Unconventional Spidroin Assemblies in Aqueous Dope for Spinning Into

pubs.acs.org/journal/abseba Article

Unconventional Spidroin Assemblies in Aqueous Dope for Spinning into Tough Synthetic Fibers § § Chun-Fei Hu, Zhi-Gang Qian, Qingfa Peng, Yaopeng Zhang, and Xiao-Xia Xia*

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ABSTRACT: Spider dragline silk is a remarkable fiber made by spiders from an aqueous solution of spidroins, and this feat is largely attributed to the tripartite domain architecture of the silk proteins leading to the hierarchical assembly at the nano- and microscales. Although individual amino- and carboxy-terminal domains have been proposed to relate to silk protein assembly, their tentative synergizing roles in recombinant spidroin storage and spinning into synthetic fibers remain elusive. Here, we show biosynthesis and self-assembly of a mimic spidroin composed of amino- and carboxy-terminal domains bracketing 16 consensus repeats of the core region from spider Trichonephila clavipes. The presence of both termini was found essential for self-assembly of the mimic spidroin termed N16C into fibril-like (rather than canonical micellar) nanostructures in concentrated aqueous dope and ordered alignment of these nanofibrils upon extrusion into an acidic coagulation bath. This ultimately led to continuous, macroscopic fibers with a tensile fracture toughness of 100.9 ± 13.2 MJ m−3, which is comparable to that of their natural counterparts. We also found that the recombinant proteins lacking one or both termini were unable to similarly preassemble into fibrillar nanostructures in dopes and thus yielded inferior fiber properties. This work thereby highlights the synergizing role of terminal domains in the storage and processing of recombinant analogues into tough synthetic fibers. KEYWORDS: spider silk, terminal domains, tough fibers, microfluidic spinning, biosynthesis

■ INTRODUCTION highly conserved terminal domains, which are composed of approximately 100 amino acids, both show a five-helix bundle Spider dragline silk exhibits extraordinary toughness that 16,17 combines high tensile strength and extensibility, thus out- structure in solution. They have been proposed to play fi 1−3 important roles in stabilizing the concentrated spidroins during competing all other natural or synthetic bers. The 18,19 remarkable silk is spun by spiders from an aqueous solution storage in the silk gland and also triggering spidroin transformation into macroscopic fibers.20,21 Growth evidence Downloaded via SHANGHAI JIAO TONG UNIV on July 15, 2021 at 06:52:53 (UTC). of the constituent proteins, termed spidroins, and this feat is largely attributed to the underlying architecture of the from extensive studies shows that structural conversions of the

See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. spidroins enabling their ordered assembly into insoluble terminal domains are smartly induced by external chemical and fi 4−6 mechanical triggers that occur through the spinning duct of the bers. The two protein components, major ampullate 22,23 7,8 spiders. For example, the correctly folded, dimeric state of spidroins 1 (MaSp1) and 2 (MaSp2), share a common CTD during storage is turned into a molten globulelike state tripartite architecture of a nonrepetitive N-terminal domain upon acidification14 and ion exchange of sodium and chloride (NTD), a long repetitive core region, and a nonrepetitive C- 9 ions by potassium and phosphate ions within the spinning terminal domain (CTD). The core region comprises up to duct.17 This partially unfolded state of CTD further mediates 100 tandem repeats, each consisting of 40−200 amino acids 10 the shear-induced alignment of repetitive segments into β- with a high content of polyalanine- and glycine-rich motifs. sheet structures.21 Meanwhile, acidification and depletion of In the process for spinning of soluble spidroins into fibers, the sodium chloride induce conformational changes in NTD polyalanine motifs of the core region tend to convert into β- sheet crystallites, responsible for the strength of spider dragline silk,11,12 whereas the glycine-rich motifs form random coil/ Received: April 12, 2021 helical amorphous regions responsible for the extensibility of Accepted: July 2, 2021 the fibers.5 The structure and function of the nonrepetitive terminal domains of spidroins have gained increasing attention in the − last decade.13 15 Fundamental structural studies reveal that the

