Structural and Mechanical Roles for the C-Terminal Nonrepetitive

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Structural and Mechanical Roles for the C-Terminal Non-Repetitive Domain Become Apparent in Recombinant Spider Aciniform Silk Lingling Xu, Thierry Lefèvre, Kathleen E. Orrell, Qing Meng, Michèle Auger, Xiang-Qin Liu, and Jan K. Rainey Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01057 • Publication Date (Web): 21 Sep 2017 Downloaded from http://pubs.acs.org on September 23, 2017

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Biomacromolecules

Structural and Mechanical Roles for the C-Terminal Non-Repetitive Domain Become Apparent in Recombinant Spider Aciniform Silk

Lingling Xu1,2, Thierry Lefèvre3, Kathleen E. Orrell1, Qing Meng2, Michèle Auger3, Xiang-Qin Liu1* & Jan K. Rainey1,4*

1. Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, NS, Canada.

2. Institute of Biological Sciences and Biotechnology, Donghua University, Shanghai, P.R. China.

3. Département de Chimie, Regroupement québécois de recherche sur la fonction, la structure et l'ingénierie des protéines (PROTEO), Centre de recherche sur les matériaux avancés (CERMA) Université Laval, Québec, QC, Canada.

4. Department of Chemistry, Dalhousie University, Halifax, NS, Canada.

* (J.K.R.) E-mail: [email protected], (X-Q.L.) E-mail: [email protected]

1 ACS Paragon Plus Environment

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Abstract

Spider aciniform (or wrapping) silk is the toughest of the seven types of spider silks/glue due to a combination of high elasticity and strength. Like most spider silk proteins (spidroins), aciniform spidroin (AcSp1) has a large core repetitive domain flanked by relatively short N- and C-terminal non-repetitive domains (the NTD and CTD, respectively). The major ampullate silk protein (MaSp) CTD has been shown to control protein solubility and fiber formation, but the aciniform CTD function remains unknown. Here, we compare fiber mechanical properties, solution-state structuring, and fibrous state secondary structural composition and orientation relative to native aciniform silk for two AcSp1 repeat units with or without fused AcSp1- and MaSp-derived CTDs alongside three AcSp1 repeat units without a CTD. The native AcSp1 CTD uniquely modulated fiber mechanical properties, relative to all other constructs, directly correlating to a native-like structural transformation and alignment.

Keywords

Spider silk, aciniform silk, spidroin, C-terminal domain, mechanical properties, polarized Raman spectromicroscopy


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1. INTRODUCTION

Spiders produce up to seven different protein-based silks or glues with distinct mechanical properties tailored for specific functions.1, 2 Aciniform silk is the toughest spider silk, used to wrap and immobilize prey, build the eggcase inner layer, and decorate the web. Moreover, aciniform silk is twice as tough (the energy absorbed before rupturing, it is a combination of strength and extensibility) as spider major ampullate (MA) silk, approximately six times tougher than Kevlar® 49 and sixty times tougher than steel.1 The main component of aciniform silk is AcSp1 (aciniform spider silk protein 1, spidroin 1), which is produced and stored in the aciniform gland as a highly soluble protein and can be readily spun into solid fibers. From solution to a solid-state fiber, AcSp1 undergoes a structural transition from globular helical domains connected by intrinsically disordered linkers3, 4 to a similar proportion of disorder alongside a mixture of oriented β-sheet and α-helical domains.5 α-helix to β-sheet conversion is believed to provide strength to silk fibers.6, 7

AcSp1 is typically a large protein (~300-430 kDa, depending upon the species) composed of iterated repeats comprising >90% of the protein sequence, flanked by short N- and C-terminal non-repetitive domains (the NTD and CTD, respectively).8-10 AcSp1 from Argiope trifasciata is composed of a 200-amino acid (aa) unit (W) repeated at least 14 times and a 125-aa CTD.11 An NTD is also likely present, but has yet to be sequenced. This sandwich-like protein architecture, with a large repetitive domain flanked by small non-repetitive domains, is typical of spidroins.10, 12, 13

Currently, understanding of structure-function relationship of spider silks is primarily limited to MA silk. In MA silk, the core repetitive domain is believed to be responsible for its mechanical properties, with the CTD controlling protein solubility and fiber formation.14-18 Although the CTDs of AcSp1 and 3 ACS Paragon Plus Environment

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MaSps are similarly structured in solution,19 the core repetitive region of AcSp1 has radically different amino acid content, primary structure and secondary structuring than MaSps.5, 9 As a result, the actual function of the CTD of AcSp1 remains unknown and may not fully extrapolate from that in MaSps. Functional characterization of the AcSp1 CTD is especially important for understanding this toughest type of spider silk’s mechanical properties and for design of artificial spider silks.8, 20

Previously, we have shown that recombinant AcSp1 proteins comprising 2-4 repetitive units (W2-4) and devoid of a CTD were soluble, stable in solution for extended periods and could form silk-like fibers upon application of shear forces.3, 21, 22 We have also previously investigated a fusion of W2 with an MaSp CTD,23 showing that construct to be capable of silk-like fiber formation, albeit with less impressive mechanical properties than those we have observed for W2 through W4.24 The solubility and fiber formation competency of W unit concatemers of varying molecular weight in the absence of a CTD suggest that the role of the CTD in controlling aciniform silk protein solubility and fiber formation may not be as significant as it is for MA silk. This introduces the possibility that there may be other functional roles for the aciniform CTD, such as tailoring of fiber mechanical properties.

