Evidence of Decoupling Protein Structure from Spidroin Expression in Spider Dragline Silks

The exceptional strength and extensibility of spider dragline silk have been thought to be facilitated by two spidroins, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2), under the assumption that protein secondary structures are coupled with the expressed spidroins. We tested this assumption for the dragline silk of three co-existing Australian spiders, Argiope keyserlingi, Latrodectus hasselti and Nephila plumipes. We found that silk amino acid compositions did not differ among spiders collected in May. We extended these analyses temporally and found the amino acid compositions of A. keyserlingi silks to differ when collected in May compared to November, while those of L. hasselti did not. To ascertain whether their secondary structures were decoupled from spidroin expression, we performed solid-state nuclear magnetic resonance spectroscopy (NMR) analysis on the silks of all spiders collected in May. We found the distribution of alanine toward β-sheet and _3,10helix/random coil conformations differed between species, as did their relative crystallinities, with A. keyserlingi having the greatest _3,10helix/random coil composition and N. plumipes the greatest crystallinity. The protein secondary structures correlated with the mechanical properties for each of the silks better than the amino acid compositions. Our findings suggested that a differential distribution of alanine during spinning could decouple secondary structures from spidroin expression ensuring that silks of desirable mechanical properties are consistently produced. Alternative explanations include the possibility that other spidroins were incorporated into some silks.     

Diversified Structural Basis of a Conserved Molecular Mechanism for pH‐Dependent Dimerization in Spider Silk N‐Terminal Domains

Conversion of spider silk proteins from soluble dope to insoluble fibers involves pH‐dependent dimerization of the N‐terminal domain (NT). This conversion is tightly regulated to prevent premature precipitation and enable rapid silk formation at the end of the duct. Three glutamic acid residues that mediate this process in the NT from Euprosthenops australis major ampullate spidroin 1 are well conserved among spidroins. However, NTs of minor ampullate spidroins from several species, including Araneus ventricosus (^AvMiSp NT), lack one of the glutamic acids. Here we investigate the pH‐dependent structural changes of ^AvMiSp NT, revealing that it uses the same mechanism but involves a non‐conserved glutamic acid residue instead. Homology modeling of the structures of other MiSp NTs suggests that these harbor different compensatory residues. This indicates that, despite sequence variations, the molecular mechanism underlying pH‐dependent dimerization of NT is conserved among different silk types.     

Exploring the backbone dynamics of native spider silk proteins in Black Widow silk glands with solution-state NMR spectroscopy

Spider dragline silk is an outstanding biopolymer with a strength that exceeds steel by weight and a toughness greater than high-performance fibers like Kevlar. For this reason, understanding how a spider converts the gel-like, aqueous protein spinning dope within the major ampullate (MA) gland into a super fiber is of great importance for developing future biomaterials based on spider silk. In this work, the initial state of the silk proteins within Black Widow MA glands was probed with solution-state NMR spectroscopy. ¹⁵N relaxation parameters, T₁, T₂ and ¹⁵N-{¹H} steady-state NOE were measured for twelve backbone environments at two spectrometer frequencies, 500 and 800 MHz. The NMR relaxation parameters extracted for all twelve environments are consistent with MA silk protein backbone dynamics on the fast sub-nanosecond timescale. Therefore, it is concluded that the repetitive core of spider MA proteins are in an unfolded, highly flexible state in the MA gland.     

Recombinant Silk Proteins with Additional Polyalanine Have Excellent Mechanical Properties

This paper explores the structures of exogenous protein molecules that can effectively improve the mechanical properties of silkworm silk. Several transgenic vectors fused with the silkworm fibroin light chain and type 3 repeats in different multiples of the ampullate dragline silk protein 1 (MaSp1) from black widow spider with different lengths of the polyalanine motifs were constructed for this study. Transgenic silkworms were successfully obtained by piggyBac-mediated microinjection. Molecular detection showed that foreign proteins were successfully secreted and contained within the cocoon shells. According to the prediction of PONDR^® VSL2 and PONDR^® VL-XT, the type 3 repeats and the polyalanine motif of the MaSp1 protein were amorphous. The results of FTIR analysis showed that the content of β-sheets in the silk of transgenic silkworms engineered with transgenic vectors with additional polyalanine was significantly higher than that of wild-type silkworm silk. Additionally, silk with a higher β-sheet content had better fracture strength and Young’s modulus. The mechanical properties of silk with longer chains of exogenous proteins were improved. In general, our results provide theoretical guidance and technical support for the large-scale production of excellent bionic silk.     