© XXXX The Authors. Published by American Chemical Society https://doi.org/10.1021/acsbiomaterials.1c00492 A ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX ACS Biomaterials Science & Engineering pubs.acs.org/journal/abseba Article accompanied by antiparallel dimerization to lock the spidroin imidazole were obtained from Sangon Biotech (Shanghai, China). into large networks,16 whereasthisdomainexistsina Phusion high-fidelity DNA polymerase, restriction endonucleases, monomeric state to inhibit premature aggregation of the antarctic phosphatase, and T4 DNA ligase were purchased from New spidroin during storage.20 Although much has been done to England Biolabs (Ipswich, MA). Chemically competent cells of E. coli DH5α and BL21(DE3), TIANprep Mini Plasmid Kit, and TIANgel explore the function of terminal domains, their roles have been Midi Purification Kit were obtained from TIANGEN Biotech mostly discovered from studies on recombinant proteins (Beijing, China). Ni-NTA agarose (Catalog No. 30230) and dialysis containing individual terminal domains or their fusions to a tubing with 3.5 kDa molecular weight cutoff were purchased from short repeat region (e.g., refs13−20), markedly different from Qiagen (Hilden, Germany) and Spectrum Laboratories (Phoenix, native spidroins with large molecular weights and the presence AZ), respectively. Amicon Ultra-0.5 mL centrifugal filters with of both terminal domains. Ultracel-3K membranes were obtained from Millipore (Billerica, To make artificial spider silk fibers with the advantageous MA). mechanical properties of their natural counterparts, it is highly Construction of Expression Plasmids. For recombinant desirable to synthesize recombinant spidroins with nativelike expression of 16 repeats of the consensus sequence architecture and solubility for subsequent spinning into solid (GRGGLGGQGAGAAAAAGGAGQGGYGGLGSQG) of spider T. fi 24−31 clavipes MaSp1, plasmid pET19b-MaSpI16 was constructed as bers. For example, Andersson et al. designed a synthetic described earlier.32 To express 16 consensus repeats N-terminally spidroin composed of an NTD and a short repetitive region fused to the CTD of T. clavipes MaSp1 (GenBank accession no. from spider Euprosthenops australis and a CTD from spider AAC38957.1), the CTD-encoding fragment was first liberated from 28 Araneus ventricosus (NT2RepCT). As a constitutive dimer plasmid pUC57-MaSpIC33 with restriction enzymes NheI and SpeI, with a molecular mass of 66 kDa, NT2RepCT could be agarose gel-purified, and then ligated with SpeIandalkaline concentrated to >500 mg mL−1 in 20 mM Tris−HCl buffer phosphatase treated-plasmid pET19b-MaSpI16, leading to plasmid fi pET19b-I16C. Plasmid pET19b-NcCT was constructed as described (pH 8.0) and spun into continuous bers with a toughness of 33 45 ± 7MJm−3 when the concentrated dope was extruded into previously, which permits recombinant expression of the CTD of T. a collection bath consisting of 500 mM sodium acetate buffer clavipes MaSp1 under the strong T7 promoter. 28 fi A synthetic gene encoding NTD of T. clavipes MaSp1 (GenBank and 200 mM NaCl (pH 5.0). However, the speci c impacts accession no. ACF19411.1) was synthesized and delivered on plasmid of individual terminal domains on the minispidroin assembly fi pUC57-MaSp1-Nterm from GenScript (Nanjing, China). The DNA and the mechanical properties of resulting bers remain to be fragment encoding mature NTD (without the secretion leader 29 explored. In another study, a recombinant spidroin with 12 peptide) was then PCR-amplified from pUC57-MaSp1-Nterm with consensus sequences of a MaSp2 protein and both terminal primers FmNNhe (5′-AATGCTAGCCAGAACACCCCGTGGAG- domains was dialyzed against 30−50 mM sodium phosphate CAG-3′ )andRmNSpe(5′ -CATACTAGTGCT- buffer (pH 7.2) to induce liquid−liquid phase separation CACTTCGTTCGCGC-3′). The amplified DNA was digested with fi (dependent on the CTD). The resulting high-density micellar enzymes NheI and SpeI, agarose gel-puri ed, and ligated with the NheI phase serving as an aqueous dope could be wet-spun and and alkaline phosphatase treated-plasmid pET19b-MaSpI16. The poststretched in 90% isopropanol baths (pH 7.7) into fibers resulting plasmid pET19b-mNI16 permitted expression of the 16 − repeats C-terminally fused to the mature NTD. Similarly, the with a toughness of 189 ± 33 MJ m 3, which even slightly fi restricted DNA was ligated into the NheI site of plasmid pET19b- exceeds the toughness (on average) of natural bers from A. I16C to construct plasmid pET19b-mNI16C, which encodes a −3 diadematus spiders (167 ± 65 MJ m ). However, the biomimetic spidroin with 16 consensus repeats flanked by both NTD “locking” role of NTD was presumably unrealized because and CTD. The orientation of each insertion was verified by double the NTD only dimerizes at acidic pH.29 To the best of our digestion with SpeI and NheI. For recombinant expression of the knowledge, the synergizing roles of terminal domains in the NTD alone, plasmid pET19b-NcNT was constructed by amplifying assembly of recombinant spidroin into synthetic fibers remain the DNA fragment with primers FmNNde (5′-CATCATATGCA- GAACACCCCGTGGAGCAG-3′)andRmNXho(5′-AATCTC- elusive. ′ Here, we determine the synergizing roles of terminal GAGGCTCACTTCGTTCGCGCTG-3 )forcloningintothe NdeI−XhoI site of plasmid pET19b (Novagen, Madison, WI). The domains in guiding a spidroin mimic to preassemble in fi fi nucleotide sequences of the plasmids containing PCR-ampli ed concentrated aqueous solutions and to coagulate for ber fragments were confirmed by dideoxy sequencing. formation. Based on the major dragline silk (MaSp1) Recombinant Protein Expression and Purification. Chemi- component of the golden orb-web spider Trichonephila clavipes cally competent cells of E. coli BL21(DE3) were transformed with (formerly Nephila clavipes), four spidroin variants were first each of the intended plasmids and plated onto selective Luria−Bertani designed to contain the same core region of 16 consensus (LB) agar plates. The desirable transformants were maintained as 15% repeats, yet differing in the presence of either one or both (v/v) glycerol stocks at −80 °C. Recombinant protein expression was terminal domains. Next, these four variants were biosynthe- performed with fed-batch cultures in a 5 L jar fermentor (Biotech- 5JG-7000; BaoXing Bio-Engineering Equipment, Shanghai, China) as sized, characterized by a combination of spectroscopic and 34 fi described previously. When the cell optical density at 600 nm microscopic analyses, and spun into bers in parallel. ∼ fi (OD600) reached 60, the culture temperature was downshifted to 16 Strikingly, the biomimetic spidroin self-assembled into bril- °C, and IPTG was added at 1 mM for inducing spidroin biosynthesis. like nanostructures in concentrated aqueous dope (other than After overnight induction, the bacterial cells were harvested by 24,25,28,29 conventional micelles ) for processing into artificial centrifugation at 7024g for 25 min at 4 °C in an Avanti J-E centrifuge fibers with nativelike toughness, whereas the variants lacking (rotor JA-10; Beckman Coulter, Inc.). The cell pellets were then one or both termini were unable to similarly preassemble and resuspended in a buffer containing 5 mM Tris−HCl (pH 8.0), 150 yield tough synthetic fibers. mM NaCl, and 5 mM imidazole. Following high-pressure homogenization and centrifugation in an Eppendorf 5804R centrifuge ° ■ MATERIALS AND METHODS with an F-34-6-38 rotor (14 866g,4 C for 20 min), the supernatant of the cell lysate was loaded onto a prepacked column with 25 mL of Chemicals and Materials. Ampicillin, isopropyl-β-D-thiogalacto- Ni-NTA agarose resin for affinity purification. The column was eluted side (IPTG), 5× protein loading buffer (Catalog No. C508320), 5× with the above buffer supplemented with 250 mM imidazole. The nonreducing protein loading buffer (Catalog No. C516031), and eluted proteins of interest were successively dialyzed at 4 °C against

