Production and properties of triple chimeric spidroins

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Production and properties of triple chimeric spidroins Yizhong Zhou, Anna Rising, Jan Johansson, and Qing Meng Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00402 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

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Biomacromolecules

Production and

properties

of

triple

chimeric

spidroins Yizhong Zhou†, Anna Rising§, ‡, Jan Johansson‡ & Qing Meng*, † †

Institute of Biological Sciences and Biotechnology, Donghua University, Shanghai 201620, P.R. China

§

Department of Anatomy, physiology and biochemistry, Swedish university of Agricultural Sciences, Uppsala, Sweden. ‡

Department of Neurobiology, Care Sciences and Society (NVS), Karolinska Institutet, Stockholm, Sweden.

KEYWORDS. spider, silk, artificial, aciniform, protein structure, mechanical properties

ABSTRACT: All spider silk proteins (spidroins) are composed of N- and C-terminal domains (NT and CT) that act as regulators of silk solubility and assembly, and a central repetitive region, which confers mechanical properties to the fiber. Among the seven types of spider silks, aciniform silk has the highest toughness. Herein, we fused NT and CT domains from major and minor ampullate spidroins (MaSps and MiSps), respectively, to 1-4 repeat domains (W) from another type of spidroin, aciniform spidroin 1(AcSp1). Although the three domains originate from distantly related spidroin types, they keep their respective characteristics in the chimeric

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spidroins. Furthermore, all chimeric spidroins could form silk-like fibers by manual-drawing. In contrast to fibers made in the same manner from W domains only, NTW1-4CT fibers show superior mechanical properties. Our results suggest that chimeric spidroins with NT, CT and repeat domains can be designed to form fibers with various mechanical properties.

Introduction The seven naturally occurring types of spider silks are comprised of specific spider silk proteins (spidroins). Most silk fibers combine high stress and strain tolerance and are therefore very tough, defined as energy absorbed before failure. Moreover, spider silks have low density and appear to be biocompatible1, 2. These features make spider silks interesting for use in a broad range of biomedical applications, including drug delivery, nerve regeneration and production of artificial skin3-7. The spidroins usually consist of three main parts; two conserved terminal domains and a core repetitive domain. The amino acid sequence of the repetitive domain is characteristic for each spidroin type and confer specific mechanical properties to different fibers8-11. Recent genomic and transcriptomic data, however, show that repetitive patterns are more complex than previously realized and that the spidroins are produced in different glands in an overlapping manner12. The N- and C-terminal domains (NT and CT) form five-helix bundles and are regulators of silk formation13-15. When the pH is lowered from neutral pH to 90%. The correct identities of all spidroins were confirmed by western-blot using an anti-His6 antibody (Figure 2B). ESI-MS after HPLC under denaturing conditions was used to verify the covalent structure of purified spidroins. ESI-MS of NTW1CT gave a main peak for [M+H]1+ of 45765.6, which is in excellent agreement with the calculated mass of 45766 Da. Also a peak for [M+2H]2+ at 22882.8 and a peak corresponding to a dimer can be observed (Supplementary S2). For NTW3CT smaller sized bands were seen in the Coomassie stained gel, which were not detected by immunoblotting (Figure 2A and B). This suggests that the extra bands are non-spidroin components or that they are degradation products that lack the His-tag.


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Biomacromolecules

Figure 2. Recombinant protein expression and identification. Migration of protein size markers are shown in lane M. Except NTW4CT, the proteins were purified by Ni-NTA after cell lysis and solubilization, and NTW4CT required urea denaturation and refolding before purification by Ni-NTA. Purified proteins were analyzed SDSPAGE followed by (A) Coomassie brilliant blue staining and (B) Western-blot with His6-tag antibody.

The NTW1CT could be concentrated to 574mg/mL in 20mM Tris-Cl, pH 8.0, 500mM NaCl without precipitation (Table 1), which is comparable to the solubility of the recently described recombinant spidroin NT2RepCT27. The highly concentrated NTW1CT solution was transparent and viscous. The number of W modules correlated negatively also to solubility (Table 1). Secondary and quaternary structures of recombinant spidroins CD spectra revealed that all four recombinant spidroins in solution at pH 7.0 had a mainly αhelical conformation (Figure 3, Table 2). Although the four constructs have different number of W modules, they all showed almost identical CD spectra, which indicates that NT, CT as well as the repeat modules, as expected, were in an α-helical conformation14,

32, 47

. Furthermore, the

similar spectra obtained for all constructs imply that the NTW4CT spidroin refolded correctly after urea denaturation.


