Modular Assembly of a Conserved Repetitive Sequence in the Spider

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Characterization, Synthesis, and Modifications

Modular Assembly of a Conserved Repetitive Sequence in the Spider Eggcase Silk: from Gene to Fiber Jianming Chen, Jinlian Hu, Sono Sasaki, and Kensuke Naka ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00428 • Publication Date (Web): 06 Jul 2018 Downloaded from http://pubs.acs.org on July 16, 2018

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ACS Biomaterials Science & Engineering

Modular Assembly of a Conserved Repetitive Sequence in the Spider Eggcase Silk: from Gene to Fiber Jianming Chen,a Jinlian Hu,*,a Sono Sasaki,b and Kensuke Nakac a

Institute of Textiles and Clothing, The Hong Kong Polytechnic University, 11 Yuk Choi Road,

Hung Hom, Kowloon, Hong Kong, China b

Faculty of Fiber Science and Engineering, Kyoto Institute of Technology, Goshokaido-cho,

Matsugasaki, Sakyo-ku, 606-8585 Kyoto, Japan c

Faculty of Molecular Chemistry and Engineering, Kyoto Institute of Technology, Goshokaido-

cho, Matsugasaki, Sakyo-ku, 606-8585 Kyoto, Japan. *Email: [email protected] (Corresponding author)

KEYWORDS: Fiber assembly, conserved repetitive sequence, spider eggcase silk, micelle, microfluidic

ABSTRACT: Spider silk features extraordinary toughness in combination with great biocompatibility and biodegradability, fascinating researchers to prepare artificial silk fibers inspired from the natural art of spinning. In addition to C- and N-terminal domain, a repeat unit from Lactrodectus Mactans spider eggcase silks displays substantial sequence conservation across species. Herein, we attempt to spin the engineered tubuliform spidroin 1 (eTuSp1) by

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microfluidics in a mode of modular assembly comprising the genetic construction, micellar formation, phase separation and further solidification. Based on the conserved gene sequence, a unique amphiphilic behavior was predicted and then verified by combined techniques of DLS, TEM and synchrotron radiation XRD to reveal the formation of micelle-like structure. Through the employ of biomimetic microfluidic devices, desolvation of eTuSp1 was simplified by the non-solvent induced phase separation in place of the conventional ions exchange and acidification. Both controlled by protein concentrations and flow rate ratios, silk fibers were assembled similar to these reported in other studies of spheres/spherical aggregates observed as intermediates. Due to the applied shear and elongational flow in microfluidic systems, these intermediates were forced to form fibrillar assemblies accompanied by the conformational transformation from α-helix to β-sheet. The resultant mechanical properties were investigated in response to the change of secondary structures and morphologies during spinning process. This work studies the sequence-structure-property relationship, providing comprehensive and systematic insight into the design rational on the preparation of artificial silk fibers from micro scale to macro scale.

1. INTRODUCTION Spiders are unparalleled in the versatility of sophisticated silks and well-equipped with up to seven silk types.1 Thereof, task-specific eggcase silk is exclusively synthesized by spiders for cocoon construction, allowing the protection of their offspring from predator, parasitoid invasion or temperature fluctuation.2 In comparison to the well-known dragline silk, eggcase silk is less tough but more resistant to the aqueous environment. Even among all types of silks, it is unique because of high serine and low glycine content. Eggcase silk proteins are initiated at sexual


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maturation and primarily secreted by the tubuliform gland that is only found in female spiders.3 Accounting for the majority of the amino acid (aa) composition, tubuliform spidroin 1 (TuSp1) is therefore considered as the major component of eggcase silks.4 Given that insoluble silk fibers in rich of β-sheets are formed via the natural refolding of water-soluble spidroins with helical structures, but silk assembly mechanism remains largely undiscovered. Due to high length and sequence conservation across species and silk types, nonrepetitive individual N- or C-terminal domain has been intensively investigated for their roles in the course of silk production. These termini were proposed to act as independent switches that are sensitive to the changes of pH and ions, respectively.5 Specifically, as a structural switch, the N-terminal domain changed conformation by dimerization along with a drop of pH, enabling a rapid formation of silk fibers.6-9 While as a molecular switch, the C-terminal domain was implicated in the protein storage by forming supramolecular assemblies, and involved in the fiber assembly by triggering the salting-out of proteins.10-11 In addition to the N-/C-terminus, studies have suggested that a single repeating unit alignment within TuSp1 exhibited substantial sequence conservation among spider superfamilies with limited variations observed.12 37 fibroin sequences from 12 spider species were examined to confirm that the repetitive architecture of TuSp1 was a general feature of the eggcase gene family. The homogeneous repeats also provided evidence on the concerted and modular evolution. In light of past brilliant achievements on the studies of conserved aa sequences, it is thereby of significance to attract attentions from popular media for this repetitive internal block repeat. The full-length of TuSp1 was predicted with a molecular weight of approximately 300 kDa, it kept in suspense whether these small molecules from the repeat unit could individually direct fiber assembly in the absence of termini. Thus in this study, this unique repetitive sequence of TuSp1 was investigated for the establishment of