Silk Spinning in Silkworms and Spiders

Spiders and silkworms spin silks that outcompete the toughness of all natural and manmade fibers. Herein, we compare and contrast the spinning of silk in silkworms and spiders, with the aim of identifying features that are important for fiber formation. Although spiders and silkworms are very distantly related, some features of spinning silk seem to be universal. Both spiders and silkworms produce large silk proteins that are highly repetitive and extremely soluble at high pH, likely due to the globular terminal domains that flank an intermediate repetitive region. The silk proteins are produced and stored at a very high concentration in glands, and then transported along a narrowing tube in which they change conformation in response primarily to a pH gradient generated by carbonic anhydrase and proton pumps, as well as to ions and shear forces. The silk proteins thereby convert from random coil and alpha helical soluble conformations to beta sheet fibers. We suggest that factors that need to be optimized for successful production of artificial silk proteins capable of forming tough fibers include protein solubility, pH sensitivity, and preservation of natively folded proteins throughout the purification and initial spinning processes.     

Molecular Dynamics of Synthetic Flagelliform Silk Fiber Assembly

In order to better understand the relationship between flagelliform (Flag) spider silk molecular structural organization and the mechanisms of fiber assembly, the Nephilengys cruentata Flag spidroin analogue rNcFlag2222 is designed and produced. The recombinant proteins are composed by the elastic repetitive glycine-rich motifs (GPGGX/GGX) and the spacer region, rich in hydrophilic charged amino acids, present at the native silk spidroin. Using different approaches for nanomolecular protein analysis, the structural data of rNcFlag2222 recombinant proteins are compared in their fibrillar and fully solvated states. Based on the results, it is possible to identify the molecular structural dynamics of NcFlag2222 prior to and after fiber formation. Overall rNcFlag2222 shows a mixture of semiflexible and rigid conformations, characterized mostly by the presence of polyproline type II helix (PPII), β-turn, and β-sheet. These results agree with previous studies and bring insights about the molecular mechanisms that might drive Flag silk fibers assembly and elastomeric behavior.     

Active control on molecular conformations and tensile properties of spider silk

The mechanical properties of spider dragline silk vary with the spinning conditions, and molecular conformation is one of the important factors for the strength and strain of materials. Four kinds of Araneus ventricosus spider dragline silk fibers, measured by Raman microscopic spectrometry, were produced under different conditions: (1) reeled at the rate of 2 cm/s; (2) secreted by a dropping spider from a 100‐cm‐high table; (3) spun by spiders raised in two different containers. The Raman spectra of these fibers showed that the spinning method and growing environment of spiders had evident influences on the molecular conformations and tensile properties of dragline silk, and the dragline silk obtained from a dropping spider contained the greatest number of molecules with highly oriented β‐sheet structures and gave higher stress/strain values. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 901–905, 2004     

The Role of Filippi’s Glands in the Silk Moths Cocoon Construction

Filippi’s glands (FGs), formerly also called Lyonet’s glands, are accessory secretory structures of the labial (silk) glands of lepidopteran caterpillars, which were implicated to play an important role in the maturation of the silk material and the construction of the cocoon. In our previous study, we have identified several species of giant silk moths that completely lack the FGs. Interestingly, the absence of FGs in these species correlates with the construction of a loose cocoon architecture. We investigated the functions of FGs by their surgical extirpation in the last instar larvae of the silkworm, Bombyx mori. We found that the absence of FGs altered the structure of the resulting cocoon, in which the different layers of silk were separated. In further experiments, we found no effects of the absence of FGs on larval cocoon formation behavior or on changes in cocoon mass or lipid content. Differential proteomic analysis revealed no significant contribution of structural proteins from FGs to silk cocoon material, but we identified several low abundance proteins that may play a role in posttranslational modifications of some silk proteins. Proteomic analysis also revealed a difference in phosphorylation of the N-terminal sequence of fibroin-heavy chain molecule. Thus, FGs appear to affect silk stickiness during spinning by regulating posttranslational modifications. This could also explain the link that exists between the absence of these glands and the formation of loose cocoons in some giant silk moth species.     