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Figure 3. Analysis of spidroin secondary structure contents. The CD mean residual ellipticity (MRE, 103×deg.dmol-1.cm2.residue-1) for NTW1CT, NTW2CT, NTW3CT and NTW4CT at pH 7.0, 10mM sodium phosphate buffer.

Table 2. The contents of each secondary structure of NTW1~4CT protein solution.a NTW1CT

NTW2CT

NTW3CT

NTW4CT

α-helix

50%

51%

54%

54%

β-sheet

10%

11%

9%

11%

β-turn

16%

15%

16%

13%

Random coil

24%

23%

21%

22%

a

The secondary structure contents were estimated from CD spectra using the CDpro and the method CDSSTR.

Next, we analyzed the effects of pH and temperature on the secondary structure. When the pH was decreased from 7.0 to 5.5 the CD spectra of NTW1CT indicated a loss of α-helical structure and an increased β-sheet content (Figure 4, Table 3). The isodichroic point at around 203 nm48 is compatible with a two-state model for pH induced unfolding. The most drastically change in secondary structure content occurred between pH 6.5 to 6.0. Spectra of NTW2CT to NTW4CT at different pH showed similar properties (Supplementary Figure S3).


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Biomacromolecules

Figure 4. Protein solution stability of NTW1CT at different pHs. CD spectra of NTW1CT was measured and calculated at pH 7.0, pH 6.5, pH 6.0 and pH 5.5 (10mM sodium phosphate buffer) to show the effect of pH at 20°C.

Table 3. The secondary structure contents of NTW1CT protein solution in different pH. NTW1CT

pH 7.0

pH 6.5

pH 6.0

pH 5.5

α-helix

50%

47%

30%

22%

β-sheet

10%

12%

21%

25%

β-turn

16%

16%

21%

23%

Random coil

24%

25%

28%

30%

Temperature induced denaturation of NTW1CT at pH 7.0 showed that the spidroin converts from α-helix to β-sheet structure, and that do not refold completely after cooling (Figure 5A). There is an isodichroic point at about 203 nm48, compatible with a two-state unfolding model14, 17, 49

, The melting curve did not show plateaus before or after melting, which was also observed

for the melting curve of W150. The apparent melting temperature (Tm) of NTW1CT was 53°C (Figure 5B), which was close to the Tm of NT (54°C ~60°C)47, but lower than the Tm for CT (65°C)14 or W modules (71°C)50.


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Figure 5. Temperature-induced unfolding of NTW1CT (A) CD spectra measured at 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C following a 1-minute incubation and at 20°C after cooling (asterisk) to show the effect of temperature at pH 7.0 in 10mM sodium phosphate buffer. (B) To obtain the melting temperature (Tm), CD signal of NTW1CT was measured at 222nm between 20°C -80°C and converted to folded fraction.

Upon size exclusion chromatography (SEC), NTW1CT eluted between calibrant proteins with masses of 60 and 120 kDa, respectively. As the calculated molecular weight of NTW1CT monomer is ~46 kDa, in agreement with SDS-PAGE analysis (Figure 2A) the SEC results indicate that NTW1CT is a dimer in solution (Supplementary Figure S4). Morphology and mechanical properties of silk-like fibers All four chimeric spidroins could form more than 10 cm long silk-like fibers by manualdrawing from aqueous solutions. Inverted phase microscopy and scanning electron microscopy were used to examine the morphology of the fibers. The fibers of NTW1CT to NTW4CT all had smooth surface and a diameter of ~1.5µm (Figure 6A, Figure 7), which is similar to the diameters of native aciniform silk fibers (0.35µm) and Wx fibers (1.5~3.8µm)16, 32. The fracture surface of fibers displayed circular cross-sections (Supplementary Figure S5). The part of the fiber closest to the solution from which the fiber was drawn showed many smaller fibrils (Figure


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Biomacromolecules

6), which suggest that the fibers were assembled from subfibrils, as previously observed for native spider silk and WX(X=2-4) fibers32, 33, 51.