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roles from individual domains, further unveiling the mystery of silk assembly. Upon passage along the spinning duct, spidroins are subjected to the physicochemical environment, which can be technically best mimicked by the microfluidic devices with precise control on the laminar and elongational flow conditions. For ions exchange and acidification, it is quite flexible in disclosing or closing specific channels on demand to effectively add phosphates and acids. A sheath-core structure is designed for the laminar flow, while elongational flow can be accomplished simply by narrowing the channel width. No matter which spinning approach is applied for the induction of soluble proteins to solid fibers, the fundamental principle of phase separation involved is: 1) change the protein concentration in solutions; 2) find ways to trigger the desolvation of proteins.13 For fiber assembly, phase separation must occur under the shear and elongation, whereas spheres can be obtained solely from the stimulus of potassium phosphate.14 In microfluidic systems, silk fibers can be well prepared for specific functional applications.15-17 It becomes feasible to mimic the natural spinning process of spiders and insects in a subtly designed microfluidic device, allowing the visualization of structural changes and analysis of independent variables.18 In this work, a spider eggcase silk protein is genetically engineered by using the highly conserved repeating unit of TuSp1 from a black widow spider, Lactrodectus Mactans. In a biomimetic manner, silk assembly is modularized as the genetic construction, micellar formation, phase separation and further solidification. Herein, efforts have been expended verifying the micelle-like structure of eTuSp1 by combined techniques of DLS, TEM and synchrotron radiation XRD, as well as discussing the underlying mechanism of fiber assembly. Moreover, we would shed highlight on the exploration of structural and morphological changes to indicate the various mechanical properties of eTuSp1 in different spinning stages.


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2. EXPERIMENTAL SECTION Gene Recombinant. The engineered spider eggcase silk protein was cloned, expressed and purified according to our previous study.19 Specifically, a single repeating unit of Tubuliform Spidroin1 (TuSp1) was engineered from a black widow spider with His6-tag attached in the absence of non-repetitive N-/C-teminus. By using this truncated TuSp1, DNA construct was designed for the following prokaryotic expression. The target gene was adopted from previously characterized TuSp1 cDNA with residues of 1-161 from Genbank (AAY28947.1) and optimized with codon usage table for maximization of the expression. To amplify this cDNA, Polymerase Chain Reaction (PCR) with seven forward and seven reverse oligonucleotides were assembled into the eTuSp1 gene. After amplification, the cDNA fragment was subjected to gel extraction, ligation with plasmid vector (pUC18-SN) and transformation with E. coli. Restriction enzyme digestion and agarose gel electrophoresis were carried out to verify whether the cDNA was correctly inserted to the plasmid. Once validated, this modified plasmid was transferred to DNA sequence analysis. In the stage of protein expression, 200 mL lysogeny broth (LB) was added as 10% inoculum. At mid-log growing phase of A600= 0.6-1, 50 µg/mL ampicillin was added and protein was induced by 2 mL isopropyl-β-D-thiogalactoside (IPTG) at 37°C for 2 h. By using a Bandelin SONOPULS ultrasonic homogenizer, the bacterial cells were lysed under sonication for 20 min (10 s/10 s). Subsequently, the supernatant was obtained by centrifugation at 6000g for 5 min and then examined by Coomassie blue staining and immunoblotting. During purification, the recombinant protein was purified by immobilized metal ion affinity chromatography (IMAC) and Ni-NTA agarose was used via selective binding with attached His-Tag according to the manufacturer’s instructions. Finally, The recombinant protein was achieved at up to 91% purity and verified by SDS-PAGE


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with a molecular mass of 16 kDa (Figure S1). The fresh-made protein was then lyophilized and stored at –20 °C.