Major Ampullate Spider Silk with Indistinguishable Spidroin Dope Conformations Leads to Different Fiber Molecular Structures

To plentifully benefit from its properties (mechanical, optical, biological) and its potential to manufacture green materials, the structure of spider silk has to be known accurately. To this aim, the major ampullate (MA) silk of Araneus diadematus (AD) and Nephila clavipes (NC) has been compared quantitatively in the liquid and fiber states using Raman spectromicroscopy. The data show that the spidroin conformations of the two dopes are indistinguishable despite their specific amino acid composition. This result suggests that GlyGlyX and GlyProGlyXX amino acid motifs (X = Leu, Glu, Tyr, Ser, etc.) are conformationally equivalent due to the chain flexibility in the aqueous environment. Species-related sequence specificity is expressed more extensively in the fiber: the β-sheet content is lower and width of the orientation distribution of the carbonyl groups is broader for AD (29% and 58°, respectively) as compared to NC (37% and 51°, respectively). β-Sheet content values are close to the proportion of polyalanine segments, suggesting that β-sheet formation is mainly dictated by the spidroin sequence. The extent of molecular alignment seems to be related to the presence of proline (Pro) that may decrease conformational flexibility and inhibit chain extension and alignment upon drawing. It appears that besides the presence of Pro, secondary structure and molecular orientation contribute to the different mechanical properties of MA threads.     

Nanostructural physical and chemical information derived from the unit cell scattering amplitudes of a spider dragline silk

Characterizing the nanostructures of spider major ampullate (dragline) silks is an important step in understanding the origin of their high mean strength and toughness, and for producing polymeric analogs that mimic these properties. Here we present transmission electron microscopy (TEM) diffraction patterns and an accompanying structure factor analysis for the dragline silk of Latrodectus hesperus (black widow spider). The chemical and physical composition of crystalline regions in this silk fiber was studied by manipulating the positions and size of amino acid side groups in a theoretical model, and comparing the expected unit cell scattering amplitudes with experimental electron diffraction patterns. The results suggest that—in addition to the smaller amino acid side groups such as alanine, glycine and serine—some of the bulkier amino acid side groups such as tyrosine and leucine are included in the crystalline fraction of the major ampullate silk. The structure factor analysis also demonstrates a marked sensitivity of the respective diffraction spot intensities to a slight change in both side group position and side group bulkiness. These observations point to a unique function for TEM in characterizing silk and other polymers.     

Investigation of Synthetic Spider Silk Crystallinity and Alignment via Electrothermal,Pyroelectric, Literature XRD,and Tensile Techniques

The processes used to create synthetic spider silk greatly affect the properties of the produced fibers. This paper investigates the effect of process variations during artificial spinning on the thermal and mechanical properties of the produced silk. Property values are also compared to the ones of the natural dragline silk of the Nephila clavipes spider, and to unprocessed (as‐spun) synthetic silk. Structural characterization by scanning pyroelectric microscopy is employed to provide insight into the axial orientation of the crystalline regions of the fiber and is supported by X‐ray diffraction data. The results show that stretching and passage through liquid baths induce crystal formation and axial alignment in synthetic fibers, but with different structural organization than natural silks. Furthermore, an increase in thermal diffusivity and elastic modulus is observed with decreasing fiber diameter, trending toward properties of natural fiber. This effect seems to be related to silk fibers being subjected to a radial gradient during production.

     

Ripening time and fiber formation of chitosan spinning dope

A study was carried out on the wet spinning of chitosan fibers using 2% acetic acid as a solvent, 10% aqueous sodium hydroxide as a nonsolvent, and 4% chitosan solution as a polymer concentration. In this study, we investigated the effect of the ripening time of the spinning dope on the ability of fiber formation (i‐value), structure, thermal, and mechanical properties (such as fineness, tenacity, elongation, work of rupture, etc.) of chitosan fibers. Based on the results, it can be seen that the ripening time of spinning dope (in days), with the same polymer concentration of spinning dope, changes from 1 to 8, and the i‐value of the spinning dope increased with an increasing of the ripening time. At the ripening time of 8 days, tensile strength, elongation, and work of rupture showed minimum value attributed to the excessive degradation of the chitosan polymer chains left from the mixing operation that took place at the same time as the ripening time of the spinning dope, which means that the optimum ripening time of the spinning dope is 1 to 7 days. However, the thermal decomposition temperature and the onset of the exothermic temperature of thermal properties decreased with an increased ripening time. On the other hand, tenacity, elongation, and toughness decreased with increasing ripening time, and these qualities radically decreased with an increasing ripening time of more than a week. This indicates that the dispersion of aggregates and the degradation of chitosan polymer chains left from the mixing operation occurred at the same time during the ripening time of the spinning dope. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 2870–2877, 2003     

