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Dec 17, 2011 - School of Engineering and Computer Science, University of the Pacific, Stockton, California 95211, United States ... Wide-angle X-ray d...
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Conserved C-Terminal Domain of Spider Tubuliform Spidroin 1 Contributes to Extensibility in Synthetic Fibers Eric Gnesa,† Yang Hsia,† Jeffery L. Yarger,‡ Warner Weber,‡ Joan Lin-Cereghino,† Geoff Lin-Cereghino,† Simon Tang,§ Kimiko Agari,† and Craig Vierra*,† †

Department of Biological Sciences, University of the Pacific, Stockton, California 95211, United States Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, United States § School of Engineering and Computer Science, University of the Pacific, Stockton, California 95211, United States ‡

ABSTRACT: Spider silk is renowned for its extraordinary mechanical properties, having a balance of high tensile strength and extensibility. To date, the majority of studies have focused on the production of dragline silks from synthetic spider silk gene products. Here we report the first mechanical analysis of synthetic egg case silk fibers spun from the Latrodectus hesperus tubuliform silk proteins, TuSp1 and ECP-2. We provide evidence that recombinant ECP-2 proteins can be spun into fibers that display mechanical properties similar to other synthetic spider silks. We also demonstrate that silks spun from recombinant thioredoxin-TuSp1 fusion proteins that contain the conserved C-terminal domain exhibit increased extensibility and toughness when compared to the identical fibers spun from fusion proteins lacking the C-terminus. Mechanical analyses reveal that the properties of synthetic tubuliform silks can be modulated by altering the postspin draw ratios of the fibers. Fibers subject to increased draw ratios showed elevated tensile strength and decreased extensibility but maintained constant toughness. Wide-angle X-ray diffraction studies indicate that postdrawn fibers containing the C-terminal domain of TuSp1 have more amorphous content when compared to fibers lacking the C-terminus. Taken together, these studies demonstrate that recombinant tubuliform spidroins that contain the conserved Cterminal domain with embedded protein tags can be effectively spun into fibers, resulting in similar tensile strength but increased extensibility relative to nontagged recombinant dragline silk proteins spun from equivalently sized proteins.

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Asp).4,6,7 Less is known regarding the specific biological function of the ECPs in cob-weavers and their interactions with TuSp1 in tubuliform silks. Because ECP molecules lack homogenized internal block repeats as well as the conserved nonrepetitive N- and C-termini, both considered defining characteristics of the traditional spidroin family members, their specific roles in tubuliform silks have been unclear. Moreover, because ECP molecules have molecular weights that are considerably smaller (∼90 kDa) relative to spidroin family members (250−350 kDa), it has not been established whether these molecules can direct fiber assembly in the absence of TuSp1. Given the presence of high levels of Ala and Gly residues within the ECP protein sequences, in particular the organization of iterations of GA couplets, it suggests that these proteins might have the intrinsic ability to assemble into fibers. One of the hallmark features of traditional spidroin family members is the presence of the C-terminal domain.8,9 In the dragline silk spidroin members, MaSp1 and MaSp2, this domain has been implicated in the control of protein solubility and fiber formation.10−12 High-resolution NMR studies of the C-terminal domains of MaSp1 and TuSp1 show these regions

INTRODUCTION Spiders spin egg case silk fibers to help protect their offspring during development. In the cob weaving black widow spider, Latrodectus hesperus, biochemical studies have revealed that egg sacs contain tubuliform and aciniform silks.1 Tubuliform silks have been shown to consist of at least three distinct proteins: Tubuliform Spidroin 1 (TuSp1), Egg Case Protein 1 (ECP-1), and Egg Case Protein 2 (ECP-2).2−4 Amino acid composition studies of raw egg sacs support the assertion that TuSp1 is the major constituent of tubuliform silk.4 Consistent with these studies, MS/MS analysis of peptides obtained from solubilized egg sacs after tryptic digestion confirms the presence of TuSp1 in tubuliform silks.4 Translation of the full-length TuSp1 cDNA predicts a large molecular weight protein with a mass of approximately 300 kDa.5 The amino acid sequence of TuSp1 reveals an architecture containing 13−15 internal block repeat modules that are approximately 180 amino acids, flanked by conserved nonrepetitive N- and C-termini.4,6,7 The typical submodules that have been identified in other spidroin protein sequences, which include Gly-Gly-X (X = Ala, Leu, Gln, and Tyr), polyalanine stretches (or Gly-Ala couplets), and Gly-ProGly-X-X (X = Gly, Gln, and Tyr), are notably absent in the TuSp1 amino acid sequence. Instead, the protein sequences of tubuliform silks have the motifs Sern, (Ser-Ala)n, (Ser-Gln)n, and GX (X = Gln, Asn, Ile, Leu, Ala, Val, Tyr, Phe, and © 2011 American Chemical Society

Received: September 9, 2011 Revised: November 16, 2011 Published: December 17, 2011 304