B https://doi.org/10.1021/acsbiomaterials.1c00492 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX ACS Biomaterials Science & Engineering pubs.acs.org/journal/abseba Article buffers containing 10 mM phosphate buffer (pH 7.4), 0.5 M urea, and KOH). The resulting solutions were cast on mica surfaces to allow NaCl at decreasing concentrations. The concentration of NaCl in the adsorption for 5 min, flushed with deionized water, and then dried buffers was decreased from 200 to 100, 80, and 40 mM consecutively. overnight at room temperature. The AFM images were collected at a Upon centrifugation (14 866g,4°C for 20 min), the resulting scan rate of 1.72 Hz and a scanning window of 600 nm and processed supernatants that contained the purified spidroins (∼2mgmL−1) with NanoNavi II analysis software (SII NanoTechnology Inc.). were used for various characterizations (described below). Alter- Fiber Spinning. Amicrofluidic spinning apparatus with a natively, these protein solutions were concentrated into spinning progressively narrowing fluidic channel was used for fiber spinning, dopes (∼200 mg mL−1) using Amicon Ultra-0.5 mL centrifugal filter which mimics the shape of spider’s major ampullate gland. The width units with Ultracel-3K membranes. of the channel decreased from an initial 2590 μm to the terminal 100 The protein concentrations were determined with a Pierce BCA μm (see the schematic diagram in the Results and Discussion Protein Assay kit (Thermo Scientific, Rockford, IL). The purity of the section). Each spinning dope was injected into the channel at a rate of − proteins was analyzed via 10% SDS-PAGE by preparing the samples 5 μL min 1 and extruded into one of two coagulation baths at room in 1× Laemmli sample buffer, which contained 1% (v/v) 2- temperature. The bath solutions (1 L) contained 90% ethanol and mercaptoethanol. Protein samples were also analyzed under non- 17.5 mM acetic acid with pH of 5.0 or adjusted to pH 7.4 using 1 M reducing conditions by the loading buffer without 2-mercaptoethanol. KOH. The resulting as-spun fibers were collected by reeling on a The gels were stained with Coomassie Brilliant Blue R-250 and rotating cylinder in the air, cut into 20 mm lengths and then imaged using a Microtek Bio-5000plus densitometer (Shanghai, immersed in 80% aqueous ethanol solution for 1 h. For drawing, one China). The molecular weights of the purified proteins were end of each as-spun fiber was fixed and the other end stretched to five − determined by a Bruker UltrafleXtreme MALDI-TOF mass times the original length continuously at a rate of 1 mm s 1. The spectrometer (Bruker Daltonics, Bremen, Germany). postdrawn fibers were kept in the ethanol solution for 2 h and then Circular Dichroism (CD). CD spectra were collected using a 1 dried in the air for 1 h under tension to maintain the extended length. mm path-length cuvette in a Jasco J-815 spectropolarimeter equipped Scanning Electron Microscopy (SEM). The cross section and with a Peltier temperature controller (Tokyo, Japan). Before analyses, surface of the fibers were observed on a model S-3400N scanning the protein samples were diluted in the final dialysis buffer containing electron microscope (Hitachi, Tokyo, Japan). Prior to SEM analyses, 10 mM phosphate buffer (pH 7.4), 0.5 M urea, and 40 mM NaCl to a the fibers were fractured in liquid nitrogen and coated with gold using concentration of 0.2 mg mL−1. The spectra were acquired at 190−260 a Leica EM SCD050 sputtering device with a water-cooled sputter nm with a bandwidth of 1 nm, speed of 100 nm min−1, and resolution head (Leica Microsystems GmbH, Vienna, Austria). The cross- of 0.5 nm at 25 °C. Three scans were averaged, and smoothing was sectional area (CSA) of the fiber specimens was imaged by ImageJ applied using OriginPro 9.0 software (OriginLab Corp., North- software version 1.8.0_112, which is freely available at website ampton, MA). The CD data were reported as mean residue ellipticity https://imagej.nih.gov/ij/. For each type of the fiber, the fiber ([θ], deg cm2 dmol−1).35 diameter was calculated from CSA and presented as an average value Tryptophan Fluorescence. Tryptophan fluorescence spectra from five independent measurements along the axis of the fiber. were acquired on an FLS1000 fluorescence spectrometer (Edinburgh Mechanical Testing. The fibers were tensile