The potential contribution of CTDs to silk fiber mechanical properties has not been extensively investigated. The CTD of recombinant tubuliform spidroin1 (TuSp1) was shown to contribute to fiber mechanical properties by increasing the extensibility of synthetic fibers.25 However, in this study, a relatively large thioredoxin fusion tag was also present and directly correlating the exact effect of the CTD upon fiber properties is challenging. The role of fusion of NTD and/or CTD upon recombinant MA silk fiber formation and properties has also been investigated, with the conclusion that fusion of a CTD has no direct impact of fiber mechanical properties; instead, it was shown to improve spinning


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dope properties and, correspondingly, the fiber forming process.26 In short, the potential function of the CTD in controlling silk fiber mechanical properties remains unclear. To the best of our knowledge, the effect of a CTD upon structural composition and orientation in fibers has also not been directly characterized.

Here, we have maintained similar protein sizes with and without the inclusion of a CTD allowing for a more direct comparison of CTD-dependent properties. Specifically, we compare similarly sized recombinant A. trifasciata AcSp1 with (W2Cac) or without a CTD (W2 and W3) under non-denaturing conditions (all protein constructs are detailed in Figure 1). Following from the hypothesis noted above that the AcSp1 CTD may have distinct functionality, two non-native CTDs from MaSps were also fused to W2 (W2Cma1 and W2Cma2; Figure 1) and compared with W2Cac. Unique contributions to fiber mechanical properties and structural alignment were observed for the native AcSp1 CTD, relative to either a repetitive AcSp1 W unit or a non-native CTD, providing a new strategy for engineering of high performance recombinant aciniform silk.


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Figure 1. (A) Schematic representation of predicted full-length native and recombinant AcSp1 constructs (W3, W2Cma1, W2Cma2 and W2Cac) (B) Comparison of amino acid composition of W repeat unit from A. trifasciata AcSp1 and CTDs from E. australis MaSp1 (Cma1), A. trifasciata MaSp2 (Cma2) and AcSp1. (C) CTD sequence alignment for indicated spidroin from A. trifasciata (A. tri), E. australis (E. aus), Araneus diadematus (A. dia) and Nephila antipodiana (N. ant). The degree of shading indicates sequence similarity, with darker shading corresponding to higher conservation.


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2. MATERIALS AND METHODS

2.1 Cloning. CTD-encoding sequences for MaSp1 (Cma1)27 and MaSp2 (Cma2)28 and aciniform silk AcSp19 from Euprosthenops australis (Cma1) and A. trifasciata (Cma2 and AcSp1) were used along with the AcSp1 repetitive domain sequence9 (“W”) from A. trifasciata (Figure 1). The codon biases of each sequence were optimized for E. coli expression and the synthetic genes were produced (GenScript, Piscataway, NJ). Open reading frames encoding Wn and WnCx (n=1, 2 or 3; x = ma1, ma2 or ac) were constructed using the same cloning strategy as in our previous studies,21, 24 with each recombinant spidroin gene inserted downstream of a His6-SUMO encoding sequence in a modified pET vector.

2.2 Protein Expression and Purification. Each of the six recombinant His6-SUMO-AcSp1 fusion proteins (His6-SUMO-W1, His6-SUMO-W2, His6-SUMO-W3, His6-SUMO-W2Cma1, His6-SUMO-W2Cma2 and His6-SUMO-W2Cac) was expressed in E. coli BL21 (DE3) (Novagen, Darmstadt, Germany) at 22 °C for ~16 hours and purified using nickel-affinity chromatography under non-denaturing conditions, following our previous protocol.24 The His6-SUMO tag was cleaved from each protein using a His6-tagged SUMO protease (SUMO sequence from Saccharomyces cerevisiae). A given fusion protein was mixed with protease at a 100:1 ratio (w/w) at 4 °C overnight (in purification elution buffer: 50 mM sodium phosphate, 300 mM NaCl, 250 mM imidazole, pH 8.0). During proteolysis, dialysis against 50 mM potassium phosphate buffer, pH 7.5 was conducted to remove imidazole from the reaction mixture and to allow for reverse purification, since proteolytically cleaved silk proteins lacking a His6-tag would not bind to the nickel affinity column and could be collected in the flow through, while cleaved His6-SUMO, SUMO protease, and any remaining uncleaved fusion protein would bind to the affinity column.


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N-enriched W1 was prepared as previously described21 for 1H-15N heteronuclear single quantum

coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy experiments. Segmentally-labeled W1Cac, with only the W domain 15N-enriched, was produced through trans-splicing mediated by Rma DnaB split-intein.23, 29, 30 For segmental labeling, two constructs containing the 15N enriched N-precursor (W1IN: W1 + intein N-fragment (IN) + His6 tag) and C-precursor at natural abundance (ICCac: His6 tag + intein C-fragment (IC) + W1) were expressed, purified, spliced and reverse purified in a similar manner to our previous study.3

All protein samples were resolved by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue R-250. Protein purity was quantified using ImageJ 1.47v.