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saturation. Saturated cultures were then combined with 8 L of fresh LB, supplemented with 0.02% arabinose for induction purposes and grown an additional 4 h. Following induction, the bacterial cells were pelleted at 6000g and then lysed in 20 mL of FastBreak Cell Lysis Reagent (Promega) per 1.5 L of culture. To promote further lysis, the solution was sonicated at maximum setting for 1 min using a VirSonic 60 instrument (VirTis). After sonication, the sample was clarified by spinning for 5 min at 8000g. The supernatant was combined with 1 mL of Ni-NTA agarose resin (50% slurry; Qiagen) and gently rocked at room temperature for 1 h to allow for binding of the fusion proteins via their C-terminal 6× histidine tag. The resin was washed using a buffer (50 mM NaH2PO4, 300 mM NaCl) supplemented with 20 mM imidazole, followed by elution of the fusion proteins with 20 mL of same buffer containing 250 mM imidazole. Elution and wash samples were examined for the presence of the fusion proteins using Western blot analyses using an antihistidine monoclonal antibody. After validation of the fusion protein in the elution fractions, the purified proteins were precipitated using 100% acetone (1:1 v/v) overnight at −20 °C. After overnight incubation, the protein was pelleted by centrifugation at 4000g for 10 min and then resuspended in 15 mL of 8 M urea. Solubilized protein samples were dialyzed against water for 2−3 days and then subject to freeze-drying using a FreeZone12Plus instrument (Labconco). Typical yields for the TuSp1 1× and TuSp1 1×C recombinant proteins after purification using 10 L of culture were approximately 48 and 72 mg, respectively. For experiments using reconstituted egg case silk fibers, the eggs were removed and the silk fibers were weighed and then dissolved overnight in 1,1,1,3,3,3hexafluoro-2-propanol (HFIP). Fiber Production and Post-Spin Draw. Synthetic or reconstituted fibers were spun using 20% (wt/vol or 200 mg/mL) of purified recombinant protein or dissolved egg sac material. Briefly, the lyophilized recombinant proteins or egg sacs were dissolved in HFIP and incubated overnight with vortexing to facilitate solubilization. Following solubilization, the samples were transferred into a 0.25 mL glass syringe (Hamilton Company) outfitted with a 26s gauge (inner diameter of 127 μm) blunt end needle. Samples were heated to approximately 58 °C, followed by extrusion of the protein solution using a syringe pump at volumetric rate of 15 μL/min (Harvard Apparatus). Fibers were spun into a 95% isopropanol bath, allowed to sit for 15 min and then motor spooled onto a custom device at a linear speed of 2.7 mm/s. During the winding process, the fibers were secured to the spooling apparatus using double-sided tape. For postspin draw experiments, the fibers were stretched in 75% isopropanol at a constant rate of 1.5 mm/s (11.5% strain rate) using a linear actuator, and the postspin ratio was calculated based on the initial length of the fibers. To further reduce experimental error, all postspin draw ratios were collected from the same fiber and used for the mechanical analysis. Mechanical Analysis. Fibers were air-dried at 25 °C for 1−2 h and then mounted on a 25.4 × 25.4 mm mechanical testing cardstock frame. Prior to mechanical analysis, diameters were measured at three points along the longitudinal axis using a light microscope at a magnification of 115× (Leica MZ16). The average diameter of each fiber was calculated and used for the mechanical analysis. Mechanical tests were carried out at room temperature with ∼45% humidity on a 305C Dual Mode Force Transducer (Aurora Scientific Inc.) with a 5 N load cell at a strain rate of 2% s−1 and a sample frequency of 100 Hz. Data were plotted in Microsoft Excel, and polynomial regression curves were fitted to a polynomial order of 6 to calculate toughness. Statistical differences were determined by an unpaired t test and were significant at p < 0.05. Scanning Electron Microscope Analysis of Silks. Synthetic fibers were coated to a thickness of 14−30 nm with gold alloy in a Pelco SC-7 autosputter coater with an FTM-2 film thickness monitor. Synthetic and reconstituted silk diameters were measured on a Hitachi S-2600 SE operated with an accelerator voltage of 3 kV. Diameters of the strands were measured to the nearest 0.01 μm at three distant places in the sample and cross-referenced with the sizes obtained by light microscopy. Experiments were conducted at ambient temperature and humidity, which was 25 °C and ∼45%, respectively.

fold into a five-helix bundle, mediating protein oligomerization.10,13 Because the C-terminal domain of TuSp1 shares similar secondary structure in solution relative to the Cterminal region of dragline silk spidroins, it seems plausible that this region performs a similar function during extrusion of tubuliform silks. However, because the extrusion process for tubuliform silks has not been characterized at the level of major ampullate silks, it remains to be determined whether these Cterminal domains serve similar functions during the spinning process. Mechanical studies using naturally extruded tubuliform silks reveal breaking strengths and strains of 630 MPa and 71%, respectively.14 Tubuliform silks represent one of the toughest fibers that cob weaver’s spin, providing a critical protective function for the developing eggs in harsh environments. To date, there have been no studies reported that describe the production of synthetic tubuliform silks and their mechanical characterization. Here we perform mechanical analyses on tubuliform fibers obtained by a wet spinning methodology and develop novel postspin draw techniques to investigate the roles of the TuSp1 internal block repeats and the C-terminal domain during extrusion of recombinant proteins. We also examine whether the ECPs, specifically ECP-2, has the potential to form synthetic fibers and whether its material properties are comparable to threads spun from recombinant TuSp1 proteins.