tested at 25 °C and Instruments, Scotland). Before analyses, the spidroin dope solutions 60% relative humidity on an Instron 5944 testing machine with a 10 ff N load cell (Instron Corporation, Norwood, MA). The gauge length were diluted with bu ers that contained 0.5 M urea, 40 mM NaCl, − and 10 mM phosphate buffer at pH 7.4 or 5.0. The resulting samples was 10 mm and the stretching rate was 2 mm min 1. The engineering with a protein concentration of 2 mg mL−1 were loaded into the stress was defined as the ratio of the instantaneous force and the cuvettes and incubated at 25 °C for 10 min. Emission spectra were original CSA of the fiber, and the engineering strain was the ratio of obtained at a wavelength range from 300 to 400 nm at an excitation the change in length and the original length. The resulting engineering wavelength of 295 nm. stress−strain curves were then used to calculate mechanical data of Dynamic Light Scattering (DLS). DLS was performed on a the fibers with Bluehill Universal software (Instron). Young’s modulus Zetasizer Nano S system equipped with a temperature controller of each fiber was obtained by measuring the slope of the initial elastic (Malvern Instruments, Worcestershire, U.K.). Before analyses, the region of the stress−strain curve, and the toughness was calculated as spidroins with varying concentrations (2−200 mg mL−1) in the the area under the stress−strain curve before breakage. Data are phosphate buffer (10 mM, pH 7.4) containing 0.5 M urea and 40 mM shown as means ± standard deviation (n = 10). Differences were NaCl were loaded into 10 mm path-length cuvettes and stabilized at considered statistically significant at P < 0.05 by one-way analysis of 25 °C for 10 min. Intensity distribution data were acquired from variance. triplicates and analyzed using Zetasizer version 7.04 software Raman Spectromicroscopy. For Raman measurements, the (Malvern Instruments). fibers were gently mounted on glass microscope slides with double- Atomic Force Microscopy (AFM). Two modes of AFM analyses sided tape. The spectra were acquired on an inVia Qontor confocal were performed. In one setup, a Dimension FastScan atomic force Raman spectrometer (Renishaw, Gloucestershire, U.K.) coupled to a microscope (Bruker, Germany) was operated in the liquid peak force Leica DM 2700 M microscope (Leica Microsystems, Wetzlar, mode for imaging the adsorbed protein assemblies at the mica/liquid Germany). The samples were irradiated with a 785 nm edge laser −1 interface. To prepare the specimens, the spidroin samples were with a grating of 1200 lines mm . diluted with the final dialysis buffer to a protein concentration of 0.2 − mg mL 1, and 90 μL of each diluted solution was cast on mica ■ RESULTS AND DISCUSSION surfaces to allow adsorption for 10 min at room temperature. The protein deposited micas were then gently rinsed with 90 μLoffinal Design and Biosynthesis of Recombinant Spidroins. dialysis buffer. The AFM images were subsequently collected with a To study the tentative synergizing roles of terminal domains, scan rate of 1 Hz and a scanning window of 1 μm and processed with we designed a silk mimic composed of an NTD and a CTD Nanoscope analysis v1.8 software (Bruker). In another setup, a bracketing 16 consensus repeats of the core region from spider Nanonavi E-Sweep microscope (SII NanoTechnology Inc., Tokyo, T. clavipes (N16C; Figure 1a). Three derivatives lacking either Japan) was employed in the taping mode to characterize the protein the CTD (N16), NTD (16C), or both terminal domains (I16) assemblies during dehydration. To prepare the specimens, the were also designed to serve as controls. These four spidroins spidroin dopes were first diluted with the final dialysis buffer to 2 mg mL−1 usinganEppendorfResearchpluspipette.These were recombinantly produced by microbial biosynthesis in bacterium E. coli and then purified using immobilized-metal semidiluted solutions were then pipetted or extruded on a ffi microfluidic apparatus (also used for fiber spinning below) for a nity chromatography (Figure 1b). The protein eluents from dilution to 0.2 mg mL−1 with 90% aqueous ethanol solutions the affinity column, which contained imidazole, were initially containing 17.5 mM acetic acid at pH 5.0 or 7.4 (adjusted with 1 M dialyzed against a range of phosphate buffers with low levels of