Spider Silk-CBD-Cellulose Nanocrystal Composites: Mechanism of Assembly

The fabrication of cellulose-spider silk bio-nanocomposites comprised of cellulose nanocrystals (CNCs) and recombinant spider silk protein fused to a cellulose binding domain (CBD) is described. Silk-CBD successfully binds cellulose, and unlike recombinant silk alone, silk-CBD self-assembles into microfibrils even in the absence of CNCs. Silk-CBD-CNC composite sponges and films show changes in internal structure and CNC alignment related to the addition of silk-CBD. The silk-CBD sponges exhibit improved thermal and structural characteristics in comparison to control recombinant spider silk sponges. The glass transition temperature (Tg) of the silk-CBD sponge was higher than the control silk sponge and similar to native dragline spider silk fibers. Gel filtration analysis, dynamic light scattering (DLS), small angle X-ray scattering (SAXS) and cryo-transmission electron microscopy (TEM) indicated that silk-CBD, but not the recombinant silk control, formed a nematic liquid crystalline phase similar to that observed in native spider silk during the silk spinning process. Silk-CBD microfibrils spontaneously formed in solution upon ultrasonication. We suggest a model for silk-CBD assembly that implicates CBD in the central role of driving the dimerization of spider silk monomers, a process essential to the molecular assembly of spider-silk nanofibers and silk-CNC composites.     

Effect of the solvent type on the formation and physical properties of polyacrylonitrile fibers via a solvent‐free coagulation bath

Polyacrylonitrile (PAN) fibers were fabricated via a dry‐jet wet‐spinning technique, and a solvent‐free coagulation bath system was adopted. The effects of different types of dope solvent on the formation and physical properties of the PAN fibers were investigated. Dimethylformamide and dimethyl sulfoxide (DMSO) were selected as the solvents and were added to a spinning solution consisting of 18 wt % PAN. The PAN fibers were examined with field emission scanning electron microscopy, differential scanning calorimetry, and thermogravimetric analysis. The field emission scanning electron micrographs revealed that the PAN fibers with the DMSO solvent exhibited a more circular shape and a smoother skin. The PAN fibers with the DMSO solvent had their glass‐transition temperature (T_g) at 121°C. This study indicated that the different types of dope solvent used in the dope preparation did not affect T_g of the PAN fibers because of the solvent‐free coagulation bath system; however, they significantly affected the physical formation of the PAN fibers. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Preparation and properties of conductive polyaniline/poly‐ω‐aminoundecanoyle fibers

Historically, polyaniline (PANI) had been considered an intractable material, but it can be dissolved in some solvents. Therefore, it could be processed into films or fibers. A process of preparing a blend of conductive fibers of PANI/poly‐ω‐aminoundecanoyle (PA11) is described in this paper. PANI in the emeraldine base was blended with PA11 in concentrated sulfuric acid (c‐H₂SO₄) to form a spinning dope solution. This solution was used to spin conductive PANI / PA11 fibers by wet‐spinning technology. As‐spun fibers were obtained by spinning the dopes into coagulation bath water or diluted acid and drawn fibers were obtained by drawing the as‐spun fibers in warm drawing bath water. A scanning electron microscope was employed to study the effect of the acid concentration in the coagulation bath on the microstructure of as‐spun fibers. The results showed that the coagulating rate of as‐spun fibers was reduced and the size of pore shrank with an increase in the acid concentration in the coagulation bath. The weight fraction of PANI in the dope solution also had an influence on the microstructure of as‐spun fibers. The microstructure of as‐spun fibers had an influence on the drawing process and on the mechanical properties of the drawn fibers. Meanwhile, the electrically conductive property of the drawn fibers with different percentage of PANI was measured. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 85: 1458–1464, 2002     

Exploring the effects of non-solvent concentration, jet-stretching and hot-drawing on microstructure formation of poly(acrylonitrile) fibers during wet-spinning

The simultaneous effects of non-solvent concentration in the spinning dope, jet-stretching and hot-drawing on porosity, morphology development and mechanical properties of wet-spun poly(acrylonitrile) fibers were studied. Addition of non-solvent to the spinning dope increased dope viscosity, the entanglement density of the polymeric solution and the number of entanglements per chain. Drawability of the as-spun fiber depended on the number of entanglements per polymer chain. Therefore, addition of non-solvent improved or spoiled drawability of the wet-spun fibers based on the concentration of the initial spinning dope. Hot-drawing and jet-stretching did not affect the fraction of nanovoids but shifted their size distribution towards smaller values. However, hot-drawing was more effective in reducing the overall porosity of the fibers in comparison with jet-stretching. Fiber tenacity increased when overall porosity decreased. Finally, strength-diameter correlation showed good agreement with the Griffith’s theory.     