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EXPERIMENTAL SECTION

DNA Constructs. Four DNA constructs were designed for expression studies to produce truncated TuSp1 and ECP-2 proteins. Two distinct TuSp1 constructs were engineered for prokaryotic expression: one containing the nonrepetitive C-terminus and a single internal block repeat (TuSp1 1×C) and the other having only a single internal block repeat (TuSp1 1×). To amplify the TuSp1 1× cDNA, we performed PCR using the forward and reverse primers 5′-CTC GAG CAT ATG CCC GGG TCC TCT TCA ACA TCA ACA ACT3′ and 5′-GGA TCC TCC GGA AGG AAC AGC AAA TCC TGC AAT-3′, respectively. The TuSp1 1×C cDNA was amplified using the same forward primer (see above) but a different reverse primer 5′GGA TCC TCC GGA AAC AAG GGG AAC AAG GTA ATT-3′. The underlined regions denote the addition of XhoI, NdeI, and XmaI sites on the forward primers and BamHI and BspE1 sites on the reverse oligonucleotides, respectively. Both the TuSp1 1× and 1×C were amplified from a previously characterized TuSp1 cDNA and correspond to residues 761−936 and 761−1058, respectively (GenBank Accession DQ109035.1).4 For the ECP-2 expression studies, two different cDNA regions were amplified from the fulllength ECP-2 cDNA (GenBank Accession DQ341220.1) using PCR. One segment encoded the N-terminal region (amino acids 24−364) whereas the other was designed to amplify the C-terminal region (amino acids 359−825). For the N-terminus (ECP-2N), the forward and reverse primers were 5′-GAA TTC TGT TTC AAC AAA TGT TTA-3′ and 5′-CTC GAG TCT CGA AGT AGC TCT TCC-3′. To obtain the C-terminus (ECP-2C), we used the forward and reverse primers 5′-GAA TTC GGA AGA GCT ACT TCG AGA-3′ and 5′CTC GAG TAA GTT TGC AAA ATG TGC-3′, respectively. Both 5′ termini of the forward and reverse primers were engineered to incorporate an EcoRI and XhoI site, respectively. Following amplification of the cDNAs, these fragments were gel extracted and ligated into the prokaryotic expression vector pBAD-Thio-Topo (Invitrogen) and transformed into E. coli. To validate the cDNAs were inserted in the correct orientation, we performed restriction digestion analysis and agarose gel electrophoresis. Plasmids that carried the cDNA inserts in the proper orientation were then subjected to DNA sequence analysis. After confirmation of the cDNA sequence in the plasmid, we used these plasmids for protein induction. Protein Purification. For protein induction experiments, 2 L of LB was inoculated with each clone and grown overnight until 305

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Figure 1. (A) Schematic representation of truncated TuSp1 (TuSp1 1× and TuSp1 1×C) and ECP-2 (ECP-2N and ECP-2C), along with the predicted TuSp1 full-length protein sequence. (B) Comparison of theoretical amino acid composition of ECP-2 relative to other fibroins synthesized by L. hesperus. The predicted amino acid compositions are from translated cDNAs L. hesperus MaSp1 (EF595246.1), MaSp2 (EF595245.1), TuSp1 (AY953070.1), MiSp1 (EU394445.1), AcSp1 (EU025854.1), ECP-2 (DQ341220.1), and PySp1 (FJ973621). Structural Analysis. Wide angle X-ray diffraction (WAXD) experiments were conducted on single fibers at the BioCARS beamline (sector 14-BM-C) of the Advanced Photon Source (APS) at Argonne National Laboratory (Argonne, IL, U.S.A.).15 The X-ray diffraction (XRD) data were recorded on a 9-chip CCD detector (ADSC Quantum-315) placed 300 mm behind the sample. The X-ray monochromatic beam was focused to 150 × 200 μm using an incident wavelength of 0.9787 Å. The beamstop and sample fibers were both mounted vertically. Data were collected with a 30 mm distance between the sample and the beamstop with an X-ray exposure time of

120 s. The background (air scattering) was subtracted from all X-ray patterns shown.

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RESULTS Recombinant Tubuliform Silk Proteins Can Be Expressed in Bacteria. To investigate the mechanical properties of synthetic tubuliform silk, we expressed and purified truncated forms of the tubuliform spidroin, TuSp1. TuSp1 expression constructs were designed to contain either a single internal block repeat fused to the nonrepetitive C-terminal 306