Figure 1. Schematic illustration of the gene recombinant for the eTuSp1 in E.coli.

Microfluidic Device. The microfluidic device was fabricated by the combination of glass and PDMS, as described elsewhere.20-21 Briefly, SU-8 was uniformly dispersed onto the clean wafer by using a spin-coating machine at 1200 rpm for 30 s. Then, SU-8 with the wafer was put in the oven for a pretreatment at 95 °C for 20 min. The channel patterns of the designed mask were transferred onto the SU-8 by photolithography method and SU-8 patterns were developed by the 1-Methoxy-2-propyl acetate. Subsequently, the PDMS slabs containing channels were prepared according to the manufacturer’s instructions. By oxygen plasma treatment, the PDMS and glass slide were quickly pressed together and then put in the oven at 120 °C for 2 h. PEEK tubes were attached to the punched holes. By mimicking shear gradients generated by the silk gland of


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silkworms,22 a microfluidic device was designed with an initial channel width decreased from 500 µm to the terminal width of 250 µm (Figure 2). Finally, the fabrication of silk fibers was carried out in this biomimetic microfluidic chip. Silk Fibers. The protein was dissolved in HFIP (1,1,1,3,3,3-Hexafluoro-2-propanol) with concentrations ranging from 0.01 to 0.3 g/mL. The spinning dopes and alcoholic solvents were separately extruded by syringe pumps into different channels of the microfluidic chip. By adjusting the concentration and relative flow rate ratios, silk fibers could be obtained. For improved mechanical properties of fibers, the post-spin drawing procedure was conducted to reel the silk fibers at a speed of 2 cm/s, 5 cm/s or 6 cm/s under a coagulation bath containing a mixture of water and methanol (90%). Dynamic Light Scattering (DLS). The size and polydispersity index of the eTuSp1 spheres was determined by DLS measurement conducted with Zetasizer Nano ZS. All samples were tested at 25 °C under the same concentration. The eTuSp1 was first dissolved in HFIP and adjusted to the concentration of 0.02 and 0.04 mg/mL. Finally, the result with the intensity as a function of size was obtained. Additionally, the eTuSp1 solution (0.04 mg/mL) was sonicated for 10 min and then transferred to quartz cuvette for measurement. The average size of eTuSp1 particles was calculated by the equation: = ∑ ⁄∑ . Thereof, D and I represent the size and intensity, respectively. Transmission Electron Microscopy (TEM). The diluted solution of HFIP-dissolved eTuSp1 was added dropwise to an electron microscopy specimen grid covered with a thin carbon support. TEM micrographs were recorded on a JEOL JEM-2100F operating at a voltage of 200 kV. Small-Angle and Wide-Angle X-ray Scattering. Synchrotron radiation experiments were


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carried out in SPring-8 (RIKEN, Hyogo, Japan). For small-angle X-ray scattering (SAXS) measurements at the BL45XU beamline having an undulator source (RIKEN Structure Biology I), the wavelength and size of the direct beam were 0.1 nm and 250 µm (V) × 300 µm (H). The distance between a sample and a detector (PILATUS 3×2M, DECTRIS Ltd.) was 2136 mm. A diluted eTuSp1 in a concentration of 0.02 mg/mL was kept in a capillary for SAXS measurement. For wide-angle X-ray scattering (WAXS) measurements, the BL44B2 beamline having a bending-magnet source (RIKEN Materials Science) was used. The eTuSp1 solution (0.25 g/mL) could be directly injected into the capillary for testing and a bundle of silk fibers made from the same solution were tightly stacked inside capillary for WAXS experiment. Intensity analysis for two-dimensional scattering patterns was made using the software package FIT2D after background intensity correction. Optical and Cross-polarized Microscopy. Silk fibers assembled in the microfluidic device can be directly moved to the widow for observation by using NIKON Eclipse E200. By changing a cross-polarized lens in the same microscopy, the ordered structure of silk fibers can be detected. Field Emission Scanning Electron Microscopy (FE-SEM). The surface morphology of silk spheres and fibers were determined using JEOL JSM-6335F. Silk spheres and fibers were coated with platinum/palladium for 90 s with an ion sputter. Attenuated Total Reflection Fourier Transformation Infrared Spectroscopy (ATR-FTIR). The secondary structure of silk spheres and fibers was recorded on Nicolet 5DXC. Before testing, the freshly prepared silk spheres and fibers were directly transferred to the oven with the set temperature of 80 °C. Upon evaporation of all the solvents, the dry spheres and fibers were put onto the crystal for measurement.