The Development of Fibers That Mimic the Core–Sheath and Spindle‐Knot Morphology of Artificial Silk Using Microfluidic Devices

Spider and silkworm produce diverse silk fibers from spinning dopes through smart spinnerets. Spider's capture silk is composed of core thread and periodic spindle‐knots, while silkworm silk consists of fibroin core and sericin outer layer. To mimic the morphologies of natural heterostructured silks, artificial fibers are dry‐spun using a multichannel microfluidic chip, served with a highly viscous core solution of regenerated silk fibroin and low viscosity sheath solution of sericin. Silk fibers with core–sheath, groove, and spindle‐knot structures are obtained by controlling the flow rates and viscosities of the two microfluids depending on the laminar flow, Kelvin–Helmholtz instability, or Plateau–Rayleigh instability.     

静电纺再生丝素纳米纤维的结构与性能

静电纺丝获得的丝素纳米级纤维可作为细胞培养支架,用于纺丝工艺及后处理能改变丝素微细结构,影响其水溶性和力学性能。本文采用XRD、FTIR、固态13CNMR和DSC研究了不同工艺下丝素纳米纤维及经甲醇处理后的微细结构,比较了不同微细结构下的水溶性和力学性能。结果表明,电纺丝的微细结构受纺丝工艺影响,高电压、纺丝液中丝素质量分数大时纺得的电纺丝结晶度高,经甲醇处理后,β化程度提高;w(丝素)=11%、15%时制备的电纺丝断裂强度分别为8.5、11.9 cN/mm;w(丝素)=11%、19%,水溶性由51.2%下降到43.3%;w(丝素)=19%、电压32 kV制得的电纺丝甲醇处理前后水溶性从43.3%下降到6.6%,说明丝素纳米纤维结晶度提高,强度增加、水溶性下降,满足了细胞支架的要求。     

Development of silk-like materials based on Bombyx mori and Nephila clavipes dragline silk fibroins

In order to develop new silk-like materials in the form of fiber and non-woven nano-fiber, this study synthesized a new silk-like protein by selecting the sequence from the crystalline region of Bombyx mori silk fibroin, (GAGSGA)₆, to imitate the processing condition of the silk fibroin with the combination of the sequence YGGLGSQGAGRG, the hydrophilic motif of spider dragline silk which was considered as the origin of supercontraction of spider dragline silk (Yang Z, et al. J Am Chem Soc 2000;122: 9019-25). The CD pattern of the silk-like protein in hexafluoroacetone (HFA) indicates that it takes helical structure. The solid-state structures of the silk-like protein before and after methanol treatment were determined to be random coil and Silk II structure (mainly β-sheet structure), respectively, using ¹³C CP/MAS NMR. The ¹³C CP/MAS NMR spectra of the protein after methanol treatment in hydrated state showed that the random coil peaks from Ala Cβ and Ser Cβ carbons become sharper compared with the corresponding peaks in the dry state. The 2D-WISE spectra in the hydrated state also showed increase of the narrow components for these carbons. Thus, water molecules are relatively easy to access at the random coil regions of the protein. The fiber formation of the silk-like protein was possible with wet-spinning or electrospinning methods using HFA as dope solvent and methanol as coagulation solvent.     

纤维素与透明质酸、肝素、丝素、甲壳素湿纺复合生物纤维的制备与性能研究

室温下,将再生纤维素与透明质酸(HA)、肝素(Hep)、丝素(SF)、甲壳素(N-ACS)等的复合纤维的纺丝液溶于NaOH溶液中,通过喷丝头(孔径0.1mm)喷入含有40%~43%硫酸铵的10%硫酸溶液中,得到产率为75%~98%的新型白色、柔韧性较好的复合生物纤维。FT-IR结果显示,复合纤维组分间存在物理吸附和氢键相互作用;少量的HA、Hep、SF和N-ACS等纤维的添加可增加复合纤维的韧能,其中纤维素-SF复合纤维在SF含量为10%时纤度为9.9旦,韧度值为1.08g/旦,伸长率为35.0%,机械性能相对最好。SEM图像中可以看出复合纤维表面呈现条纹、鳞状、填充和均匀包覆的结构,复合纤维间存在一定的相容性;复合纤维直径为19~55μm,密度为0.1~0.36旦/μm。