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Materials and Methods section. The yields of the purified I16, N16, 16C, and N16C were approximately 1.1, 1.4, 0.8, and 1.2 gL−1 of bacterial culture. Remarkably, the purified spidroins in the final dialysis buffer (10 mM phosphate, 0.5 M urea, 40 mM NaCl, pH 7.4) could be concentrated into 200 mg mL−1 dope solutions without obvious precipitation. Denaturing protein electrophoresis revealed that all of the purified spidroins had purity greater than 95% (Figure 1c), and the identities of these spidroins were further verified by mass spectrometry (Figure S1). The reducing SDS-PAGE (boiling with reducing agent) also revealed a monomeric state for each of the four spidroins. This monomeric state was also observed for I16 and N16 under the nonreducing condition, whereas the CTD- containing spidroins (16C and N16C) mainly existed as a dimer without the prior reduction to break disulfide bonds (Figure 1c). These results implied that both N16C and 16C fi Figure 1. Schematic overview of the biomimetic design and were able to homodimerize through disul de bonding when recombinant biosynthesis of spidroins for fiber spinning. (a) Scheme stored in aqueous solutions without the addition of a of the recombinant spidroins. The amino acid sequences of the reductant. consensus repeat of the core domain and the amino- and carboxy- Characterization of Spidroins in Aqueous Dopes and terminal domains (NTD and CTD) are derived from major ampullate upon Coagulation. Then, we characterized the spidroins in spidroin 1 of spider T. clavipes. (b) Illustration of spidroin the concentrated aqueous dopes with a combination of biosynthesis, purification, and concentration into dope solutions. fi spectroscopic and microscopic methods. We started by (c) Coomassie-stained 10% SDS-PAGE gel analysis of the puri ed investigating the secondary structures of the spidroins at the spidroins under reducing and nonreducing conditions. 2-Mercaptoe- thanol (a reducing agent) is omitted for protein sample preparation neutral pH by far-UV circular dichroism (CD) spectroscopy under the nonreducing condition, and thus, the cysteine-containing (Figure 2a). As expected, the control spidroin with consensus spidroins (16C and N16C) were predominantly oxidized into dimers repeats only (I16) showed a typical random coil structure, and (indicated by solid arrows), the monomers of which were indicated by recombinant proteins of the NTD and CTD alone showed a dotted arrows. typical α-helical pattern with pronounced double minima at 208 and 222 nm (Figure S2). The typical α-helical secondary NaCl. The motivation to decrease NaCl concentration in the structures were also observed for both N16C and N16 in the dialysis buffer is triggered by the fact that even a moderate level aqueous dopes containing 0.5 M urea. The 16C variant of the salt (150 mM) would adversely affect fiber formation essentially showed spectra of an α-helical pattern yet with a from a concentrated aqueous solution of the I16 protein.36 slight blue shift of the 208 nm minimum to 206 nm, which However, a substantial proportion of the spidroins precipitated indicated the formation of type I β-turns or disordered during dialysis against the phosphate buffer with 40 mM NaCl, structures.33 This reflected partial unfolding of the native five- possibly due to the formation of insoluble protein aggregates. helix bundle structure of CTD17 upon fusion with the 16 This result reflected aggregation-prone characteristics of the consensus repeats, a phenomenon also observed upon heat or four recombinant spidroins. low pH treatment of a recombinant construct with CTD To tackle the daunting obstacle known for decades in only.14,33 In contrast, the native five-helix bundle structure of handling recombinant spidroins, we included 0.5 M urea (a NTD16 was retained upon fusion with the 16 consensus hydrogen-bonding disruptor) into the dialysis buffer to repeats, reflecting the highly soluble characteristic of NTD that minimize spidroin aggregation while maintaining the expected has recently been proposed to function as a “solubilizer” at the secondary structures for the silk mimic in a soluble state. physiological pH.22,37 Indeed, N16C and the other three variants could be facilely We also studied whether the secondary structures of the prepared in a sequential dialysis process as described in the recombinant spidroins changed in response to pH decrease