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mL and attempted to spin them into fibers. We also solubilized the natural fibers from egg sacs collected from female black widow spiders. The reconstituted egg case fibers were capable of being spun into threads (Figure 3A). Analysis of the fibers by

domain (TuSp1 1xC) or a single internal block repeat (TuSp1 1x). The thioredoxin tag was placed upstream of the internal block repeat (∼180 amino acids) to enhance solubilization during expression experiments and a 6x-his tag was added to the C-terminus to facilitate purification (Figure 1A). Because tubuliform silks in cob weavers have been shown to be composite materials that contain TuSp1 and ECP proteins, we also attempted to express ECP-2. Expression of full-length ECP-2 resulted in low levels of protein (data not shown). However, expression of the N- and C-terminal region of ECP-2 yielded sufficient amounts of product (ECP-2N or ECP-2C; Figure 1A). The N-terminal region of ECP-2 has been shown to be cysteine-rich, containing 16 cysteine residues within the first ∼150 amino acids, whereas the C-terminus of ECP-2 is glycine-alanine rich (Figure 1A). Overall, the predicted amino acid composition profile of ECP-2 was similar to MaSp1, MaSp2, and the minor ampullate spidroin, MiSpl-like, in particular, for the residues glycine and alanine (Figure 1B); however, the predicted molecular weight of ECP-2 is considerably smaller relative to other spidroin family members and ECP-2 lacks the nonrepetitive conserved N- and C-termini, which is one of the hallmark features of these structural proteins. All four recombinant fusion proteins (TuSp1 1x, TuSp1 1xC, ECP-2N and ECP-2C) were purified using affinity chromatography and then analyzed by SDS-PAGE analysis under denaturing conditions, followed by visualization of the proteins using silver staining methodology. Analysis of the proteins revealed they were purified to homogeneity and exhibited migration patterns similar to the predicted molecular masses (Figure 2). To confirm the purified proteins corresponded to

Figure 3. SEM analysis of fibers spun from reconstituted and recombinant egg case proteins. Images are representative of at least five different samples from each fiber type. (A) Reconstituted egg case fibers at 1000×. (B) TuSp1 1× fibers at 500×. (C) TuSp1 1×C fibers at 1000×. (D) TuSp1 1×C fibers at 6000× after forcibly breaking. (E) ECP-2C fibers at 500×. (F) ECP-2C fibers at 2500× after forcibly breaking. (G) TuSp1 1×C with ECP-2C at 1000×. (H) TuSp1 1×C with ECP-2N at 1000×.

scanning electron microscopy showed the reconstituted fibers displayed creases along the longitudinal axis (Figure 3A). Both the TuSp1 1× and TuSp1 1×C recombinant proteins could also be spun into fibers, but showed profound differences with their surface structure (Figure 3B and C, respectively). The TuSp1 1× spun fibers contained wrinkles, similar to the reconstituted egg case fibers, while the TuSp1 1×C fibers had a smooth surface with uniform deep indentations that extended parallel to the longitudinal axis of the fibers (Figure 3C). Analyses of the TuSp1 1×C fibers after fracture revealed uniform channels and holes on the interior of the threads, which likely formed during the evaporation of the HFIP or isopropanol during fiber production (Figure 3D). Because ECP-2 has been shown to be a constituent of tubuliform silks, we also investigated whether ECP-2C could form fibers. Using

Figure 2. Size fractionation of purified recombinant egg case proteins using SDS-PAGE analysis under reducing conditions, followed by protein visualization via silver staining: lane 1, molecular markers with sizes indicated in kDa; lane 2, purified ECP-2C; lane 3, purified ECP2N; lane 4, purified TuSp1 1×C; lane 5, purified TuSp1 1×.

the expected products, we performed a Western blot analysis using an antihistidine monoclonal antibody. As expected, the antihistidine monoclonal antibody recognized the purified products (data not shown). Synthetic Fibers Produced from Different Tubuliform Recombinant Silk Proteins Reveals Differences in Morphological Features. To examine whether artificial silk fibers could be generated from the recombinant proteins, we solubilized the proteins in HFIP at a concentration of 200 mg/ 307

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Table 1. Summary of the Averaged Mechanical Data Collected for the Fibers at Select Post-Spin Draw Ratiosa reconstituted egg case TuSp1 1× TuSp1 1×C TuSp1 1×C/ECP-2N TuSp1 1×C/ECP-2C ECP-2C TuSp1 1×C TuSp1 1×C/ECP-2C natural egg case a

post-spin draw

ultimate tensile strength (MPa)

% ultimate strain (mm/mm)

toughness (MJ/m3)

3.5× 3.5× 3.5× 3.5× 3.5× 3.5× 6.0× 6.0×

203.8 ± 8.0 80.0 ± 12.0 95.1 ± 3.3 88.8 ± 8.5 82.8 ± 2.5 121.9 ± 5 158.6 ± 7.5 101.2 ± 18.7 630.0

9±1 9±3 25 ± 4 18 ± 2 16 ± 3 18 ± 1 13 ± 1 8±2 70

12.7 ± 1.5 6.5 ± 2.2 20.7 ± 3.8 11.6 ± 0.8 10.7 ± 2.4 17.4 ± 1.2 11.9 ± 1.6 6.3 ± 2.7

diameter (μm) 35.3 ± 60.7 ± 30.5 ± 51.0 ± 39.1 ± 47.1 ± 24.5 ± 35.9 ± ∼4−5

0.8 3.0 0.5 2.1 0.9 1.1 0.3 1.7

Ultimate strength, strain, and toughness were calculated assuming a constant diameter.