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Atomic Force Microscopy (AFM) Indentation. Bruker Nanoscope SPM8 with an ACTA-20 silicon probe was performed for the compressive modulus measurement of silk spheres. Determined by the thermal tune method, the pyramid-tipped cantilever had an average spring constant k of 8.90 ± 0.11 N/m. The force-distance curves were recorded by the indent mode under ambient conditions (20 °C and 50% relative humidity). Prior to use, silicon wafer was thoroughly cleaned, allowing the silk spheres to settle on its surface. Under the flow rate ratio of core/sheath as 0.25, silk spheres could be prepared by the microfluidic device in a concentration of 0.01 g/mL. Rinsed three times using DI water, silk spheres were placed on the clean silicon wafer. During measurement, cantilever was positioned over a single microsphere. By assuming a Poisson ratio of 0.5, compressive modulus of spheres was calculated using Hertz model. Mean values of the modulus were obtained by fitting 20 approaching indentation curves from different silk spheres. Tensile Testing. Silk fibers were allowed to dry in the air for 2 h and then mounted on a 20 × 30 mm cardstock frame. Prior to mechanical testing, the average diameter of each fiber was measured under the optical microscopy. Four measurements were performed per sample. The tensile-strain curve was recorded under ambient conditions on the Instron 5566 with a 10 N load cell at a rate of 10 mm/min.

3. RESULTS AND DISCUSSION It is generally accepted by the silk community that a highly ordered structure of protein molecules (liquid crystals23 or micelles24) exists in the spinning dope with good kinetic and thermodynamic stability. A typical micelle-based silk formation process is demonstrated in Figure 2.24 By employing isopropanol (IPA) as the sheath flow, the desolvation of spidroins from


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HFIP is controlled by microfluidics. In the following, we modularize the fiber assembly inspired from the natural spinning process.

Figure 2. Schematic of the silk fiber assembly by a biomimetic microfluidic device. A spider’s gland comprising tail, sac, duct and tapper is illustrated with corresponding function of protein secretion, micellization, induced aggregation and solidification. On the lower left, the recombinant protein eTuSp1 consists of approximately 60% hydrophobic and 40% hydrophilic moieties. On the lower right, a microfluidic chip is designed with two inlet channels and one outlet channel, where isopropanol is taken as the sheath flow and HFIP with spidroins dissolved is used as the core flow. The table inset shows the miscibility features of isopropanol (IPA), ethanol (EtOH) and methanol (MeOH) towards the HFIP and spidroins.


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Genetic construction of a conserved single repeat unit within eTuSp1. For the primary structure, spider silks can be regarded as amphiphilic multiblock copolymers with a repetitive core domain flanked by highly conserved non-repetitive N- and/or C-terminus. Garb and coworkers found that TuSp1 genes retained sequence conservation in a repeat unit with individual repeats readily aligned among species in a couple of million evolution.12 Thus, the eTuSp1 variant was made by recombinant technology in the absence of both terminus. Similar to eggcase silks, this truncated eTuSp1 is distinct from dragline silks due to low glycine (9%) and high serine (24%) contents. How the silk molecules exist in the solution and if they can direct the fiber assembly is not yet fully understood. By using Bioedit, the hydropathy behavior of eTuSp1 was plotted under the scan window size of 13 (Figure S2). Obviously, the total 167 aa were divided into distinguishing domains. The aa from 1 to 100 are attributed to the hydrophobic domain, while the remaining aa from 101 to 167 can be assigned to the hydrophilic domain. It is assumed that the coexistence of distinct hydrophobic and hydrophilic regions endows the silk molecules to form micelle-like structure in a polar solvent. Verification of micelle formation by combined techniques. So far, it remains ambiguous how the dope inside spider glands exists in such a high concentration but no aggregation observed. In spite of two theories established to explain this phenomenon, there is a consensus that spider silk proteins consist of relatively hydrophobic core flanked by hydrophilic N-/Cterminus.5 As previously reported,25 the TuSp1 containing either terminus formed micelle-like structure. According to another research,26 vesicle-like assemblies were observed only for the recombinant spider silk protein with C-terminus, but not for the one without C-terminus. All these studies imply the significance of distinct amphiphilic domains on the formation of micelles. Thereby, it is kinetically favorable for the eTuSp1 to assemble into micelles according to the