Figure 6. Inverted phase microscopy images of a tail end of fiber from NTW1CT spidroin. The boxed area in (A) image (100X magnification) is shown at a higher magnification in (B) image (1000X magnification). Scale bars: (A) 40µm (B) 4µm

Figure 7. Scanning electron microscopy images of fibers formed from NTW1CT to NTW4CT. The fibers from NTW1CT (Φ1.00µm) (A), NTW2CT (Φ1.37µm) (C), NTW3CT (Φ1.21µm) (E) and NTW4CT (Φ2.72µm) (G) were


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made by manual-drawing. (B) (D) (F) (H) are zoomed-in images (10000X magnification) of boxed areas in (A) (C) (E) (G) images (5000X magnifications), respectively. Scale bars: (A) (C) (E) (G) 2µm ;(B) (D) (F) (H) 1µm.

The secondary structure contents of the fibers were determined by FTIR spectroscopy. All fibers gave similar spectra irrespective of which construct that was analyzed. A representative spectrum is shown in Figure 8. FTIR of NTW1-4CT fibers indicated a lower fraction of α-helical structure and a slightly increased β-sheet conformation compared to Wx fibers, W2Cx(x=ma1, ma2, ac) fibers and native aciniform silk33, 34 (Table 4). A comparison of the secondary structure contents of NTW1CT fibers (Table 4) and NTW1CT protein solution (Table 2) shows that in the fibers the contents of α-helical and random coil conformations are decreased, while the contribution of βsheet and β-turn conformations are increased.

Figure 8. Secondary structure contents of silk-like fibers determined by FTIR spectroscopy. Decomposition of NTW1CT fiber spectrum in the amide I region (1600-1700cm-1). Red peaks correspond to α-helix; green peaks correspond to β-turn; pink peeks correspond to random coil; and blue peaks correspond to β-sheet. Black curve corresponds to the original curve and yellow curve corresponds to the sum of the decomposed spectra.

Table 4. Secondary structure contents of NTW1~4CTa, W2b, W2Cacb fibers and natural A.aurantia aciniform silk b.


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Biomacromolecules

NTW1CT NTW2CT NTW3CT NTW4CT

W233

W2Cac34

A.aurantia silk33

α-helix

23%

23%

22%

22%

32%

30%

31%

β-sheet

32%

28%

28%

31%

28%

27%

27%

β-turn

30%

34%

31%

34%

nd

nd

nd

Random coil

15%

15%

19%

13%

nd

nd

nd

a

The secondary structures contents of NTW1~4CT fibers are based upon FTIR spectral decomposition of Amide I band (Figure 8). bThe percentage of α-helix and β-sheet in W2, W2Cac fibers and natural A.aurantia aciniform silk are calculated from the decomposition of Amide I band from Raman spectra. nd, Not determined.

Typical stress-strain curves of triple chimeric spidroins fibers are shown in Figure 9. The maximum tensile strength and the toughness of manual-drawing fibers increased with increasing numbers of W modules. The stress at break for the NTW4CT fibers is 245±34MPa (Table 5), which is superior to the other three types of artificial silk fibers now examined. This is about one third of the tensile strength of native aciniform silk from Argiope trifasciata (687±56MPa)16 that contains at least 14 repetitive regions. The breaking strain of NTW4CT fibers was 28%±11%, which is one-third that of the natural aciniform silk (86%±3%)16. The Young’s module of NTW4CT fibers was 7±2GPa, which is close to that of native aciniform silk (9.8±3.8GPa)16.


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Figure 9. Stress-strain curves of representative fibers of NTW1CT to NTW4CT.