Figure 4. Mechanical analysis of synthetic silk fibers. (A) Stress−strain curves of all TuSp1 1×C spun fibers with various postspin draw ratios. (B) Typical stress−strain curves of ECP-2C spun fibers with various postspin draw ratios. (C−F) Bar graphs representing averaged values in tensile strength, ultimate strain, toughness, and diameter size for fibers tested in each group. Error bars represent the standard error of the mean. All values were statistically significant for tensile strength and ultimate strain when comparing the initial and final postspin draw ratio for each synthetic fiber type, except for the TuSp1 1× spun fibers. Additionally, when comparing the postspun fibers from the TuSp1 1× and TuSp1 1×C constructs, there was a statistical difference between the ultimate strain and toughness. For each postspin draw ratio and fiber type, approximately five individual fibers were used to compute the averaged mechanical data (exception was for the fibers experiencing postspin draw ratios of 6, where two samples were analyzed for each fiber type).

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fiber extensibility, adding approximately 200% extensibility to the fibers (Table 1). Because tubuliform silks are naturally spun as a composite material, we tested the mechanical properties of composite fibers containing TuSp1 1×C mixed with ECP-2N or ECP-2C. Fibers spun from TuSp1 1×C and ECP-2C mixtures (TuSp1 1×C/ECP-2C) were capable of being postspun drawn to 6× their original length; however, the composite threads containing TuSp1 1×C and ECP-2N could only be postspun drawn to 4.5× their initial lengths (Figure 4). Across the different postspin draw ratios, the TuSp1 1×C/ECP-2C composite threads displayed similar tensile strength, extensibility and toughness relative to TuSp1 1×C fibers, except when the fibers were stretched to 6× their original lengths, where the composite fibers showed reduced tensile strength, extensibility, and toughness (Figure 4C−E). Similar material properties were obtained for the TuSp1 1×C/ECP-2N composite fibers relative to the TuSp1 1×C spun fibers (Figure 4C−E). Collectively, these results suggest the addition of TuSp1 1×C to ECP-2C or ECP-2N (20:1 mixture ratios) produced little, if any, dramatic change to the material properties of the fibers spun from the recombinant proteins. Next, we examined the stress strain curves produced from fibers spun exclusively from purified ECP-2C proteins. Postspin draw ratios ranging from 1 to 3.5 were successfully used to collect ECP-2C recombinant fibers. Attempts to collect fibers that exceeded postspin draw ratios beyond 3.5 resulted in the fibers breaking during the procedure. Relative to fibers spun from TuSp1 1×C recombinant proteins (postspin draw ratios of 3.5), the stress−strain curves revealed the ECP-2C fibers displayed higher breaking strengths at 121.9 MPa. Interestingly, when the ECP-2C fibers were examined as “as spun” fibers, these threads showed remarkable extensibility, displaying ∼80% ultimate strains (Figure 4D). When comparing the mechanical properties of our synthetic tubuliform silk fibers to reconstituted respun egg case silk fibers, our fibers routinely had lower breaking strengths. For example, at postspin draw ratios of 3.5, reconstituted respun egg case silk fibers displayed breaking strengths approaching 204 MPa. Relative to natural tubuliform silk fibers, which have been shown to have breaking strengths at 630 MPa,14 the reconstituted tubuliform silks, TuSp1 1×C and ECP-2C threads were approximately 3-, 4-, and 5-fold lower, respectively (highest postspin draw ratios). Natural tubuliform silks have also been reported to be highly extensible, having 71% ultimate strains. This is much higher relative to the reconstituted respun egg case silk fibers at postspin draw ratios of 3.5, which showed ultimate strains less than 10%, suggesting the reconstituted fibers also lost a high degree of their extensibility upon respinning. In addition to measuring breaking strength and strain, we noted particular differences in the diameter sizes of the spun fibers. During the spinning procedure, we extruded the fibers through needles with inner diameter sizes of ∼127 μm. Synthetic fibers spun from TuSp1 1× consistently had the largest diameter size measuring ∼60 μm (Figure 4F). In contrast, the TuSp1 1×C fibers produced the smallest diameter size of all the spun fibers, having sizes of ∼36 μm. Although the TuSp1 1×C fibers produced the smallest diameter size, it is still larger relative to natural dragline silks, which are ∼5 μm. Taken together, this supports the assertion that the C-terminal domain is essential for the assembly process during the extrusion of the fibers under shear (Figure 4F).