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hydropathy profile (Figure 2). To date, the prediction for the formation of micelles has been reported through the analysis of aa sequence and 3D structure. As earlier studied by David L. Kaplan’s group,24 natural silk proteins of spiders and insects enable fibroin molecules to adopt a micelle-like structure, which was predicted by the hydrophobic plot of the primary sequence. Upon exposure to aqueous solution or polar solvents, the more hydrophilic moiety formed the outer layer with the less hydrophilic one embedded as the core. However, the results were different in case of the 3D structure analysis of TuSp1 reported elsewhere.25 To be specific, the more hydrophobic component was considered as the sheath because of the hydrophobic interaction induced by the hydrophobic residues exposed to the protein surface. Notably, the repetitive sequence was not capable of forming micelles in the aqueous solution unless covalently linked with at least one terminal domain. As shown in Figure S3, Tyndall effect was observed for the eTuSp1 in HFIP, suggesting a potential micellar colloid system. To determine the eTuSp1 structure in HFIP, small angle X-ray scattering (SAXS) experiment was performed (Figure S4). The high intensity generated by this synchrotron radiation facility (SPring-8, Japan) enables the rapid and accurate measurement of the protein structure in terms of the molecular shape and radius of gyration (Rg).27 The eTuSp1 solution with a diluted concentration of 0.02 mg/mL was recorded for ten times with an interval of 0.1 s, and the average SAXS data is depicted in Figure 3A. Guinier law allows determining the Rg of a particle with unknown shape and size. The intensity of SAXS, I(q), for a particle is given by the equation (eq. 1) below:

= exp−

(1)

However, this law is valid only under condition: a, q is much smaller than 1/Rg; b, the system


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should be dilute and isotropic. Apparently, the scattering profile did not reach the Guiner range. This result bears a similarity to the formation of large-scale silk aggregates on other studies.28 It is hypothesized that spherical micelles are formed in HFIP, thus spheres model is provided here to fit the corresponding plot by the equation (eq. 2) below:29 Iq = ρ υ

!" # $

(2)

Noteworthily, the intensity profile of SAXS after background intensity correction wasn’t fitted perfectly with any sphere model of a protein particle. In a q range smaller than ca. 0.1 nm-1, however, the profile could be fitted well with a sphere model with the Rg of 26 nm. The slope of a double-logarithmic plot of I(q) vs q for protein particles was ca. -3.0 in a q range of ca. 0.1 ~ 0.2 nm-1. This indicates the shape of silk particles might be an ellipsoid that was between a perfect sphere and a perfect disk. It is found that the size of protein monomers with similar molecular weights normally ranges from 1 to 3 nm, which is much smaller than 26 nm measured in this study. To understand the prominent difference, DLS was utilized to verify the dimension of protein molecules under the same concentration. Comparable to the SAXS result, majority of silk particles were determined by DLS with a size of ca. 26.7 nm in radius (Figure 3B). We contribute the difference to the formation of soluble aggregates.


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Figure 3. Molecular structure and dimension of eTuSp1 in HFIP under the concentration of 0.02 mg/mL. A: Double-logarithmic plot of I(q) vs q for protein particles. The shape of silk particles might be an ellipsoid. The scattering intensity in the higher q range was relatively high due to insufficient background correction for the nontransparent solution sample. B: Particle size distribution examined by DLS. Majority of protein particles were 53.4 nm in diameter. As shown in Figure 4, the solution structure of eTuSp1 is further demonstrated with an increased protein concentration from 0.02 to 0.04 mg/mL. Accordingly, the average size calculated by the intensity was increased from 54.1 to 102.2 nm. This result was consistent with the relationship between micellar size and solution concentration. Sonication is an alternative to evaluate the hydrophobic interaction, which plays pivotal role on the study of protein molecular structures in the solution. As revealed by DLS, after sonication, the increased peak numbers and peak-shifting phenomenon became pronounced. This result indicates an enhanced hydrophobic interaction on octamers and soluble aggregates evidenced by the peaking shifting from 10 to 20 nm and from 100 to 200 nm, respectively. Similarly, soluble aggregates can be induced from increased hydrophobic interaction between silk micelles by the elevated temperature.25 Probably


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because of cavitation effect, a small amount of monomers with a size of ~1.3 nm was observed to separate from loosely packed micelles.