Table 5. Mechanical properties of chimeric spidroins fibers and natural aciniform silks. Data for the triple chimeric spidroins are from this work, while data for W2, W3 and W4 are from references 33, 51 and 32, respectively, as indicated.

a

Fiber Type

Diameter (µm)

Stress at Break Strain at Break (MPa) (mm/mm)

Young’s Modulus (GPa)

Toughness (MJ/m3)

NTW1CT

1.4±0.4

51.5±20

0.12±0.06

2.8±0.8

4.80±3.2

NTW2CT

1.6±0.6

114±31

0.19±0.07

4.8±0.8

18.0±9.4

NTW3CT

1.3±0.4

170±43

0.31±0.14

5.7±1.0

40.8±28

NTW4CT

1.5±0.4

245±34

0.28±0.11

6.7±1.8

50.7±20

W233

1.5±0.1

66.7±16

0.31±0.11

1.7±0.7

18.4±10.4

W352

1.8±0.1

91.6±22

0.23±0.11

2.9±0.6

16.0±6.9

W432

3.4±0.3

115.0±24

0.37±0.11

2.4±0.6

33.8±13

Native silka

0.35±0.01

687±56

0.86±0.03

9.8±3.8

376±39

Aciniform silk from A. trifasciata16. All error bars represent s.d. (n=9).

The mechanical properties of manual-drawing NTWX(X=2-4)CT fibers are similar or clearly superior to WX(X=2-4) fibers32-34, 52 which have the same number of repeat regions (Table 5). We also compared NTWX(X=1-3)CT fibers with WX(X=2-4) fibers, since the constituent spidroins have


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Biomacromolecules

similar molecular masses(about 8kDa difference in molecular weight). This showed that except strain at break, mechanical properties of NTWX(X=1-3)CT fibers were almost the same or still higher than WX(X=1-3) fibers Table 5. With increasing number of W units, more pronounced differences in mechanical properties are seen, especially in tensile strength and Young’s modules.

Discussion Herein we designed four triple chimeric spidroins by fusing an NT from E. australis MaSp1 and a CT from A. ventricosus MiSp1 with one to four repetitive parts from A. trifasciata AcSp1. This is the first study of chimeric spidroins that are composed of three domains from different spidroins and spider species. The yields and solubility of NTW1CT to NTW4CT are all higher than for W2Cx(x=ma1,ma2,ac)34 . This might be caused by the high solubility mediated by the NT and CT domains13, 14, 53. The secondary structures of NTW1CT to NTW4CT determined by CD spectroscopy agree well with a combination of the secondary structures of the three constituent domains17,

33, 47

,

supporting the concept that NT, CT and repetitive domains from different spidroins are interchangeable and that their secondary structure are minimally influenced by surrounding domains. This applies also to NTW4CT, even though it was purified after denaturation and refolding. For NTW1CT transition of secondary structure can be induced by a decrease in pH or increase in temperature, and the structural conversion upon thermal denaturation is irreversible. These phenomena are in contrast to the situation observed for W1, which is insensitive to pH change


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and can refold after thermal denaturation50. These results support the view that NT and CT domains confer pH sensitive secondary structure changes to the triple chimeric spidroins. Furthermore, previously studies show that when pH is decreased below ~6.5, NT will dimerize and CT will get destabilized, which may lead to structural conversion of the repeat domains into β-sheet conformation38. The pH and temperature induced secondary structure transformations of our spidroins illustrate that NT, CT and the repetitive parts keep their respective properties also when fused. Previous studies have shown that W1 alone cannot form fibers32, 50. Likewise, the NT or CT in isolation cannot be assembled into silk-like fibers47. In here we show that a triple chimeric NTW1CT can form fibers by manual drawing, thus the MaSp1 NT from E. australis and the MiSp CT from A. trifasciata in combination with AcSp W enables fiber formation. Fiber formation was accomplished without lowering the pH and accompanied by a transition of the secondary structure from α-helical to β-sheet conformation. Possibly, this is mediated by shear forces that may cause the CT to unfold and go into β-sheet aggregates15. Although it previously has been shown that the NT and CT domains can increase the mechanical properties of biomimetically wet-spun fibers35, it remains unclear whether the two domains play a similar role in fibers formed by manual-drawing. NTW2CT to NTW4CT fibers displayed higher mechanical properties than W2 to W4 fibers, suggesting that, with identical number of repeat W domains, the presence of NT and CT contribute to the mechanical properties of fibers. We also compared the properties of fibers made from proteins of similar molecular weights of the respective subunits, ie not taking the non-covalent dimeric nature of the CT containing spidroins into account. NTW1CT to NTW3CT fibers still showed higher breaking stress, toughness and Young’s modulus, but displayed a slightly lower breaking strain than W2 to