a spinning dope containing recombinant ECP-2C proteins, this protein segment was capable of forming synthetic fibers (Figure 3E). Inspection of the surface of the ECP-2C fibers revealed wrinkles along the longitudinal axis of the fibers, but these indentations were less pronounced relative to the reconstituted egg case silk, TuSp1 1× and TuSp1 1×C (compare Figure 3A− C to Figure 3E). Fracture of the ECP-2C fibers showed a densely packed interior (Figure 3F), indicating the ECP-2C proteins were capable of assembling into a tightly packed structure despite the absence of a traditional nonrepetitive Cterminus common to spidroin family members. We also spun composite fibers using TuSp1 1×C supplemented with ECP2C or ECP-2N. Because Q-PCR studies indicate TuSp1 mRNA levels are ∼20-fold higher relative to ECP-2, we spun fibers from a mixture of these two components at a 20:1 ratio (final protein concentration 20%). Interestingly, these fibers were similar in their morphological appearance relative to the reconstituted egg case fibers, indicating that mixing either truncated ECP-2N or ECP-2C proteins with TuSp1 1×C results in fibers with wrinkles along the longitudinal axis (Figure 3G,H). Mechanical Studies of the Synthetic Tubuliform Silks Reveal the C-Terminal Domain Adds Extensibility to the Fibers. To investigate the material properties of the artificial silk fibers, we analyzed the mechanical features of the spun fibers. Because previous studies with artificial silk fibers have demonstrated that postspin drawing substantially improves the quality of the fibers after spinning, we spun our fibers into an alcohol bath and subjected them to different postspin draw ratios ranging from 2.5 to 6. By building a custom mechanical device to better control for the postspin draw procedure, we made large advances to reduce the variations that are introduced by using a hand-drawn postspin methodology (Table 1).16 Different TuSp1 1×C fibers collected from the same postspin draw ratios displayed similar mechanical profiles (Figure 4A). Because of the reduced variation, we were able to collect mechanical data to help elucidate the function of specific fibroin domains. Analysis of the TuSp1 1×C or ECP-2C fibers revealed that these threads could withstand a postspin draw ratio up to either 6 or 3.5, respectively (Figure 4A,B). The TuSp1 1×C fibers had a tensile strength of 158.6 MPa and an extensibility of 13%. At lower postspin draw ratios, which ranged from 2.5 to 3.5, the TuSp1 1×C recombinant fibers displayed extensibilities that ranged from 25 to 35% (Figure 4A; Table 1). Inspection of the stress−strain curves for the TuSp1 1×C fibers at different postspin draw ratios revealed a basic trend for these fibers: increases in the postspin draw ratio increased tensile strength but decreased ultimate strain (Figure 4A). However, the toughness, which was calculated from the area under the stress−strain curve, remained the same for these fibers. In comparison, the maximum observed postspin draw ratios for the TuSp1 1× fibers were 3.5 (Figure 4C−F). At this postspin draw ratio, the tensile strength was 80 MPa and the ultimate strain was 9% (Figure 4C,D; Table 1). Overall, the tensile strength and ultimate strain were lower at the different postspin draw ratios for the TuSp1 1× fibers relative to the TuSp1 1×C threads (Figure 4C,D). Moreover, the TuSp1 1× fibers were significantly less tough relative to the TuSp1 1×C fibers at the same postspin draw ratios (Figure 4E). The maximum toughness determined for the TuSp1 1×C fibers was 20.7 MJ/m3 at a postspin draw ratio of 2.5−3.5, whereas the TuSp1 1× fibers were 6.5 MJ/m3 (Table 1). Collectively, these data support the C-terminal domain functions to help increase 309

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Figure 5. Wide angle X-ray diffraction (WAXD) of recombinant silk fibers: (A) TuSp1 1×; (B) TuSp1 1×C single fibers. The air scattering background was removed from the data. The double arrow indicates the direction of both fiber axis and beam stop. The data were collected with a detector distance of 300 mm and a beamstop distance of 30 mm. The grid lines are the physical gaps between detectors (9 CCD array detector).

Structural Analyses of TuSp1 1×C Spun Fibers Reveal More Amorphous Components Relative to TuSp1 1× Fibers. Wide angle X-ray diffraction (WAXD) of TuSp1 1× and TuSp1 1×C single fibers that were postspun drawn shows two diffuse anisotropic rings centered at 0.6 and 1.4 inverse Å (Figure 5). This is consistent with oriented β-sheet crystallites within a partially disordered matrix. There is a clear increase in Bragg diffraction in the TuSp1 1× fiber (Figure 5A). The equatorial Bragg reflections (0.6 and 1.4 A−1) are similar to what is observed silkworm silk fibers, and results from β-sheet nanostructures oriented along the fiber axis.17,18 The WAXD patterns are typical for seminanocrystalline morphology, which is characterized by Bragg reflections and diffuse diffraction from amorphous components (Figure 5). The strong equatorial reflections (quantified by the azimuthal plots, not shown directly) in the TuSp1 1× and TuSp1 1×C fibers clearly indicates that both fibers have their hydrogen bonding beta stands oriented perpendicular to the fiber axis. This is the typical orientation for β-sheet nanocrystalline domains in spider silk fibers, with the h-bonding perpendicular to the fiber axis (beta stands) and the sheets parallel to the fiber axis. The azimuthal width is also similar to what is found in most spider silk fibers and indicates that the β-sheets are nanocrystalline. However, the WAXD intensity difference between TuSp1 1× and TuSp1 1×C fibers indicates an increase in β-sheet content for the TuSp1 1× fibers (or a higher amorphous fraction in TuSp1 1×C). Traditional spider silk spidroins have very large molecular weights, ranging from 250 to 350 kDa.19,20 Recent evidence supports that protein size plays an important role in determining the mechanical properties of synthetic dragline silk fibers spun in vitro from purified recombinant native sized MaSp1 proteins.21 Presumably, the increased length of the protein chains allow for more intermolecular interactions and fewer chain termini, leading to a fiber with improved material properties. It has also been reported that a (MaSp1)24 synthetic silk protein, which has a predicted molecular weight of 70 kDa, outperformed a smaller version of the same construct (MaSp1)16 that has a molecular weight of 46 kDa.16 The average performance of (MaSp1)16 fibers after postspin stretching to 3× their original length have been reported to yield tensile strengths of ∼54 MPa. Interestingly, the molecular

