Spider Dragline Silk Research Articles

A new cell-sized support for 3D cell cultures based on recombinant spider silk fibers.

It is now generally accepted that 2D cultures cannot accurately replicate the rich environment and complex tissue architecture that exists in vivo, and that classically cultured cells tend to lose their original function. Growth of spheroids as opposed to 2D cultures on plastic has now been hailed as an efficient method to produce quantities of high-quality cells for cancer research, drug discovery, neuroscience, and regenerative medicine. We have developed a new recombinant protein that mimics dragline spidersilk and that self-assembles into cell-sized coils. These have high thermal and shelf-life stability and can be readily sterilized and stored for an extended period of time. The fibers are flexible, elastic, and biocompatible and can serve as cell-sized scaffold for the formation of 3D cell spheroids. As a proof of concept, recombinant spidersilk was integrated as a scaffold in spheroids of three cell types: primary rat hepatocytes, human mesenchymal stem cells, and mouse L929 cells. The scaffolds significantly reduced spheroid shrinkage and unlike scaffold-free spheroids, spheroids did not disintegrate over the course of long-term culture. Cells in recombinant spidersilk spheroids showed increased viability, and the cell lines continued to proliferate for longer than control cultures without spidersilk. The spidersilk also supported biological functions. Recombinant spidersilk primary hepatocyte spheroids exhibited 2.7-fold higher levels of adenosine triphosphate (ATP) continued to express and secrete albumin and exhibited significantly higher basal and induced CYP3A activity for at least 6 weeks in culture, while control spheroids without fibers stopped producing albumin after 27 days and CPY3A activity was barely detectable after 44 days. These results indicate that recombinant spidersilk can serve as a useful tool for long-term cell culture of 3D cell spheroids and specifically that primary hepatocytes can remain active in culture for an extended period of time which could be of great use in toxicology testing.

Supercontraction of spider dragline silk for humidity sensing.

The spider dragline silk (SDS) has a supercontraction characteristic, which may cause the axial length of the SDS to shrink up to 50% when the SDS is wet or the relative humidity is higher than 58% RH. In this manuscript, we employ the supercontraction characteristic of the SDS to measure relative humidity. We connect two sections of a single-mode fiber (SMF) and a section of multimode fiber (MMF) with a sandwich structure to fabricate a single-mode-multimode-single-mode (SMS) interferometer. Then we fix the SDS on two SMFs to configure a bow-shaped sensing unit. The increase of environmental humidity will cause the supercontraction of the SDS, which will cause the change of the SDS length. The excellent mechanical properties of the SDS will generate a strong pulling force and change the bending of the arch, whose interference spectrum will shift correspondingly. In this way, we may perform relative humidity sensing. In the relative humidity range of 58% RH to 100% RH, the average sensitivity is as high as 6.213 nm/% RH, higher than most fiber-based humidity sensors. Compared with the traditional sensing structure with humidity-sensitive materials, the proposed sensor improves the sensitivity with environmental friendliness. The results suggest that the SDS can be used for high-sensitivity humidity sensors, and its degradability and biocompatibility also have a vast development space in biochemical sensors.

Multicomponent nature underlies the extraordinary mechanical properties of spider dragline silk

Dragline silk of golden orb-weaver spiders (Nephilinae) is noted for its unsurpassed toughness, combining extraordinary extensibility and tensile strength, suggesting industrial application as a sustainable biopolymer material. To pinpoint the molecular composition of dragline silk and the roles of its constituents in achieving its mechanical properties, we report a multiomics approach, combining high-quality genome sequencing and assembly, silk gland transcriptomics, and dragline silk proteomics of four Nephilinae spiders. We observed the consistent presence of the MaSp3B spidroin unique to this subfamily as well as several nonspidroin SpiCE proteins. Artificial synthesis and the combination of these components in vitro showed that the multicomponent nature of dragline silk, including MaSp3B and SpiCE, along with MaSp1 and MaSp2, is essential to realize the mechanical properties of spider dragline silk.

Scalable Spider Silk Inspired Materials with High Extensibility and Super Toughness

Spiders silks have extraordinary strength and toughness simultaneously, thus has become dreamed materials by scientists and industries. Although there have been tremendous attempts to prepare fibers from genetically manufacture spider silk proteins, however, it has been still a huge challenge because of tedious procedure and high cost. Here, a facile spider-silk-mimicking strategy is reported for preparing highly scratchable polymers and supertough fibers from chemical synthesis route. Polymer films with high extensibility (>1200%) and supertough fibers (~387 MJ m-3) are achieved by introducing polypeptides with β-sheet and α-helical structure in polyureathane/urea polymers. Notabley,the toughness of the fiber is more than twice the reported value of a normal spider dragline silk, and comparable with the toughest spider silk, aciniform silk of Argiope trifasciata.