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Biomacromolecules

W4 fibers. More pronounced differences in mechanical properties were seen with increasing number of W units. This strongly suggests that in manual-drawn fibers, the presence of NT and CT domains regulates assembly of the repetitive W domains in a manner that reflects their physiological functions and that results in mechanically superior fibers. The triple chimeric nature of the presently analysed spidorins hints that mechanical properties are independent of any potential influences from co-evolution of three domains in natural spidroins. The secondary structures of NTW1~4CT fibers differ from W2Cx(x=ma1,ma2,ac) fibers and native aciniform silk by having a lower proportion of α-helical structure and increased β-sheet conformation (Table 4). Previous studies suggest that secondary structures that are close to those in native silk result in improved mechanical properties34. However, mechanical properties of NTW2CT are similar to W2Cma1 and W2Cma2 fibers34 although their secondary structures are different (Table 4). This may suggest that additional factors are important as well, or that different methods for measuring secondary structures can give somewhat different results. The correlations between mechanical properties of fibers and the number of W units of the spidrions were analyzed. The mechanical properties of NTW1CT to NTW4CT fibers correlate with the number of W units. Except for the breaking strain (R2=0.84), the breaking stress, Young’s modulus and toughness of NTW1CT to NTW4CT fibers are linearly correlated (R2>0.9) to the number of W units (Figure 10). Also non-linear regression analysis give strong correlations (R2>0.9) between mechanical parameters and the number of W units. This is in contrast to W2 to W4 fibers that do not show any correlation between mechanical properties and number of W units34. Mechanical properties of previously engineered spider silks did not show any linear dependence on molecular weight34, 36. These results indicate that under non-denaturing


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conditions and the presence of NT and CT result in spidroins in which the number of repeat units correlate with mechanical properties.

Figure 10. Mechanical properties correlate with number of repeat units in triple chimeric spidroins. The graph shows linear regression analysis of mechanical properties versus number of W units in NTW1~4CT fibers.

Conclusions In this study, we constructed triple chimeric minispidroins NTW1~4CT and studied their properties. As expected, the expression level and solubility were very high for the shortest spidroins and decreased with increased molecular weight. All three modules NT, W and CT kept their secondary structures in fusion although they are derived from different spidroin types and spider species. All chimeric spidroins, including NTW1CT, could form silk-like manual-drawn fibers at neutral pH. These results reveal that the presence of NT and CT domains in chimeric spidroins can promote the formation of fibers also in a fiber forming process based mainly on shear forces. Fibers were composed of subfibrils and had smooth surfaces and showed circular cross-sections after breakage. In general, the mechanical properties of NTW1~4CT fibers correlated with the number of W units, as observed for WX(X=2-4) fibers32, and the mechanical properties of NTWX(X=1-4)CT fibers were better than WX(X=2-4) fibers. This supports that the


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Biomacromolecules

presence of NT and CT domains regulates the assembly of W units and contribute to the fibers’ mechanical properties, and offers a possible way to design chimeric spidroins that give rise to silk fibers with defined mechanical properties.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge. The complete amino acid sequence of NT, W, CT units; the ESI-MS spectrum of NTW1CT spidrions; detailed information concerning CD data analysis; CD spectra of NTW2~4CT at different pH; Size-exclusion chromatograms of purified NTW1CT spidroins and calibrant proteins; the fracture surface of a chimeric spidrion fiber; the FTIR spectrum of NTW1CT fibers(PDF). AUTHOR INFORMATION Corresponding Author *e-mail: [email protected] Author Contributions AR, JJ, QM conceived the study. YZ did all experimental work. All authors analyzed the results and contributed to writing the manuscript. All authors have approved to the final version of the manuscript. Funding Sources


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This work was supported by grants from the Ministry of Education of the People's Republic of China (No. TS2011DHDX025), Science and Technology Commission of Shanghai Municipality (No. 14521100700 and No. 14520720200), National Natural Science Foundation of China (No. 31570721), the Swedish Research Council and CIMED (Stockholm county council and Karolinska Institutet). Notes The authors declare no competing financial interest

ACKNOWLEDGMENT We are grateful to Dr. Nina Kronqvist, Prof. Mingwu Shen, Prof. Xiangyang Shi, Prof Xiumei Mo, Tonghe Zhu, Dr. Lan Li, Prof. Changrui Lu, Shanshan Feng, Dr. Rong Wang, Dr. Xiaohua Zhang, Weiwei Li and Anguo Sun for constructive advice. This work was supported by grants from the Ministry of Education of the People's Republic of China (No. TS2011DHDX025), Science and Technology Commission of Shanghai Municipality (No. 14521100700 and No. 14520720200), National Natural Science Foundation of China (No. 31570721) and the Swedish Research Council and CIMED (Stockholm county council and Karolinska Institutet).