masses of (MaSp1)16 and TuSp1 1×C are similar (46 vs 45 kDa, respectively); however, our studies reveal that the TuSp1 1×C fibers were almost twice as strong and ∼17× more extensible relative to the synthetic dragline silk fibers. Our TuSp1 1×C fiber could also be stretched up to a postspin draw ratio of 6.0. On a comparative basis, this observation supports that the integration of the C-terminal domain and/or differences within the internal block repeat modules between the recombinant MaSp1 and TuSp1 fibroins are responsible for the alterations in the mechanical properties of the spun fibers. Because our TuSp1 1× construct is similar in molecular mass relative to the TuSp1 1×C construct (33 vs 45 kDa) and these spun fibers only differ by the absence or presence of the Cterminal domain, our data provide the first insight into the importance of the integration of the C-terminal domain into spun fibers to achieve higher performance threads during the in vitro spinning process. In particular, the internal block-repeat from TuSp1 produced fibers with more extensibility and higher toughness relative to fibers spun from the same protein lacking the conserved nonrepetitive C-terminus. One interesting question that emerges from our studies involves the C-terminus and its relationship to increased extensibility in the synthetic tubuliform fibers. This increased elasticity might be due to the gain of helical structure contributed from the addition of the Cterminal domain.10,13 Our X-ray diffraction data collected from postdrawn TuSp1 1× and TuSp1 1×C fibers are qualitatively similar to those seen in natural silkworm and spider silk fibers.17,18 The weaker diffuse scattering from the TuSp1 1×C spun fibers indicates a more amorphous structure relative to TuSp1 1× fibers that lack the C-terminal domain (Figure 5). This increased amorphous structure might explain, in part, the additional extensibility observed in the TuSp1 1×C spun fibers since in natural spider silks the amorphous region has been shown to contain α-helical structures.22 In our tested fusion proteins, the thioredoxin appendage represented approximately 20, 28, and 40% of the molecular mass of the ECP-2C, TuSp1 1×C, and TuSp1 1× recombinant fibroins, respectively. Addition of the thioredoxin tag increased protein solubility without abrogating the assembly of the tubuliform fibroins during the in vitro spinning process. In future experiments, it will be interesting to investigate the mechanical properties of fibers spun from recombinant proteins 310

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aggregation is occurring until postspin stretching. The structural organization of the Gly-Ala couplets could suggest these sequences give rise to β-sheets during the extrusion process, similar to those reported from silkmoth fibers.24 Although we attempted to enhance the material properties of the TuSp1 1×C fibers by supplementing the spinning dope with two different ECP-2 truncations (ECP-2N and ECP-2C), our stress−strain curves failed to show statistically significant differences in the material properties relative to the TuSp1 1×C spun fibers alone. Several reasons could explain the lack of the ECP-2 truncations to alter the material properties of the TuSp1 1×C fibers when spun as composites. First, our TuSp1 1×C construct lacks the N-terminal region of TuSp1, which has been shown to have cysteine residues.4 In dragline silk fibroins, conserved cysteine residues in the C-termini have been proposed to play an important role in inter- or intramolecular cross-linking of spidroin molecules.25,26 Given the observation that ECP-2 contains 16 cysteine residues clustered within its initial 153 amino acids, it seems plausible that the N-termini of ECP-2 and TuSp1 could physically interact to influence the mechanical properties of tubuliform silks. Because our studies do not include the N-terminus of TuSp1 or account for different oxidization states, our spinning dope contents or chemical conditions may not be optimal for intermolecular interactions. Second, our studies relied on mixing TuSp1 1×C with the truncated ECP-2 at ratios of 20:1, which was based upon Q-PCR and mRNA levels. Thus, translational regulation with the either fibroin mRNA could shift the protein mixtures. Future work will aim to identify optimal compositions of these proteins to maximize mechanical functions.