2.3 Fiber Production and Morphological Characterization. Fibers were hand-drawn24 and fixed on paper frames as follows. A U-shaped paper frame with a 1 cm × 0.5 cm (l × w) window was prepared. On one side of the paper, transparent tape was attached on one side of the frame, providing a waterproof surface for protein solution drop deposition, and double-sided tape was affixed to the other side, for fiber attachment. Drops (~10 µL) of purified W2, W3, W2Cma1, W2Cma2, W2Cac or W1Cac protein dissolved (~40 µM) in 50 mM potassium phosphate (pH 7.5) were deposited on the transparent tape substrate at room temperature (RT; 22 ± 2 °C). For each spidroin type, a pipette tip (200 µL plastic pipette tip, Axygen Scientific, Union City, CA, USA) was submersed into the droplet and drawn upwards out of solution, allowing silk-like fibers to be pulled from solution. The resulting fiber was then drawn across the window of the paper frame and fixed onto the double-sided tape. Fibers were subsequently air-dried at RT. After drying, fiber ends were secured using tape. The portion of each fiber spanning the frame (i.e., not in contact with tape) was employed for subsequent characterization.


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Fiber surface morphologies were characterized by scanning electron microscopy (SEM; S-4700, Hitachi, Tokyo, Japan) at an accelerator voltage of 3 kV. Fibers were fixed on conductive adhesive tape glued onto SEM stubs and coated with gold particles by an SC7620 mini sputter coater (Quorum Technologies, East Sussex, UK) before SEM imaging. An inverted optical microscope (OM; Zeiss Axiovert 200M Inverted Microscope; 400× magnification) was used to screen fibers to eliminate ones having artifacts or uneven diameters and to measure diameters for tensile testing, as previously described.24

To confirm reliable diameter estimation by OM measurements, another three to five fibers for each construct were observed using SEM. Two to three locations on each fiber were imaged and diameters were measured and averaged on micrographs acquired at a 5000× magnification. For W2Cac fibers, which often have smaller diameters, five fibers were measured by OM (×400) followed by SEM (2000×). For tensile strength testing, fibers with diameters less than 1.5 µm were excluded due to inaccuracy of OM measurements.

2.4 Tensile testing. The cross sectional area of each fiber was calculated using the averaged diameter from nine locations along the fiber. Tensile strengths were measured at 22 ± 2 °C and ~40% humidity, using an Agilent T150 UTM, following previously reported procedures.24

Breaking strength (σ), or the maximum applied stress without failure, was calculated as:31

σ = F/A

(1)

where F is the tensile force applied to the specimen and A0 is the original cross-sectional area of the specimen. Strain (ℇ) was calculated as:31


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ℇ = ∆L/L0

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(2)

where ∆L is length change of the specimen and L0 is its original length. The Young’s modulus (E), or stiffness, was determined as the slope of the stress-strain curve during the initial elastic phase:

E = σ/ℇ

(3)

Finally, toughness was calculated as the area under the stress-strain curve. One-way ANOVA tests were used to compare means of different properties, including strength, extensibility, toughness and stiffness, diameter and secondary structure orientation of four types of silk and Tukey’s honest significant difference tests for unequal sample sizes were used to compare differences between types of silk fiber.

2.5 Solution-State Protein Structural Characterization. Far-UV circular dichroism (CD) spectra of recombinant aciniform silk proteins (~0.6 mg/ml in 50 mM potassium phosphate buffer, pH 7.5) were recorded at 100 nm/min in 0.1 nm intervals from 195 to 260 nm in a 0.05 cm pathlength quartz cuvette (Hellma, Müllheim, Germany) using a J-810 spectropolarimeter (Jasco, Easton, MD) at 22 ± 2 °C. All samples were analyzed in duplicate, with the resulting spectra blank corrected, averaged and converted to mean residue ellipticity [θ]32. Protein concentration (~0.4-0.6 mg/mL) was determined by the Beer–Lambert law using absorbance at 210 nm using molar absorptivities estimated33 to be 270858 M-1cm-1, 543596 M-1cm-1, 816334 M-1cm-1, 676398 M-1cm-1, 757241 M-1cm-1, 687086 M-1cm-1 and 414348 M-1cm-1 for W1, W2, W3, W2Cma1, W2Cma2, W2Cac and W1Cac, respectively.