Figure 2. Characterization of spidroins. (a) Far-UV CD spectra of various spidroins (0.2 mg mL−1) at pH 7.4. (b) Tryptophan fluorescence of the spidroins (2 mg mL−1). Before analyses, the spidroin dope solutions were diluted with buffers that contained 0.5 M urea, 40 mM NaCl, and 10 mM phosphate buffer at pH 7.4 or 5.0.

D https://doi.org/10.1021/acsbiomaterials.1c00492 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX ACS Biomaterials Science & Engineering pubs.acs.org/journal/abseba Article that occurs in the spinning duct. CD measurements revealed that the α-helical secondary structures of the individual terminal domains of spider T. clavipes showed no significant changes when the pH was downshifted to 5.0 (Figure S2). In addition, the pH acidification did not change the respective secondary structures of the four spidroins (Figure S3). These results were not surprising and coincided with previous reports that the CD spectra of recombinant MaSp1 NTD of spider E. australis did not show significant changes in the pH interval 7− 3 (ref 16) and that recombinant MaSp1 CTD of T. clavipes well retained its α-helical structure from pH 7.4 to 5.0 (ref 14). Next, we examined the conformational change of biomimetic spidroin in response to the pH decrease. As previously proposed, NTD functions as a pH sensor that undergoes a conformation change in response to duct acidification.16,22,37 Such conformational change can be facilely analyzed exploiting the single tryptophan residue in NTD (absent in the repetitive core and CTD) as a fluorescent probe. When excited with light at 295 nm, the fluorescence emission of N16C dramatically decreased in intensity when the solution pH was downshifted from 7.4 to 5.0, and such fluorescence change was also observed in N16 but not in the two variants lacking the NTD (Figure 2b). The decrease in fluorescence intensity was because the Trp residue became more solvent-exposed at the Figure 3. Dynamic light scattering (DLS) size distribution profiles of lower pH, which is likely associated with altered molecular the spidroins in (a) semidilute and (b) concentrated dope solutions. − contacts between monomer units that strengthen their The semidilute solutions with proteins at approximately 2 mg mL 1 interaction and contribute directly to stabilize NTD dimeriza- and the resulting 100-fold concentrated dopes were analyzed. tion for physically cross-linking the spidroins into an 13,38 interconnected network. Therefore, the pH-dependent behaviors (Figure S5), two main characteristics of native conformational change of the biomimetic spidroin suggests the dope that have recently been recapitulated for recombinant potential suitability of acidic pH coagulation in synthetic fiber NT2RepCT aqueous solutions.25 Albeit shear thinning, the spinning (see the results below). control dope of I16 showed the dominance of the elastic (or Furthermore, we conducted dynamic light scattering (DLS) storage) modulus over the viscous (or loss) modulus across the analysis to monitor the hydrodynamic diameter (Dh) of the entire frequency range investigated. These results indicate that spidroins in the concentrated dopes and the preceding the presence of both terminal domains was also critical to semidilute solutions with a protein concentration of 2 mg fl − determine the ow properties of recombinant dopes. mL 1 (Figure 3). All of the spidroins existed as nanostructures To study how the spidroin assemblies in dope transition with a small hydrodynamic diameter size of approximately 7 upon coagulation, which is an important step in fiber spinning, nm, which was suggestive of the presence of single protein we diluted the spidroin dopes into solutions of neutral or molecules in the semidilute aqueous solutions. After being acidified 90% ethanol and characterized morphologies of the concentrated to dopes, the biomimetic silk N16C was found to resulting aggregates by AFM (Figure 4b). As expected, I16 self-assemble into larger nanostructures with an average experienced a structural transition from micellar-like particles diameter size of about 28 nm, whereas the other three into dispersed fibrils of hundred nanometers upon ethanol spidroin variants did not significantly change their hydro- treatments at both neutral and acidic pH, and similar dynamic diameter sizes. nanofibrils were also obtained for variant 16C. In contrast, To examine the formed nanostructures, atomic force the coagulation pH showed a markedly different effect on the microscopy (AFM) analysis was performed for the four two spidroins harboring NTD. The micellar-like particles of spidroins in liquid conditions (Figure 4a). The two variants N16 transitioned into irregular aggregates with varying sizes lacking the CTD (I16 and N16) in the aqueous state existed as from 10 to 60 nm at pH 7.4 but into mostly nanofibrils with a micellar-like particles with a diameter of 10−17 nm. Such high aspect ratio by decreasing the coagulation pH to 5.0. particles were also observed for 16C, with the coexistence of Notably, the N16C fibrils preassembled in dope also turned small amounts of fibril-like nanostructures (20−80 nm), which into irregular precipitates in a neutral-pH coagulation bath and might arise from the coalescence of smaller micellar-like instead into nanofibrils with a length of approximately 90 nm particles due to enhanced interaction of the 16C dimers in the in an acidic coagulation bath. To study assembly more relevant concentrated dope. Surprisingly, N16C formed uniform fibril- to fiber spinning (see the results below), we also analyzed the like nanostructures with a length of 30−80 nm, reflecting a spidroin morphologies by microfluidic extrusion of diluted concerted outcome of both terminal domains in guiding dope into the ethanol coagulation baths (Figure S6). Again, spidroin preassembly under similarly crowded conditions. In ordered fibrillar structures were observed for N16 and N16C addition, such preassembly of biomimetic silk was found to be in the acidic ethanol bath, and the shear force imposed by highly dependent on protein concentration, as revealed by microfluidic extrusion enabled the formation of micrometer- combined DLS and AFM analyses (Figure S4). Furthermore, scale fibrils, approximately tenfold longer than those achieved rheological analyses revealed that the fibrillar dope solution of by simple dilution without the microfluidic device. In contrast, N16C showed shear-thinning viscosity and viscoelastic N16 and N16C transitioned into irregular aggregates in the

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Figure 4. Morphologies of various spidroins in dope and coagulation baths. (a) Representative liquid AFM images of spidroin microstructures in diluted dope at room temperature. (b) AFM images of spidroin aggregates upon coagulation in 90% ethanol at pH 7.4 or 5.0.