Figure 4. DLS plots of eTuSp1 with/without sonication. The concentration of eTuSp1 is 0.04 mg/mL. As confirmed by TEM, both silk micelles and aggregates were observed. Figure 5 shows the coalescence and aggregation of micelles, leading to the formation of larger micelles and micellar aggregates.


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Figure 5. TEM result of eTuSp1 micelles and aggregates. A, the coalescence of two micelles; B, aggregation of several micelles. Taken together, the formation of eTuSp1 micelles is verified by combined techniques of SAXS, DLS and TEM. The hydrophobic sequence pattern in eTuSp1 reveals an amphiphilic behaviour, initiating the hydrophilic component to form the outer layer of micelles, but leaving the hydrophobic one embedded inside as the inner core. It is noteworthy that serine and threonine residues are very abundant (accounting for ~30%) with significant contribution to the intermolecular hydrogen bonds. The micelle structure is therefore driven and stabilized both by hydrophobic interactions and hydrogen bonds. Silk micelles are crucial to stabilize spidroins in the solution and micellar aggregates are critical for the fiber assembly. Induced phase separation from micelles to fibers. The phase separation of eTuSp1 was explored by microfluidics to study the morphological and structural transformation. It is recognized that the fluids with low surface tension tend to generate high laminar shear. HFIP has


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a lower surface tension of 14.6 dynes/cm than water (72.8 dynes/cm). Therefore, eTuSp1 dissolved in HFIP generates reinforced laminar shear for the formation of silk fibers. Despite lacking ion- and pH-sensitive terminal switches, the desolvation of eTuSp1 is simplified by the non-solvent induced phase separation (NIPS).30 Specifically, three HFIP-miscible alcoholic solvents, IPA, EtOH and MeOH are studied as the sheath flow. Upon contact with the sample flow, the phase separation of eTuSp1 was induced because of limited miscibility between solvents and spidroins. IPA, usually used in other spinning system, was finally selected with optimized results. As unveiled by the micelle-theory for the fiber assembly, micelles can be driven by hydrophobic interactions to form silk globules along with an increased protein concentration in solutions.22 These globules can directly converted into silk spheres after a complete phase separation. Alternatively, silk globules interact with each other in a higher protein concentration to form globule aggregates, which are subsequently induced for the fiber assembly in the presence of shear stress and elongation. In conformity to this theory, spherical aggregates and the corresponding induced fibrillar assemblies were observed by the optical and crosss-polarized microscopy (Figure S5). As expected, spherical aggregates were isotropic but the fibrillar assemblies adopted ordered structure, mainly attributed to the structural transition from α-helix to β-sheet. The critical silk proteins for the fiber assembly varies from case to case due to a plenty of factors, including the silk types, spinning methods, solvent systems, and molecular weights. Compared with the spinning dope of spiders, silk protein concentration for artificial silk fibers has been reported in a broad range.31 As exhibited in Figure 6, protein concentrations and flow rate ratios are exploited for diverse silk morphologies. Higher than most microfluidic systems,


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the critical protein concentration for silk fiber formation is 0.18~0.20 g/mL, predominantly due to low molecular weights of eTuSp1. Below this critical concentration, silk spheres can be obtained. Here, the shear strength is practically mediated by the relative flow rate. Even under an enhanced shear stress, only silk aggregates can be observed. Above the critical concentration, it is easier to form silk fibers under higher shear strength. Thus, silk spheres can be solely formed upon exposure to IPA without shear, whereas a high protein concentration is essential for the formation of silk aggregates that are favorable for the fiber assembly in the presence of shear stress. To detect the surface structure by FE-SEM, silk spheres (Figure 6A) are dense and compact, free of substructures. While, silk fibers (Figure 6C) obtain a grainy structure, which is distinct to the smooth surface of natural spider silks. We assume that the interaction between silk aggregates (Figure 6B) and the following turbulent flow make considerable contribution to the texture of silk fibers. By changing flow rate ratios, different silk fibrillar morphology can be obtained (Figure 6D).