Uniformly 15N-enriched W1 and segmentally 15N-enriched W1Cac (~0.1mM) were dissolved in an L-arginine (50 mM)/L-glutamate (50 mM) buffer34 containing 1 mM NaN3 and 1 mM

2,2-dimethyl-2-sila-pentane-5-sulfonic acid (DSS) in H2O:D2O (9:1, v:v). 1H-15N HSQC experiments


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were acquired at 30 °C using sensitivity enhancement35 and with 15N decoupling during acquisition. For W1, a 16.4 T Avance III spectrometer equipped with a 1.7 mm TCI cryoprobe (Bruker Canada, Milton, ON; 2048×128 points, 1.5 s recycle delay, 24 scans) was employed. For W1Cac, an 11.7 T Avance spectrometer equipped with a 5 mm BBFO SmartProbe (Bruker Canada; 1024×64 points, 1.5 s relaxation delay, 64 scans) was employed. The data were processed using NMRPipe36 and visualized using CcpNMR Analysis 2.3.37

2.6 Fiber-State Protein Structural Characterization. Raman spectromicroscopy was carried out at 22.0 ± 0.5 °C and 20 ± 5% RH using a LABRAM 800HR Raman spectrometer (Horiba Jobin Yvon, Villeneuve d’Ascq, France), as previously described.38 An Ar+ laser (514 nm; 50 mW) was orientated either parallel (Z) or perpendicular (X) to the fiber axis by a half-wave plate (Melles Griot, Carlsbad, CA). Scattered light intensity was observed at parallel (Z) or perpendicular (X) polarizations to the fiber axis. Polarized spectra XX, XZ, ZX and ZZ were recorded, where the first and second letter indicate the polarization of the incident and scattered radiation, respectively. In total, 2×15 sec acquisitions were collected 3-6 times at 2-6 positions on 3-4 recombinant fibers. The spectra were baseline-corrected, smoothed, and averaged for each fiber.

The orientation-insensitive spectrum was calculated and the amide I band decomposed to estimate the content of α-helices and β-sheets, as described previously.38, 39 In order to characterize the potential variation arising from the peak fitting parameter, four sets of fitting parameters were chosen (Supporting Information, Table S1), and peak fitting was performed for each fiber type with each set of parameters. The widths for α-helix and β-sheet bands were set to be < 17.5 cm-1 and each band position was allowed to vary by a maximum of 0.5 cm-1 between all fiber types. The structural content was then


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determined on the basis of each fit for each fiber type. The average structural content was determined for the four separate fitting processes and the average deviation determined.

Orientations of α-helical and β-sheet structures in recombinant and native aciniform silk from A. trifasciata were determined using the ratio of intensities IZZ/IXX at ~1657 cm-1 (α-helix) and 1240 cm-1 (β-sheet) for the amide I and amide III bands, respectively. Baseline correction was performed using points at 1190 and 1380 cm-1 for the amide III band.

3. RESULTS AND DISCUSSION

3.1 Recombinant Spidroin Production and Purification. A His6-SUMO tag was fused to the N-terminus of each silk protein to enhance protein expression and solubility and to facilitate purification.24 All His6-SUMO tagged precursor proteins exhibited ~80% solubility upon expression. The His6-SUMO tag was effectively removed by SUMO protease digestion in each case, allowing reverse purification to ~95% purity for W1-3 and W2Cma1, ~85% for W2Cma2 and ~80% for W2Cac (Figure 2). Yields of each protein construct were similar, with ~20 mg of purified spidroin obtained per liter of E. coli culture.

3.2 Recombinant Spidroins With or Without a CTD Formed Fibers With Indistinguishable Diameter and Morphology. Fibers drawn from solutions of W2Cma1, W2Cma2, W2Cac and W3 were homogeneous in diameter (~2 µm), as measured by OM (Table 1). One-way ANOVA statistical tests showed no significant difference in fiber diameter between the four types of recombinant silk. SEM was also employed to measure fiber diameters to ensure accurate strength determination by tensile testing


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(Eq. 1). No significant difference was observed between microscopy techniques for W3, W2Cma1, W2Cma2 and W2Cac (P > 0.05; Supporting Information, Figure S1). For each type of fiber, a similar surface morphology composed of fibrillar structures with diameter ~60-200 nm running parallel to the fiber long axis was observed using SEM (Figure 3). This is consistent with the morphology previously observed using SEM and atomic force microscopy for recombinant hand-drawn fibers of W23, W4,24 and W2Cma2.23

Figure 2. SDS-PAGE (Coomassie blue stained) demonstrating spidroin purification, SUMO cleavage, and dimer formation. (A) His6-SUMO-W3 purification; (B) SUMO protease cleavage of His6-SUMO-W3; (C) flow-through of reverse purification for each of the four purified recombinant aciniform silk proteins; and (D) resolution of purified recombinant proteins under non-reducing conditions. Lane markers in (A)-(B): S: soluble protein in cell lysate; P: insoluble protein in cell lysate; U: flow-through (unbound protein); E: eluted protein; C: mixture after SUMO cleavage.


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Figure 3. Scanning electron microscopy of indicated fiber type. Scale bar: 2 µm.