Figure 5. Processing of spidroin dopes into fibers. (a) Schematic illustration of microfluidic wet spinning and postspun stretching. The diagram does not follow the actual scale for an intuitive illustration of the microfluidic chip. Mechanical properties of the postdrawn fibers with coagulation at pH 5.0 or 7.4: (b) toughness, (c) extensibility, (d) strength, and (e) Young’s modulus. neutral ethanol bath even in the presence of shear force. These previously unprecedented feature with implication in artificial results demonstrated that the NTD enabled both N16 and fiber spinning involving an organic solvent for coagulation. N16C to respond to acidic pH for assembly into ordered Nonetheless, it would be interesting to investigate why acidic nanostructures even in the presence of 90% ethanol, a and neutral ethanol baths differentially affect the assembly of

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Figure 6. Representative Raman spectra of the postdrawn ﬁbers with coagulation at pH 5.0 or 7.4. The peaks at 1651 cm−1 (vertical dashed lines) are indicative of the α-helix structure, and those at 1667 cm−1 (vertical dotted lines) are indicative of the β-sheet/β-turn structure.

Figure 7. Scanning electron micrographs of the postdrawn fibers. The four spidroins as indicated above the corresponding micrographs were spun into coagulation baths with pH at 5.0 (a, b) or 7.4 (c, d). (a, c) Analysis at breakpoint to examine the fiber interior core. (b, d) Analysis of the fiber surface, illustrating fibrillar structure. Scale bars, 1 μm. the NTD-containing spidroins, and the underlying mecha- processed by drawing to improve fiber mechanical perform- nisms remain yet to be discovered in future studies. ance, which was inspired by stretching of the nascent fibers by Microfluidic Spinning and Fiber Mechanical Proper- spiders.40,41 It was found that the postdrawn fibers were ties. Having characterized the spidroins in dopes, we then homogeneous along the fiber length (Figure S7). pumped each spinning dope into a coagulation bath using a The engineering stress−strain curves of the postdrawn fibers microfluidic device for fiber spinning. The microfluidic are shown in Figure S8, and the tensile mechanical properties channel, which mimics the tapered spinning duct of spiders’ of these fibers are summarized in Table S1. Intriguingly, the major ampullate silk39 (Figure 5a), served to impose a shear N16C fiber with acidic pH coagulation exhibited a toughness force on the spinning dope, and 90% ethanol with pH at either of 100.9 ± 13.2 MJ m−3 (Figure 5b), which was 2.3-fold higher 7.4 or 5.0 was used for spidroin coagulation. Continuous fibers than that of the N16C fiber with neutral-pH coagulation and were obtained (Video S1, Supporting Information), and the as- comparable to the value reported for native T. clavipes dragline spun fibers were collected on a reeling roller in air and silk (111.19 ± 30.54 MJ m−3).42 Such a toughing effect by