Figure 6. Various morphologies of eTuSp1 under controlled by protein concentrations and flow rate ratios. The flow rate ratio of core/sheath is set up from 0.125 to 1 at a fixed core flow rate of


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0.8 mL/h. Zebra region refers to the transition zone. A) eTuSp1 spheres and fibers can be prepared by different combinations of sample concentrations and the flow rate ratio of core/sheath. The FE-SEM image of silk proteins in form of: A, spheres; B, spherical aggregates; C, semi-cylindrical fiber; D, flat fiber. A bending magnet beamline BL44B2 (Figure S6) is used to investigate the crystalline structure of eTuSp1 fibers. The conformation of silk solutions is normally studied by circular dichroism (CD) spectrum but not X-ray diffraction. In this work, eTuSp1 dissolved in HFIP can be directly transferred to a glass capillary for measurement through synchrotron radiation WAXS facility.32 The samples kept rotating so that the detector could collect all structural information within several minutes. Silk proteins have been reported to obtain three crystalline structures.33 Silk I occurs in glands with a metastable state between α-helix and β-sheet and Silk II in spun state obtains β-sheet structure. While, Silk III forms at the air/water interface with helical structure. Typically, the diffraction peak of Silk I is mainly present at 2θ= 12.2 °C and 28.2 °C in correspondence with Silk II present at 2θ= 18.9 °C and 20.7 °C.34 As shown in Figure 7, a sharp peak at 2θ= 12.9 °C and relatively weak peak at 2θ= 28.2 °C were assigned to Silk I, while another strong peak at 2θ= 17.7 °C was attributed to Silk II. This result indicates the structural transition of β-sheet formed from soluble proteins to solid fibers, resembling the natural spinning process.


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Figure 7. WAXS data of eTuSp1 in HFIP and eTuSp1 fibers by using the synchrotron radiation facility of the BL44B2 beamline.


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Figure 8. ATR-FTIR spectrum of eTuSp1 spheres and fibers. The dashed black line and solid grey line represent silk spheres and silk fibers, respectively. The red lines are corresponded to βsheet, whereas the orange lines are attributed to α-helix/random coil and the green line represents the β-turn. The secondary structure largely lies behind the properties and potential applications of silk-1

based materials, even in any morphology. Within amide-I band (1600 to 1700 cm ) of FTIR spectra, the peak for silk fibers was detected with a shift to lower wavenumbers compared with silk spheres, indicating the shear-induced structural conversion from α-helix to β-sheet (Figure 8). In line with the results in Figure S5, silk spheres predominantly retained the helical -1

conformation (Figure S7) with the peak centering 1642 cm . However, significant difference was found for the chemically induced phase separation by addition of the kosmotropic salts,35 where the obtained silk spheres possessed a β-sheet-rich structure. For fiber assembly, no clear requirement was made for the spheres with certain conformation. Distinct form spheres, silk -1

fibers at 1631 cm were assembled under the physically activated phase separation with the βsheet content of ~44% (Table 1), which is quite comparable to the natural spider eggcase silks (~46%).36

Table 1. Distribution of secondary structure elements in eTuSp1 spheres and fibers as determined by deconvolution of the amide I bands after FTIR-spectroscopy.

α-helix

Random Coil

β-turn

β-sheet

eTuSp1 Spheres

19.8

24.4

20.1

35.7

eTuSp1 Fibers

17.0

24.1

15.3

43.6


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Further solidification of silk fibers by post-spin drawing. The deformation of individual eTuSp1 spheres was measured with force-displacement curve by AFM. According to the Hertz model,37 silk spheres were determined with a modulus of 3.95 ± 1.98 MPa under an assumption of the Poisson ratio of 0.5 (Figure 9A). Taken into consideration cocoon silk spheres with a reported modulus of 1.46 ± 0.75 KPa,38 silk spheres could be classified as tough materials. Intriguingly, the asymptotic fitting of modulus in each indentation depth may reveal a diffusion gradient of protein molecules in the formation of silk spheres. As for silk fibers, previous studies demonstrated that the mechanical features of artificial fibers could be substantially improved after post-spin drawing.39-40 With the said post-treatment, better orientation of protein molecules and higher proportion of the β-sheet crystalline structure can be induced.41 By building a custom-made device, it is of good feasibility in precisely controlling the post-spin procedure at different drawing ratios. As shown in Figure 9B, tensile strength and the strain were found to exhibit opposite results with regard to the increase of drawing ratios. The eTuSp1 fibers had maximum strain of ~ 40% and tensile strength of 78.3 MPa at the ratio of 1.0× and 3.0×, respectively. As expected, they were not superior enough in comparison to natural spider eggcase silks (Table 2). We attribute this mechanical feature to the low molecular weight and the absence of terminal domains. As studied, the C-terminal domain revealed its function by an increased extensibility and toughness of the TuSp1 fiber when compared to the one lacking C-terminus.26 However, it becomes quite reasonable for eTuSp1 to have remarkably higher elastic modulus in form of fibers (1.80 ± 0.88 GPa) than spheres.