Table 1. Summary of the Mechanical Data for Recombinant and Native Fibers Fiber type

Protein c

Strength

Extensibility

Toughness -3

Young's

Diameter by

(number)

size (kDa)

(MPa)

(mm/mm)

(J.cm )

modulus (GPa)

OM (µm)

W2 (9)3

38.0

66.7 ± 15.8

0.31 ± 0.11

18.4 ± 10.4

1.7 ± 0.7

1.5 ± 0.1

W3 (7)22

57.1

78.9 ± 27.9

0.21 ± 0.10

14.3 ± 7.6

2.8 ± 0.8

1.8 ± 0.1

W4 (10)24

76.1

115.1± 24.4

0.37 ± 0.11

33.8 ±13.5

2.4 ± 0.5

3.4 ± 0.3

W2Cma1 (5)

49.3

124.8 ± 25.9

0.19 ± 0.07

19.2 ± 8.3

3.9 ±0.7

2.2 ± 0.2

W2Cma2 (6)

52.8

135.3 ± 36.6

0.36 ± 0.20

41.9 ± 28.5

4.2 ± 0.7

2.1 ± 0.5

W2Cac (9)

50.3

175.1 ± 29.0

0.29 ± 0.13

41.8 ± 24.1

5.5 ± 0.5

2.1 ± 0.3

>270

687 ± 56

0.86 ± 0.03

376 ± 39

9.8 ± 3.8

0.35 ± 0.01

200-350

1290 ± 29

0.22 ± 0.01

145 ± 5

9.4 ± 1.0

3.24 ± 0.10

Native aciniform silka Native MA silkb

a: aciniform silk from A. trifasciata9 b: major ampullate silk from A. trifasciata9 c: monomeric molecular weight


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While it is possible that the observed surface features arise from drying-induced shrinkage during the fibril deposition process, similar morphology is observed in atomic force microscopy of W2 fibers where the fiber is never fully dehydrated and is imaged in a humid environment.3 Furthermore, a completely distinct surface morphology composed of micellar-type structures was observed with hand-drawn W4 fibers handled similarly and imaged by SEM.24 Finally, hand-drawn fibers formed from fusion proteins composed of both W units and MaSp repetitive domain similarly dried exhibited a smooth surface morphology.23 Hence, although the potential impact of the drying process on fiber morphology cannot be ignored, this does not appear to be the primary cause of the observed fibrillar surface morphology or, at least, does not exclusively give rise to a fibrillar morphology.

3.3 Fibers Produced from Spidroins With a CTD Show Increased Strength and Young’s Modulus. Swapping a repetitive domain for the CTD of AcSp1 from A. trifasciata (i.e., conversion from W3 to W2Cac) significantly increased fiber strength, toughness and Young’s modulus (Figure 4, Table 1). The average breaking strength values of W2Cac fibers were ~2.6×, ~2.2× and 1.5× greater than those previously observed for W2, W3 and W4 fibers, respectively, while the Young’s moduli observed for W2Cac fibers were ~2-3× those of W2, W3, and W4 fibers. Although adding a CTD did not significantly increase fiber extensibility, the average toughness observed for W2Cac fibers is more than double those observed for W2 and W3 fibers and ~1.2× that for W4 fibers.

Interestingly, exchanging the native AcSp1 CTD with cysteine-containing non-native CTDs (W2Cma1 and W2Cma2) rendered the toughness statistically indistinguishable for each type of fiber. The strength of W2Cma1 and W2Cma2 fibers was significantly increased relative to fibers of W2 and W3, but not W4, while the Young’s modulus was greater than those of W2, W3 or W4 (Figure 4, Table 1). Surprisingly, fibers


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formed by W2Cac were significantly stronger and stiffer by ~1.3-1.4× than those formed from either W2Cma1 or W2Cma2. Average toughness measurements were more variable, due in large part to variability in extensibility. It should be noted that W2Cma2 fibers were previously produced by hand-pulling, but that the resulting fibers were much weaker than those produced in the present study.23 This discrepancy may arise from differences in experimental conditions, including the protein concentration from which fibers were drawn, the manner in which fibers were handled, and/or the pulling speed.40 Herein, all fiber types being compared have been consistently prepared and handled to allow for direct comparison of properties as a function of the protein construct being employed.

Figure 4. Recombinant aciniform silk mechanical properties. (A) Stress-strain curves of three representative fibers formed from each indicated type of recombinant spidroin. (B) Expansion of area demonstrating elastic modulus (boxed region in (A)). (C) Representative optical micrograph of a W2Cac fiber employed in tensile testing.

Spider silk mechanical properties are determined by hierarchical structuring at various levels, including protein conformation; size and distribution of crystalline units; and, orientation of secondary


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structure units.6, 39, 41, 42 Given the observed differences in behavior between CTDs, the protein structuring in both the soluble and fibrous states was characterized to test for CTD-dependent variation as a potential source of altered mechanical properties.