containing the C-terminal domain and different numbers of internal block repeats. Because our threads were spun from recombinant proteins containing thioredoxin tags, it would suggest that chemical signals of at least 13 kDa can be embedded into the fibers. Engineering protein signals into the N-termini of recombinant spider silk proteins without inhibiting synthetic fiber production or compromising their mechanical properties could help revolutionize the field of bioengineering, leading to the development of next generation materials that serve a wide range of different biological applications. Attempts to remove the thioredoxin tags from our TuSp1 constructs using enzymatic digestion with enterokinase resulted in poor efficiency of tag removal, even with overnight digestion (data not shown). With the recent reports of enhanced material properties for synthetic spider silks spun from fibroins that have higher molecular weights, it will be interesting to determine whether the mechanical properties of TuSp1-spun fibers can be improved using proteins with increased molecular weights. Based upon our mechanical data from reconstituted respun egg case material, we believe this upper limit will approach ∼220 MPa for the tensile strength using our current spinning methodology. The reconstituted respun egg case spinning dope approached ∼33% of the tensile strength of the natural tubuliform fibers. Because SEM and MS/MS analyses demonstrate that the major protein component of raw egg sacs is largely TuSp1,2−4 it seems to suggest that further improvements in the spinning process, in addition to our mechanical spooling and postspin draw device, will need to be developed before higher performance synthetic fibers can be spun from recombinant tubuliform fibroins. Our studies are the first to show that a 52 kDa C-terminal portion of ECP-2 can be spun into a fiber, adding evidence that this protein has fibroin properties. ECP-2C has the ability to form synthetic fibers that have comparable mechanical properties to fibers spun from truncated recombinant major ampullate spidroins. Because the amino acid sequence of ECP-2 lacks well-defined internal block repeats and the conserved nonrepetitive C-terminus signature that defines the spidroin family, it raises the question whether the ECPs represent a different gene family encoding fibroins that evolved by convergent evolution. Recently, it has been reported that cDNAs encoding the N-termini of the ECPs have been discovered in the genus Liphistius, a very ancient spider, suggesting the ECPs are evolutionarily ancient fibroins.23 Interestingly, the mechanical properties of ECP-2C fibers after postspin stretching to 3.5× its original length produced threads with an averaged tensile strength of 121.9 MPa (Table 1). Because the predicted molecular weight of full-length ECP2 is ∼80 kDa, it demonstrates that this fibroin does not follow the conventional large molecular sizes of traditional spidroin family members. Similar to other reported studies, the postspin draw altered the material properties of the ECP-2C fibers, leading to an increase in the tensile strength, but a decrease in the extensibility (Figure 4B−D). The protein sequences that are responsible for the high tensile strength are unknown. Analysis of the C-terminal region of ECP-2 shows a high content of Gly and Ala residues, but the absence of the conserved C-terminal domain that is characteristic of traditional fibroin members. Our studies reveal that the as-spun ECP-2 fibers display the largest diameter sizes relative to other as-spun fibers produced from different recombinant proteins (data not shown), indicating that during the spinning process unspecific

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CONCLUSION Our findings reveal that integration of the C-terminal domain into TuSp1-spun fibers enhances extensibility and toughness relative to fibers lacking this domain. Moreover, the tensile strength and extensibility of these fibers can be inversely modulated by different postspin draw ratios, but without changes to toughness. These results suggest that recombinant tubuliform silks containing the C-terminal domain can have their mechanical properties tailored for a specific application that require either high tensile strength and low extensibility or vice versa, without the sacrifice of toughness. Furthermore, we demonstrate that strain-hardening at postspin draw ratios approaching 6× results in tensile strengths that are comparable to recombinant dragline silk fibroins spun into artificial silk fibers.16 We also show that the C-terminal region of ECP-2, which has iterations of GA couplets, can form synthetic silk fibers with tensile strengths that are on the same order of magnitude as natural tubuliform silk fibers.14 Taken together, the synthetic tubuliform silks that contain the C-terminal domain regions are mechanically comparable to the artificial fibers spun from truncated dragline silk fibroins. The ability to regulate the material properties of the synthetic tubuliform silks offers an additional direction to pursue for the production of new biomaterials with a wide range of applications. Our studies also demonstrate that truncated recombinant spider silk proteins containing the C-terminal domain of spidroins can be spun into synthetic fibers with mechanical properties that rival larger molecular weight recombinant fibroins that lack the C-terminal domain. Furthermore, our findings highlight that the C-terminal domain functions to add extensibility to synthetic tubuliform silk fibers. Future directions will be focused at expressing larger molecular weight 311

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TuSp1 recombinant proteins that contain the C-terminus to investigate whether the mechanical properties can be further enhanced to approach mechanical values from natural spider silk fibroins. Taken together, our studies support the importance of the integration of the C-terminal domain into synthetic spider silk production. The ability to enhance the extensibility of synthetic fibers by the addition of the Cterminal domain provides new insight into advancing the mechanics of artificially spun silk threads, providing a new strategy for the development of a next generation of materials for bioengineering applications.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 209-946-3024. Fax: 209-946-3022. E-mail: cvierra@ pacific.edu.

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ACKNOWLEDGMENTS This work was supported by NSF RUI Grants MCB-0950372 and DMR-1105310 entitled Molecular Characterization of Black Widow Spider Silks and Mechanical Behavior of Spider Glue Silks, respectively. The X-ray analysis was supported by the National Science Foundation, Division of Materials Research, under Award No. DMR-0805197 (J.L.Y.) and the Department of Defense Air Force Office of Scientific Research (AFOSR) Award No. FA9550-10-1-0275 (J.L.Y.). We would also like to acknowledge Robert Henning for the assistance with the BioCARS beamline and his work is supported by the U.S. DOE, Argonne National Laboratories. under Contract No. DEAC02-06CH11357. Lastly, we thank Dr. Kristin Kohler for her critical reading of the manuscript.

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ABBREVIATIONS MS, mass spectrometry; MS/MS, mass spectrometry/mass spectrometry; MaSp1 and MaSp2, major ampullate spidroin 1 and 2; MiSp1, minor spidroin 1; AcSp1-like, aciniform spidroin 1; PySp1, pyriform spidroin 1; TuSp1, tubuliform spidroin 1; ECP-2, egg case protein 2; Q-PCR, quantitative polymerase chain reaction; ECPs, egg case proteins; HFIP, 1,1,1,3,3,3hexafluoro-2-propanol; Hz, hertz; MPa, mega Pascal; GPa, giga Pascal

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REFERENCES

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