22

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Figure 9. Mechanical properties of eTuSp1 spheres and fibers. A) Compressive modulus of silk spheres is measured by AFM indentation. Both of the force and modulus data are well fitted by linear and asymptotic equations; B) Tensile modulus of silk fibers is tested under various postspin draw ratios.

Table 2. Mechanical data from eTuSp1 fibers and natural eggcase silks.

Post-drawing ratio

Young’s modulus

Ultimate tensile strength (MPa)

Ultimate strain (%)

Toughness (MJ/m3)

(GPa)

eTuSp1 Fibers

1.0x

0.9±0.5

21.9±8.1

40.4±5.2

6.1±1.3

2.5x

1.9±0.3

66.4±6.7

25.8±2.1

13.6±2.1

3.0x

2.4±0.4

78.3±4.2

17.5±1.6

11.5±0.8

–

6.0±0.7

225.5±44.5

63.5±20.5

–

Natural Eggcase Silks*

*Data are obtained from reference 36. In summary, we have present modular assembly of a recombinant conserved repeat unit from spider eggcase silks in a biomimetic microfluidic system. The micellar structure of eTuSp1 was predicted according to its amphiphilic characteristic and subsequently verified by the


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Page 24 of 30

synchrotron radiation SAXS, DLS and TEM. Without N- and C-terminus, phase separation of the eTuSp1 in HFIP is driven by addition of IPA in combination with the shear and elongation. Silk spheres could be easily obtained, however silk fibers can only be prepared above a critical protein concentration in the presence of physical stress. Similar to previous studies, silk aggregates were considered as significant intermediates for the fiber assembly. During spinning process from soluble proteins to solid fibers, micelle-sphere-fiber structure transition was evaluated by WAXS and FTIR. The mechanical properties of silk spheres and fibers were demonstrated by the AFM indentation and tensile testing, respectively. The facile fabrication of eTuSp1 in the microfluidic device provides explicit strategies to further investigate fiber assembly mechanism and establishes a fascinating platform for preparation of artificial silk fibers in a modular assembly mode. ASSOCIATED CONTENT Supporting Information The following files are available free of charge. SDS-PAGE results and Hydropathy plot of the purified eTuSp1, Tyndall effect of protein solution, synchrotron radiation SAXS and WAXS facilities, optical and cross-polarized microscopy of silk aggregates and fibers, as well as the conformational elements after deconvoltion of ATR-FTIR results (PDF) AUTHOR INFORMATION Notes The authors declare no competing financial interest. ACKNOWLEDGMENT


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This work is supported by the National Natural Science Foundation of China (51373147), Science and Technology Planed Project of Guangdong Province (2016A050503013) and Research Grants Council, University Grants Committee (PolyU 5158/13E). The synchrotron radiation experiments were performed at BL45XU and BL44B2 in SPring-8 with the approval of RIKEN (Proposal No. 20170094) and supported by Dr. Hiroyasu Masunaga of Japan Synchrotron Radiation Research Institute (JASRI). REFERENCES 1.

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core fibers: trimeric complexes assembled from TuSp1, ECP-1, and ECP-2. Biochemistry 2006, 45, 3506–3516. 3.

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For Table of Content Use Only

Manuscript title: Modular Assembly of a Conserved Repetitive Sequence in the Spider Eggcase Silk: from Gene to Fiber Authors: Jianming Chen, Jinlian Hu,* Sono Sasaki, and Kensuke Naka


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Modular Assembly of a Conserved Repetitive Sequence in the Spider

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