), AFOSR (FA9550-11-1-0199), and NIH (5U01EB014976). P.F. and A.M. are grateful for support by the Alexander von Humboldt Foundation and the Max Planck Society in the framework of the Max Planck Research Award funded by the Federal Ministry of Education and Research. A.M. is grateful for 9756

DOI: 10.1021/acsnano.7b01532 ACS Nano 2017, 11, 9750−9758

Article

ACS Nano financial support from the German Research Foundation (DFG) within the priority programme 1420.

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