Unconventional Spidroin Assemblies in Aqueous Dope for Spinning into Tough Synthetic Fibers.

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

Investigating the Atomic and Mesoscale Interactions that Facilitate Spider Silk Protein Pre-Assembly.

Black widow spider dragline silk is one of nature's high-performance biological polymers, exceeding the strength and toughness of most man-made materials including high tensile steel and Kevlar. Major ampullate (Ma), or dragline silk, is primarily comprised of two spidroin proteins (Sp) stored within the Ma gland. In the native gland environment, the MaSp1 and MaSp2 proteins self-associate to form hierarchical 200-300 nm superstructures despite being intrinsically disordered proteins (IDPs). Here, dynamic light scattering (DLS), three-dimensional (3D) triple resonance solution NMR, and diffusion NMR is utilized to probe the MaSp size, molecular structure, and dynamics of these protein pre-assemblies diluted in 4 M urea and identify specific regions of the proteins important for silk protein pre-assembly. 3D NMR indicates that the Gly-Ala-Ala and Ala-Ala-Gly motifs flanking the poly(Ala) runs, which comprise the β-sheet forming domains in fibers, are perturbed by urea, suggesting that these regions may be important for silk protein pre-assembly stabilization.

Super Bacteria: A New Hope of Manufacturing Spider Silk in an Efficient Way

The important properties of spider dragline silk and other protein polymers will find many applications. We have demonstrated the production of spider silk, which has many important properties, are produced from the bacteria including Escherichia coli. The productions of high molecular weight spider drag line encoded by synthetic genes. Silk protein can be efficiently produced by the microbial system has become an advantageous method like quick secretion and simple product recovery has become an efficient method .From the observation of various experiments done by several scientists has shown silk made in laboratory. The study of RIKEN centre for sustainable resource science has shown that spider silk can be produce huge amount. Observation shown that joining of the fragments by split intein sequence which then cut itself to yield full name protein .Spun into fibers make the microbial spider silk tough , stretchable and stronger. Better modification of bioengineering can increase the amount of production.

Programmable Contractile Actuations of Twisted Spider Dragline Silk Yarns.

The contraction behavior of spider dragline silk upon water exposure has drawn particular interest in developing humidity-responsive smart materials. We report herein that the spider dragline silk yarns with moderate twists can generate much improved lengthwise contraction of 60% or an isometric stress of 11 MPa when wetted by water. Upon the removal of the absorbed water, the dried and contracted spider silk yarns showed programmable contractile actuations. These yarns can be plastically stretched to any specified lengths between the fully contracted state and the state before supercontraction and return to the fully contracted state when wetted. Moreover, the generated isometric stress of these yarns is also programmable, depending on the stretching ratio. The mechanism of the programmable reversible contraction is based on the plastic mechanical property of the dried and contracted spider silk yarns, which can be explained by the variation of the hydrogen bonds and the secondary structures of the proteins in spider dragline silk. Humidity alarm switches, smart doors, and wound healing devices based on the programmable contractile actuations of the spider silk yarns were demonstrated, which provide application scenarios for the supercontraction of spider dragline silk.

Structure of Animal Silks.

As an abundant fibrous protein, animal silks have received a variety of interests in both traditional and high-tech industries, such as textiles, decoration, and biomedicine, due to their unique advantages in mechanical performance, sustainability, biocompatibility, and biodegradability. While developing applications of animal silks, the structure of animal silks has also received more and more attention in these decades. Briefly, most animal silks can be considered as semicrystalline fibers, which are composed of β-sheet nanocrystals and amorphous regions. However, different animal silks have similarities and also have obvious differences at different structural levels. In this chapter, we will introduce the structures of the three most representative animal silks, that is, spider dragline silk, tussah silk, and mulberry silk. The similarities and differences in their structures will be highlighted, so as to provide fundamental guidance for the research and use of these animal silks.

Structural Changes in Spider Dragline Silk after Repeated Supercontraction-Stretching Processes.