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for Table of Contents use only

Production and

properties

of

triple

chimeric

spidroins Yizhong Zhou†, Anna Rising§, ‡, Jan Johansson*, §, ‡ & Qing Meng*, †


33


Figure 1. Schematic illustration of NTW1~4CT proteins. His6-tags are located N-terminally of NT in all constructs. The molecular weight and the number of amino acid residues of each triple chimeric spidroin are shown. 251x119mm (300 x 300 DPI)


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Figure 2. Recombinant protein expression and identification. Migration of protein size markers are shown in lane M. Except NTW4CT, the proteins were purified by Ni-NTA after cell lysis and solubilization, and NTW4CT required urea denaturation and refolding before purification by Ni-NTA. Purified proteins were analyzed SDSPAGE followed by (A) Coomassie brilliant blue staining and (B) Western-blot with His6-tag antibody. 143x93mm (300 x 300 DPI)



Figure 3.Analysis of spidroin secondary structure contents. The CD mean residual ellipticity (MRE, 103×deg.dmol-1.cm2.residue-1) for NTW1CT, NTW2CT, NTW3CT and NTW4CT at pH 7.0, 10mM sodium phosphate buffer. 251x179mm (300 x 300 DPI)


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Figure 4.Protein solution stability of NTW1CT at different pHs. CD spectra of NTW1CT was measured and calculated at pH 7.0, pH 6.5, pH 6.0 and pH 5.5 (10mM sodium phosphate buffer) to show the effect of pH at 20℃. 251x178mm (300 x 300 DPI)



Figure 5. Temperature-induced unfolding of NTW1CT (A) CD spectra measured at 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃ following a 1-minute incubation and at 20℃ after cooling (asterisk) to show the effect of temperature at pH 7.0 in 10mM sodium phosphate buffer. (B) To obtain melting temperature(Tm), CD signal of NTW1CT was measured at 222nm between 20℃-80℃ and converted to folded fraction. 215x88mm (300 x 300 DPI)


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Figure 6. Inverted phase microscopy images of a tail end of fiber from NTW1CT protein. The boxed area in (A) image (100X magnification) is shown at a higher magnification in (B) image (1000X magnification). Scale bars: (A) 40µm (B) 4µm 209x77mm (300 x 300 DPI)



Figure 7. Scanning electron microscopy images of fibers formed from NTW1CT, NTW2CT, NTW3CT and NTW4CT. The fibers from NTW1CT (Φ1.00µm) (A), NTW2CT (Φ1.37µm) (C), NTW3CT (Φ1.21µm) (E) and NTW4CT (Φ2.72µm) (G) were made by manual-drawing. (B) (D) (F) (H) are zoomed-in images of boxed areas in (A) (C) (E) (G), respectively. Scale bars: (A) (C) (E) (G) 2µm ;(B) (D) (F) (H) 1µm. 227x335mm (300 x 300 DPI)


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Figure 8. Secondary structure content of silk-like fibers determined by FTIR spectroscopy. Decomposition of NTW1CT fiber spectrum in the amide I region (1600-1700cm-1). Red peaks correspond to α-helix; green peaks correspond to β-turn; pink peeks correspond to random coil; and blue peaks correspond to β-sheet. Black curve corresponds to the original curve and yellow curve corresponds to the sum of the decomposed spectra. 221x164mm (300 x 300 DPI)



Figure 9. Stress-strain curves of representative fibers of NTW1CT to NTW4CT 189x146mm (300 x 300 DPI)


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Figure 10. Mechanical properties correlate with number of repeat units in triple chimeric spidroins.The graph shows linear regression analysis of mechanical properties versus number of W units in NTW1~4CT fibers. 297x210mm (300 x 300 DPI)


Production and properties of triple chimeric spidroins

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