3.4 Spidroin Structural Comparison in Solution. Far-UV CD spectroscopy was used to compare secondary structure for each recombinant spidroin in solution. We previously observed identical global structuring by CD spectroscopy for constructs with 1-4 W units.24 The modularity of W unit structuring was upheld at the atomic-level by NMR spectroscopy for W2 in comparison to W1, with W3 also exhibiting a very similar 1H chemical shift pattern.3 Echoing our previous studies of W1-W4, each of the proteins investigated herein also exhibited nearly identical CD spectra (Figure 5A). Prominent negative bands at 208 and 220 nm, along with strongly positive ellipticity at 195 nm, indicate that all proteins contain significant α-helical content in solution. This is consistent with our solution-state NMR structures of W1 and W2, showing each W unit to contain a globular domains of 5 α-helices connected to its neighbor by an intrinsically disordered linker.3, 4, 21 All CTD structures reported to date also, coincidentally, contain 5 helices followed by a disordered segment.16, 19, 43, 44 CD spectra representative of a convolution of helical and disordered structures is therefore fully expected, but the exceptional degree of similarity of mean residue ellipticity for all four protein constructs is striking and would not be predicted a priori. As a whole, it is clear that swapping of a W unit for a CTD does not perturb the structuring or independence of the repetitive units in solution and also that CTDs and W unit have a similar overall structural composition.


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Figure 5. (A) Far-UV circular dichroism spectra of indicated proteins (50 mM potassium phosphate buffer, pH 7.5, at 22 °C). (B) 1H-15N HSQC spectra of 15N-enriched W1 vs. segmentally labeled W1Cac (50 mM L-Arg, 50 mM L-Glu, pH 6.7, 30 °C) with only the W unit in W1Cac being 15N-enriched.

To test for perturbation of W unit structuring by the CTD at the atomic-level, NMR spectroscopy was employed. 1H-15N HSQC spectra for W1 and W1Cac segmentally enriched with 15N in the W-unit (i.e., effectively NMR-invisible in 15N-based experiments in the Cac domain) demonstrate a practically identical peak pattern for W1 and for the W unit in W1Cac (Figure 5B). This is entirely consistent with the behavior we observed for W1 vs. W2, where W unit structure and intrinsic disorder were unperturbed by concatenation.3, 4


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3.5 Spidroin Structure and Anisotropy in Fibers. In the transition from the soluble to the fibrous state, native aciniform silk undergoes a partial conversion from α-helix to β-sheet, retaining the same proportion of disorder in both states.5 A likewise transition has also been shown for recombinant W2-4.3, 22, 24

To determine the effect of CTD addition upon this structural transition, polarized Raman

spectromicroscopy was applied to investigate protein composition, secondary structure content and orientation in each type of fiber. Raman bands attributable to amino acid side chains45 are listed in Table 2 and annotated directly on the spectral overlay shown in Figure 6A. The intensities and shapes of these bands were consistent with all types of fibers, implying very similar amino acid composition. This echoes the fact that each of the three CTDs and the W unit share high proportions of Ser, Ala and Gly and a lack of Trp (Figure 1).

Table 2. Position and assignment of main Raman bands. Band position (cm-1)

Assignment5, 45

525

α-helix

1604, 1587, 1003, 1032, 621

Phe

1615, 855, 829, 643

Tyr

921

Pro

1452

CH2 asymmetric bend

~1658

α-helix

~1671, ~1240

β-sheet


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Figure 6. Secondary structure and structural orientation of different types of fiber. (A) Orientation-insensitive Raman spectra of indicated fibers. Amide I decomposition was based on five bands: α-helix (red); β-sheet (purple); random coil and turns (3× green). (B) Boxed areas in (A) showing amide I and amide III bands of indicated fibers. (C) Izz/Ixx ratios of amide I α-helix and amide III β-sheet (spectra shown in Figure S2). The β-sheet structure of W2Cac and native aciniform silk is statistically more oriented (indicated by “*”; P < 0.05).


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The secondary structure composition of each fiber type was determined through amide I band decomposition of the orientation-insensitive Raman spectra (e.g., expansion in Figure 6A).39 Each fiber type demonstrates a mixture of α-helical and β -sheet content, ranging from ~30-33% α-helical and ~24-27% β-sheet character (Table 3). This is highly consistent with aciniform silk fibers produced by A. aurantia, which contain ~ 33% α-helix and ~ 27% β-sheet.3 This, in particular, is well-matched to the

β-sheet content of W2Cac fibers (~27%), versus the other three types of fiber (~24% for W3 and ~25% for W2Cma1 and W2Cma2). The difference in β-sheet content determined through Raman spectral decomposition is also qualitatively demonstrated in the shape of the amide I (β -sheet shoulder at ~1670 cm-1) and amide III bands (β-sheet band at ~1240 cm-1) in the orientation-insensitive spectra (Figure 6B). Interestingly and correspondingly, the α-helical content is less in both native aciniform silk fibers (~31%) and W2Cac fibers (~30%) compared to W3, W2Cma1 or W2Cma2 (all ~33%) fibers. The higher

α-helical content in these latter recombinant fibers might imply an incomplete structural transformation. Conversely, the agreement in both α-helical and β -sheet content between native aciniform silk and W2Cac fiber indicates that the aciniform silk CTD may assist in the structural transformation from an exclusively α-helical precursor (Figure 6A and B) to a mixed α-helix- and

β-sheet-containing fiber more efficiently.

The transition from α-helix to β-sheet during fiber formation is important for fiber strength. Although W2Cac fiber has more β-sheet and less α-helix than the rest of the recombinant fibers, the difference is small, only ~2%. Hence, this seems insufficient to fully give rise to the observed mechanical property differences between these fibers.