Spider dragline silk is well-known for its excellent combination of strength and extensibility as well as another unique property called supercontraction. In our previous work, the changes in conformations of the Nephila edulis spider dragline silk when subjected to different supercontraction processes were extensively investigated. When a native spider dragline silk had free supercontraction, and then restretched to its original length, the content and molecular orientation of different conformations (β-sheet, helix, and random coil) changed but the mechanical properties remained almost the same. Therefore, herein, further supercontraction-stretching treatment was performed up to three cycles, and the corresponding structural changes were investigated. In addition to the synchrotron radiation FTIR (S-FTIR) microspectroscopy employed in our previous study, synchrotron radiation small-angle X-ray scattering (S-SAXS) and atomic force microscopy (AFM) were also used in this work to determine the structural changes of spider dragline silk in different scales. The results show that by repeating the supercontraction-stretching treatment, the β-sheet structure content in spider dragline silk was slightly increased, but its orientation degree remained almost the same. Also, with the increase in cycle of supercontraction-stretching treatments, a 10.5 nm long period perpendicular to the silk fiber axis gradually appeared, endowing the spider dragline silk with periodic structure both along (6.6 nm, already existed in native silk and did not change with the supercontraction-stretching treatment) and perpendicular to the silk fiber axis. After the third supercontraction-stretching cycle, the AFM images displayed a clear 210 nm × 80 nm corn kernel-like structure on the surface of nanofibrils in spider dragline silks, which may be related to the aggregation of 10.5 nm × 6.6 nm periodic structure observed via S-SAXS. Finally, although the structure of spider dragline silk became increasingly regular with the rise in supercontraction-stretching cycles, mechanical properties remained constant after every cycle of the supercontraction-stretching treatment. These findings can aid in further understanding the structural changes that are related to the supercontraction of spider dragline silk and provide useful guidance in fabrication of high-performance regenerated or artificial silk fibers.

Mechanical and structural properties of major ampullate silk from spiders fed carbon nanomaterials.

The dragline silk of spiders is of particular interest to science due to its unique properties that make it an exceptional biomaterial that has both high tensile strength and elasticity. To improve these natural fibers, researchers have begun to try infusing metals and carbon nanomaterials to improve mechanical properties of spider silk. The objective of this study was to incorporate carbon nanomaterials into the silk of an orb-weaving spider, Nephila pilipes, by feeding them solutions containing graphene and carbon nanotubes. Spiders were collected from the field and in the lab were fed solutions by pipette containing either graphene sheets or nanotubes. Major ampullate silk was collected and a tensile tester was used to determine mechanical properties for pre- and post-treatment samples. Raman spectroscopy was then used to test for the presence of nanomaterials in silk samples. There was no apparent incorporation of carbon nanomaterials in the silk fibers that could be detected with Raman spectroscopy and there were no significant improvements in mechanical properties. This study represents an example for the importance of attempting to replicate previously published research. Researchers should be encouraged to continue to do these types of investigations in order to build a strong consensus and solid foundation for how to go forward with these new methods for creating novel biomaterials.

Selective One-Dimensional 13C-13C Spin-Diffusion Solid-State Nuclear Magnetic Resonance Methods to Probe Spatial Arrangements in Biopolymers Including Plant Cell Walls, Peptides, and Spider Silk.

Two-dimensional (2D) and 3D through-space 13C-13C homonuclear spin-diffusion techniques are powerful solid-state nuclear magnetic resonance (NMR) tools for extracting structural information from 13C-enriched biomolecules, but necessarily long acquisition times restrict their applications. In this work, we explore the broad utility and underutilized power of a chemical shift-selective one-dimensional (1D) version of a 2D 13C-13C spin-diffusion solid-state NMR technique. The method, which is called 1D dipolar-assisted rotational resonance (DARR) difference, is applied to a variety of biomaterials including lignocellulosic plant cell walls, microcrystalline peptide fMLF, and black widow dragline spider silk. 1D 13C-13C spin-diffusion methods described here apply in select cases in which the 1D 13C solid-state NMR spectrum displays chemical shift-resolved moieties. This is analogous to the selective 1D nuclear Overhauser effect spectroscopy (NOESY) experiment utilized in liquid-state NMR as a faster (1D instead of 2D) and often less ambiguous (direct sampling of the time domain data, coupled with increased signal averaging) alternative to 2D NOESY. Selective 1D 13C-13C spin-diffusion methods are more time-efficient than their 2D counterparts such as proton-driven spin diffusion (PDSD) and dipolar-assisted rotational resonance. The additional time gained enables measurements of 13C-13C spin-diffusion buildup curves and extraction of spin-diffusion time constants TSD, yielding detailed structural information. Specifically, selective 1D DARR difference buildup curves applied to 13C-enriched hybrid poplar woody stems confirm strong spatial interaction between lignin and acetylated xylan polymers within poplar plant secondary cell walls, and an interpolymer distance of ∼0.45-0.5 nm was estimated. Additionally, Tyr/Gly long-range correlations were observed on isotopically enriched black widow spider dragline silks.