G https://doi.org/10.1021/acsbiomaterials.1c00492 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX ACS Biomaterials Science & Engineering pubs.acs.org/journal/abseba Article acidic pH coagulation was also observed for the fibers spun surfaces for these two types of fibers due to the absence of from N16 but not for the fibers from the two spidroins lacking obvious spherical particles within the microfilaments. In the NTD. Similarly, acidic pH coagulation improved the particular, the N16C fiber with acidic coagulation was extensibility of the N16C and N16 fibers (Figure 5c). These appreciably smooth in surface, densely interconnected, and pH-dependent beneficial effects on fiber toughness and finely aligned (Figure S10), which might explain why this fiber extensibility were attributed to the unique NTD, which was the finest and possessed the best overall mechanical might direct spidroin assembly via noncovalent dimerization properties. to form finely interconnected fibers as long as the pH is It has long been difficult to recapitulate the extreme aqueous lowered in the aqueous ethanol coagulation bath. Nonetheless, solubility of native spinning dopes. According to Andersson et acidic pH coagulation showed different effects on the strength al., the aqueous solubility and pH responsiveness of the of the fibers derived from the two NTD-harboring spidroins as terminal domains differ in spidroins from different spider an improving effect was only observed for N16C but not for species and silk types, and the sources of the terminal domains N16 (Figure 5d). On the other hand, regardless of the would greatly affect the overall solubility of the resulting coagulation bath pH, the existence of CTD alone was found to minispidroin.28 In our study, we biosynthesized recombinant consistently increase the fiber strength as compared to the I16 spidroins of T. clavipes MaSp1 consisting of a very soluble fibers, which might be attributed to the tentative role of this NTD, a central repetitive region, and a moderately soluble terminal domain as nucleation seeds for the conversion of the CTD. As the four spidroin variants were only moderately repetitive region into β-sheet crystals.14,17,21 With regard to the soluble in aqueous solutions, the addition of solubilizing agents stiffness of the fibers, manipulation of the coagulation pH had (e.g., NaCl, urea) was necessary to keep them soluble in dilute no appreciable effect (Figure 5e). Among the four types of aqueous solutions and concentrated dopes without precip- postdrawn fibers, the 16C fibers exhibited an optimal Young’s itation or gelation. Our previous study has shown that the modulus at approximately 5 GPa, which coincided well with addition of 20.7 wt % NaCl helped to prepare the aqueous the superior degree of crystallinity within the fibers according dope of spidroin I16 yet adversely affected the uniformity and to wide-angle X-ray diffraction analysis (Figure S9). mechanical properties of the resulting fibers.36 Therefore, 0.5 Collectively, these results indicated that the spidroin M urea was added into the low-salt dialysis buffer, which architecture markedly yet profoundly affected the resulting ultimately led to the preparation of concentrated dopes for fiber mechanical properties. subsequent fiber spinning. Notably, the low level of urea To examine the structural features of the postdrawn fibers, improved the solubility of the various spidroins while we performed Raman spectroscopy analysis for the single fibers maintaining their expected secondary structures in the aqueous derived from the four spidroins (Figure 6). The most dopes and capabilities for spinning into more uniform and prominent Raman-active bands were located at approximately tougher fibers. Our study thus suggests an alternative approach −1 1651 and 1667 cm , which are indicative of α-helical and β- to handle recombinant spidroins in vitro, albeit their instinctive 43 sheet/β-turn structures, respectively. Interestingly, lowering aggregation-prone feature. the pH of the coagulation bath led to the formation of more β- β fi sheet/ -turn structures for the N16 and N16C bers, which is ■ CONCLUSIONS supposed to contribute to the improved fiber extensibility obtained from NTD-harboring spidroins by acidic pH We have biosynthesized a recombinant spidroin with the coagulation (Figure 5c). In contrast, the Raman band at biomimetic tripartite architecture for spinning into an artificial approximately 1733 cm−1, which has previously been assigned fiber with a toughness that equals that of natural spider to the side-chain vibration ν(COOH) of residues Asp and dragline silk. This desirable outcome was enabled by the Glu,43 was weakened in intensity, possibly because part of coexistence of the nonrepetitive amino- and carboxy-terminal these residues located in NTD became buried under the acidic domains, which synergistically affect the spidroin solubility, pH condition. preassembly in concentrated aqueous dope, and the resulting Finally, we examined the microstructures of the postdrawn fiber morphology and mechanical properties. Of particular fibers by scanning electron microscopy (Figure 7). Analysis of interest, we developed a designer storage buffer that includes a the fractured fibers revealed the existence of many irregular small amount of urea to prevent immature aggregation and voids in the I16 and N16 fibers, whereas such defects were improve the solubility of the spidroins even in the highly absent in the 16C and N16C fibers, reflecting the formation of concentrated aqueous dopes. By doing so, we have thus a dense, interconnected network structure for the fibers spun demonstrated a processing strategy to tackle one of the most from CTD-harboring spidroins (Figure 7a,c). In addition, the daunting challenges in preparing soluble recombinant existence of void defects was not affected by the coagulation spidroins of diverse spider species and silk types.26,28,29 Indeed, pH. Analysis of fiber surface revealed that all of the fibers had a the biomimetic spidroin N16C and the related variants lacking fibrillar structure (Figure 7b,d). This fibrillar morphology has one or both termini could be comparably concentrated in the also been observed for native spider dragline silk44 and fibers aqueous storage buffer. The resulting aqueous dope of N16C spun from aqueous dopes of other minispidroin con- specifically allowed the formation of fibril-like nanostructures, structs.26,28,29 As expected, coagulation pH did not affect the which are unique as these preassembled structures comple- surface morphology of the I16 and 16C fibers, which appeared ment the canonical micellar structures observed in natural to be hierarchically assembled from microfilaments with a spidroin dope solution45 and recombinant aqueous dopes.28,29 string of particles having a diameter of 200−400 and 100−300 The suitability of the mesoscale fibrillar assemblies for spinning nm, respectively. In contrast, the surfaces of the fibers derived into macroscale fine fibers was also demonstrated via ordered from NTD-harboring spidroins were highly dependent on the alignment and coagulation of these fibrils upon extrusion into coagulation pH. When compared with coagulation at the an acidic coagulation bath. This work thereby highlights the neutral pH, the acidic coagulation gave rise to much smoother synergizing role of terminal domains in the storage and

H https://doi.org/10.1021/acsbiomaterials.1c00492 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX ACS Biomaterials Science & Engineering pubs.acs.org/journal/abseba Article processing of recombinant biomimetic spidroin into synthetic All authors have given approval to the final version of the tough fibers. manuscript. Notes ■ ASSOCIATED CONTENT The authors declare no competing financial interest. *sı Supporting Information The Supporting Information is available free of charge at ■ ACKNOWLEDGMENTS https://pubs.acs.org/doi/10.1021/acsbiomaterials.1c00492. Financial support was provided by the National Key Research Diameters and tensile mechanical properties of the fibers and Development Program of China (2016YFE0204400 and (Table S1), mass spectroscopy analyses of the purified 2020YFA0907702), the National Natural Science Foundation spidroins (Figure S1), characterization of recombinant of China (22075179 and 32071414), and the Natural Science spidroins NcNT and NcCT with individual terminal Foundation of Shanghai (21ZR1432100). X.-X.X. acknowl- domains (Figure S2), CD spectra of the spidroins at pH edges the Program for Professor of Special Appointment at 7.4 and 5.0 (Figure S3), concentration-dependent Shanghai Institutions of Higher Learning. assembly of N16C (Figure S4), rheological properties of the dope solutions (Figure S5), images of spidroin ■ REFERENCES fl aggregates obtained by micro uidic extrusion of diluted (1) Yarger, J. L.; Cherry, B. R.; van der Vaart, A. 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