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Table 3. Content of α-helix and β-sheet in all four artificial and native aciniform silk fibers based upon Raman spectral decomposition of amide I band. The uncertainties on the values are the average deviation of the four sets of curve-fitting parameters. Fiber type

W3

W2Cma1

W2Cma2

W2Cac

Native3

α-helix (%)

32.6 ± 0.5

32.9 ± 0.4

33.1± 0.4

29.9 ± 0.4

30.7 ± 0.2

β-sheet (%)

24.3 ± 0.3

24.8 ± 0.2

24.6 ± 0.5

26.9 ± 0.4

26.7 ± 0.2

The orientation of secondary structure elements is also known to affect fiber mechanical properties, with greater β-sheet alignment typically being correlated with increased fiber strength.46-48 Recombinant and native aciniform silks were analyzed by polarized Raman spectromicroscopy recorded perpendicular (XX) and parallel (ZZ) to the long axis of the fibers. Overlay of the amide I and amide III bands for each fiber in the ZZ and XX directions clearly demonstrates greater similarity in band shape and intensities between W2Cac fibers and native aciniform silk than between W3, W2Cma1 or W2Cma2 fibers and native silk (Supporting Information, Figure S2). Across all fiber types, the Izz/Ixx ratio for the amide I α-helical band at ~1657 cm-1 was similar (Figure 6C). In contrast, the Izz/Ixx ratios for the amide III β-sheet band at 1240 cm-1 were significantly higher for W2Cac and native fibers than the other fibers (Figure 6C). Also notably, this ratio was statistically indistinguishable between W2Cac and native fibers. These results imply that α-helices are similarly aligned in all cases, while β-sheets show greater alignment in W2Cac and native fibers. The differences in mechanical properties between W2Cac and the other three recombinant fibers (W3, W2Cma1 and W2Cma2) may, therefore, arise from a greater degree of β-sheet alignment. 22 ACS Paragon Plus Environment

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3.6 Correlating Protein Structure and Fiber Properties. Based upon previous studies,14-16 spidroin non-repetitive CTDs are thought to act like a molecular switch between soluble or aggregated states, depending on the conditions. However, it remains unclear whether CTDs play any role in fiber mechanical properties because they represent 90%.49-51

The only available experimental data, as summarized in the introduction, was the demonstration by Gnesa et al. that the CTD of recombinant TuSp1 contributed to fiber mechanical properties by increasing the extensibility of synthetic fibers.25 However, that study compared a single 180-aa repeat unit of TuSp1 either in isolation or fused to a 120-aa CTD. Furthermore, the proteins compared were flanked with N-terminal thioredoxin (105-aa) and C-terminal hexahistidine (His6) tags; therefore, directly correlating the effect of the CTD in these constructs to fiber properties is challenging because of the inclusion of the relatively large thioredoxin tag (~28% or 40% of total protein mass with or without CTD, respectively) and the corresponding ~36% increase in total protein size upon CTD fusion.

More recently, Heidebrecht et al. investigated the effect of fusion of NTD and/or CTD upon recombinant wet-spun MaSp silk fiber formation and properties.26 Differences in mechanical properties were observed, with dependence upon the size of the repetitive domain, the nature of the spinning dope employed, and the nature of post-spin stretching applied during fiber formation. It was concluded that fusion of a CTD did not directly influence fiber mechanical properties; rather, this improved spinning dope properties and fiber formation, with CTD dimerization being critical.26

Contrasting with both of these studies, our findings indicate that exchange of a W domain for a CTD in AcSp1 enhances fiber mechanical properties, despite similar spidroin size. A clear dependence upon 23 ACS Paragon Plus Environment

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the CTD employed was observed, with the native AcSp1 CTD resulting in better mechanical properties than non-native CTDs from MaSp1 and MaSp2.

It should also be noted that the MaSp-derived CTDs employed (Figure 1) both contain a single Cys, likely to facilitate disulfide-linked dimer formation.16 Cys-devoid CTDs have also been shown to dimerize in solution. Specifically the CTDs of MaSp1 from A. diadematus, MaSp2 from N. antipodiana and AcSp1 from N. antipodiana were shown to have similar, domain-swapped dimer topologies even though the latter is devoid of Cys,19 implying that dimerization does not rely on disulfide formation. This is key because dimerization would be anticipated to improve mechanical properties through an increase in spidroin molecular weight (e.g., trends for recombinant W2-W4 lacking a CTD22). Corresponding to previous studies, the potential for CTD-induced dimerization of W-CTD fusion proteins is high; specifically, >80 % of the population of cysteine-containing W2Cma1 and W2Cma2 dimerize (Figure 2D). Domain-swapped non-covalent CTD dimer formation by W2Cac lacking cysteine is possible, and the high degree of similarity between the CD spectra for all W2-CTD fusion proteins could be considered as evidence for this (Figure 5A). However, W3 (with no CTD) does not appear significantly different by CD spectroscopy. This implies that CD spectroscopy may not be sufficiently sensitive to distinguish between subtleties in AcSp1 domain structuring. Indeed, perturbation of the W2 CD spectrum upon addition of the detergent dodecylphosphocholine was minimal, despite significant perturbation at the atomic-level characterized by triple-resonance NMR experiments and evidenced by concomitant disruption of fiber formation capability.3

Conversely, and in direct contrast to the behavior of the cysteine-devoid N. antipodiana AcSp1 CTD,19 W2Cac did not show significant evidence of dimer formation by SDS-PAGE (Figure 2D).