Biomimetic nuclear lamin fibers with remarkable toughness and stiffness

The native hagfish slime threads, which are made up of two intermediate filament (IF)-like proteins, exhibit mechanical properties comparable to dragline spider silk fiber, the toughest fiber in nature. However, unlike silk, the design of artificial IF-protein-based fibers has been rarely studied, possibly because the unique hierarchical organization of the keratin-like proteins within these threads is challenging to mimic, and consequently, extraordinary fiber mechanics has not been shown in slime threads from recombinant IF-protein-based system. Here, we have reported the synthesis and properties of recombinant type V IF-protein, based on the Caenorhabditis elegans (Ce) lamin gene. The protein was solubilized and wet-spun into aqueous solutions to prepare Ce-lamin fibers by varying injection flow rates and Ca+2 ion concentrations in the coagulation buffer. At specific set of conditions, Ce-lamin fibers demonstrated remarkable toughness and stiffness, comparable to hagfish slime threads and natural dragline spider silk. Transmission electron microscopy analysis showed that paracrystals were the main nanometric structure within the fibers. This study demonstrates that outstanding mechanical properties can be achieved with recombinant IF-proteins through self-organization. Thus, these results have broadened the pool of fibrous proteins that can be used in functional materials for a diverse range of applications.

Nearly complete 1H, 13C and 15N chemical shift assignment of monomeric form of N-terminal domain of Nephila clavipes major ampullate spidroin 2.

Spider dragline silk is well recognized due to its excellent mechanical properties. Dragline silk protein mainly consists of two proteins, namely, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2). The MaSp N-terminal domain (NTD) conformation displays a strong dependence on ion and pH gradients, which is crucial for the self-assembly behavior of spider silk. In the spider major ampullate gland, where the pH is neutral and concentration of NaCl is high, the NTD forms a monomer. In contrast, within the spinning duct, where pH becomes more acidic (to pH ~ 5) and the concentration of salt is low, NTD forms a dimer in antiparallel orientation. In this study, we report near-complete backbone and side chain chemical shift assignment of the monomeric form of NTD of MaSp2 from Nephila clavipes at pH 7 in the presence of 300mM NaCl. Our NMR data demonstrate that secondary structure of monomeric form of NTD MaSp2 consists of five helix regions.

Tensegrity Modelling and the High Toughness of Spider Dragline Silk.

This work establishes a tensegrity model of spider dragline silk. Tensegrity systems are ubiquitous in nature, being able to capture the mechanics of biological shapes through simple and effective modes of deformation via extension and contraction. Guided by quantitative microstructural characterization via air plasma etching and low voltage scanning electron microscopy, we report that this model is able to capture experimentally observed phenomena such as the Poisson effect, tensile stress-strain response, and fibre toughness. This is achieved by accounting for spider silks’ hierarchical organization into microfibrils with radially variable properties. Each fibril is described as a chain of polypeptide tensegrity units formed by crystalline granules operating under compression, which are connected to each other by amorphous links acting under tension. Our results demonstrate, for the first time, that a radial variability in the ductility of tensegrity chains is responsible for high fibre toughness, a defining and desirable feature of spider silk. Based on this model, a discussion about the use of graded tensegrity structures for the optimal design of next-generation biomimetic fibres is presented.

Reviewing Thermophysical Properties of Silk Fibers: A Case Study for the Need for Complementary Measurement Techniques

A recent interest in the mechanical properties of spider silk has also brought about investigations into the thermal properties of thin fibers, often with contradictory results. Because of the high length-to-diameter ratio of fibers, two key obstacles arise during their thermal characterization: the need to develop non-standard techniques to measure properties and the lack of reliable methods to confirm the validity of those measurements. This is especially important since standard reference materials are difficult or impossible to produce at these small scales. This paper presents a review on the thermal conductivity and diffusivity of spider dragline silk and how the use of a suite of measurement techniques that complement each other can bound the uncertainty of the measured property. The properties of silkworm silks are then presented as evidence of a field where complementary techniques could be applied to resolve property discrepancies. This complementary approach to validating novel techniques will increase the confidence in measured properties and will open new avenues of thermal science applications.