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Furthermore, the solution-state NMR spectroscopic behavior of W1Cac was consistent with a monomeric ~31 kDa species rather than a dimeric ~62 kDa species (Figure 5B); however, in depth hydrodynamics characterization3, 4 would be necessary to make this conclusion unambiguously.

As a whole, the effect of the potential for dimerization, and a corresponding doubling of spidroin size, must therefore be considered on mechanical properties. Our previous studies on fibers formed by W2 and W4 show that those formed from W4 are almost twice as strong as those from W2 (~115 MPa vs. ~67 MPa; Table 1). Correspondingly, W3 fibers exhibit intermediate strength (~79 MPa). It is, therefore, possible that the improved properties of fibers formed from W2-CTD fusion proteins are more related to a spidroin size increase arising from dimerization than to the CTD itself. However, even if W2Cac dimerizes, doubling the spidroin size, this does not explain the source of the difference in the mechanical properties observed for W2Cac relative to W2Cma1 and W2Cma2 fibers, since all three of these proteins would form ~100 kDa dimers falling within 7 kDa of each other.

Fibers formed from recombinant spidroin lacking a CTD (W3) or with each of three different CTDs (W2Cma1, W2Cma2 and W2Cac) showed similar trends in structural transition, from a mainly α-helical state in solution to a mixed α-helical/β-sheet containing fiber. Despite this similarity, the CTD from aciniform silk appears to augment this process, as W2Cac fibers exhibited a more complete structural conversion and improved structural alignment relative to the other recombinant silks (Figure 6 and Table 3). Strikingly, the secondary structure content and the degree of alignment in W2Cac are comparable to those observed in native aciniform silk. The observed increased stiffness of W2Cac fibers relative to the other three types of recombinant silk fiber, thus, likely arises from the combined effects


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of increased β-sheet structure content and the improved alignment of β -sheet structuring induced by the native aciniform CTD.

4. CONCLUSIONS

This work highlights the impact of the non-repetitive CTD on aciniform silk fiber properties, revealing the potential for integration of the C-terminal domain as a route to deliver fibers with higher performance. Specifically, the relatively short W2Cac improved fiber properties relative to similarly sized W2 or W3 proteins correlated to an improvement in both β -sheet content and structural alignment similar to those observed in native aciniform silk. The importance of the native CTD was further demonstrated upon exchange of the AcSp1 CTD with non-native CTDs from MA silks, leading to fibers with lower stiffness. In summary, the native aciniform silk CTD appears to play important roles both in enhancing the requisite structural transition and the orientation of structural units during the silk fiber formation process. This provides a straightforward means, distinct from other reports on silk CTD behavior, to enhance desirable recombinant aciniform silk properties.

Supporting Information Available

One PDF file is available containing Table S1: Raman spectromicroscopy peak fitting parameters for amide I; Figure S1: comparison of diameter measurements by SEM and OM; and, Figure S2: Polarized Raman spectra (XX and ZZ directions) of amide I and amide III for recombinant and native fibers. This material is available at free of charge via the Internet at http://pubs.acs.org 26 ACS Paragon Plus Environment

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Acknowledgements

Thanks to Prof. Michel Pézolet for helpful discussions during the early stages of this work; Dr. Mike Lumsden (NMR3 Facility, Dalhousie University) and Ian Burton (National Research Council (NRC), Halifax NS) for NMR instrument support; Bruce Stewart for technical assistance; Dr. David Waisman for CD spectropolarimeter access; Patricia Scallion for SEM assistance; and, Jingna Zhao for tensile testing assistance. This work was supported by Discovery Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC, to M.A., X-Q.L, and J.K.R.); Dalhousie Medical Research Foundation Capital Equipment Grants (to X-Q.L. and J.K.R.); an NSERC Research Tools & Instruments Grant (to J.K.R.); an NSERC Discovery Accelerator Supplement (to J.K.R.); the National Natural Science Foundation of China (No. 31570721 to Q.M.); the Science and Technology Commission of Shanghai Municipality (No. 14521100700 and No. 14520720200 to Q.M.); the Regroupement québécois de recherche sur la fonction, l’ingénierie et les applications des protéines (PROTEO), the Centre de recherche sur les matériaux avancés (CERMA) and the Centre québécois sur les matériaux fonctionnels (CQMF) (to M.A.); a Canadian Institutes for Health Research New Investigator Award (to J.K.R.); and, an NSERC Undergraduate Student Research Award (to K.E.O.). The TCI probe for the 16.4 T NMR spectrometer at the NRC was provided by Dalhousie University through an Atlantic Canada Opportunities Agency Grant.


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Table of Contents Graphic:


Structural and Mechanical Roles for the C-Terminal Nonrepetitive

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