Structural Characterization of Black Widow Spider Dragline Silk Proteins CRP1 and CRP4.

Spider dragline silk represents a biomaterial with outstanding mechanical properties, possessing high-tensile strength and toughness. In black widows at least eight different proteins have been identified as constituents of dragline silk. These represent major ampullate spidroins MaSp1, MaSp2, MaSp’, and several low-molecular weight cysteine-rich protein (CRP) family members, including CRP1, CRP2, and CRP4. Molecular modeling predicts that CRPs contain a cystine slipknot motif, but experimental evidence to support this assertion remains to be reported. To advance scientific knowledge regarding CRP function, we recombinantly expressed and purified CRP1 and CRP4 from bacteria and investigated their secondary structure using circular dichroism (CD) under different chemical and physical conditions. We demonstrate by far-UV CD spectroscopy that these proteins contain similar secondary structure, having substantial amounts of random coil conformation, followed by lower levels of beta sheet, alpha helical and beta turn structures. CRPs are thermally and pH stable; however, treatment with reagents that disrupt disulfide bonds impact their structural conformations. Cross-linking mass spectrometry (XL-MS) data also support computational models of CRP1. Taken together, the chemical and thermal stability of CRPs, the cross-linking data, coupled with the structural sensitivity to reducing agents, are experimentally consistent with the supposition CRPs are cystine slipknot proteins.

A marine photosynthetic microbial cell factory as a platform for spider silk production

Photosynthetic microorganisms such as cyanobacteria, purple bacteria and microalgae have attracted great interest as promising platforms for economical and sustainable production of bioenergy, biochemicals, and biopolymers. Here, we demonstrate heterotrophic production of spider dragline silk proteins, major ampullate spidroins (MaSp), in a marine photosynthetic purple bacterium, Rhodovulum sulfidophilum, under both photoheterotrophic and photoautotrophic growth conditions. Spider silk is a biodegradable and biocompatible material with remarkable mechanical properties. R. sulfidophilum grow by utilizing abundant and renewable nonfood bioresources such as seawater, sunlight, and gaseous CO2 and N2, thus making this photosynthetic microbial cell factory a promising green and sustainable production platform for proteins and biopolymers, including spider silks.

Spider Silk-Based Improved Multimode Interference Structure for Humidity Sensing

We propose and demonstrate an improved multimode interference (MMI) structure based on a spider dragline silk (SDS) for relative humidity (RH) sensing. We configure the MMI structure by connecting a section of single-mode fiber, a section of no-core fiber (NCF), and a section of single-mode fiber sequentially, and then obtain the interference spectrum. We spirally wrap the dragline silk on the NCF to improve the MMI and help the interference dips to be significant and identifiable. The refractive index and diameter of the SDS will change due to the change of the RH around the SDS, which will cause the improved interference dips to shift. The experimental results indicate that the average sensitivity of the proposed SDS-based sensor is 1.15 nm/%RH in the range of 33%RH-98%RH and the maximum sensitivity is 2.02 nm/%RH in the range of 83% RH-98%RH. The larger the RH, the larger the sensitivity. The proposed sensor also performs good repeatability with a fluctuation of less than 2.6% and a response time of 558ms. Spider dragline silk has proven itself the right candidate for RH sensing application and promises a way to fabricate environmental-friendly, bio-absorbable and biocompatible protein-based devices for biochemical sensing that uses natural materials as their constituent.

Spider silk-based tapered optical fiber for humidity sensing based on multimode interference

We develop a novel humidity sensor by wrapping the spider dragline silk (SDS) on a tapered single-mode fiber (TSMF) to configure a multimode interference (MMI) structure and obtain the optical spectrum. The TSMF is fabricated by heating a single-mode fiber (SMF) while applying tension. SDS is employed as humidity-sensing material, thus the SDS wrapped on the TSMF enable TSMF structure to be sensitive to the changes of environmental relative humidity (RH). SDS refractive index changes with the change of environmental RH, which causes the shift of multimode (MMI) interference spectrum. Experimental results indicate that the average sensitivity of the proposed SDS-based sensor is 0.532 nm/%RH in the range of 30 %RH–89 %RH and the maximum sensitivity is 0.789 nm/ %RH in the range of 70 %RH-89 %RH. The humidity-sensing curve is approximately fitted by the linear method. The response time is 374.46 ms. We provide a new method for the fabrication of natural protein-based environmental friendly sensor.