Effects of Mini-Spidroin Repeat Region on the Mechanical Properties of Artificial Spider Silk Fibers.

Spiders can produce up to seven different types of silk, each with unique mechanical properties that stem from variations in the repetitive regions of spider silk proteins (spidroins). Artificial spider silk can be made from mini-spidroins in an all-aqueous-based spinning process, but the strongest fibers seldom reach more than 25% of the strength of native silk fibers. With the aim to improve the mechanical properties of silk fibers made from mini-spidroins and to understand the relationship between the protein design and the mechanical properties of the fibers, we designed 16 new spidroins, ranging from 31.7 to 59.5 kDa, that feature the globular spidroin N- and C-terminal domains, but harbor different repetitive sequences. We found that more than 50% of these constructs could be spun by extruding them into low-pH aqueous buffer and that the best fibers were produced from proteins whose repeat regions were derived from major ampullate spidroin 4 (MaSp4) and elastin. The mechanical properties differed between fiber types but did not correlate with the expected properties based on the origin of the repeats, suggesting that additional factors beyond protein design impact the properties of the fibers.

Strategies for Making High-Performance Artificial Spider Silk Fibers.

Artificial spider silk is an attractive material for many technical applications since it is a biobased fiber that can be produced under ambient conditions but still outcompetes synthetic fibers (e.g., Kevlar) in terms of toughness. Industrial use of this material requires bulk-scale production of recombinant spider silk proteins in heterologous host and replication of the pristine fiber's mechanical properties. High molecular weight spider silk proteins can be spun into fibers with impressive mechanical properties, but the production levels are too low to allow commercialization of the material. Small spider silk proteins, on the other hand, can be produced at yields that are compatible with industrial use, but the mechanical properties of such fibers need to be improved. Here, the literature on wet-spinning of artificial spider silk fibers is summarized and analyzed with a focus on mechanical performance. Furthermore, several strategies for how to improve the properties of such fibers, including optimized protein composition, smarter spinning setups, innovative protein engineering, chemical and physical crosslinking as well as the incorporation of nanomaterials in composite fibers, are outlined and discussed.

Tuneable Recombinant Spider Silk Protein Hydrogels for Drug Release and 3D Cell Culture.

Hydrogels are useful drug release systems and tissue engineering scaffolds. However, synthetic hydrogels often require harsh gelation conditions and can contain toxic by-products while naturally derived hydrogels can transmit pathogens and in general have poor mechanical properties. Thus, there is a need for a hydrogel that forms under ambient conditions, is non-toxic, xeno-free, and has good mechanical properties. A recombinant spider silk protein-derived hydrogel that rapidly forms at 37 °C is recently developed. The temperature and gelation times are well-suited for an injectable in situ polymerising hydrogel, as well as a 3D cell culture scaffold. Here, it is shown that the diffusion rate and the mechanical properties can be tuned by changing the protein concentration and that human fetal mesenchymal stem cells encapsulated in the hydrogels show high survival and viability. Furthermore, mixtures of recombinant spider silk proteins and green fluorescent protein (GFP) form gels from which functional GFP is gradually released, indicating that bioactive molecules are easily included in the gels, maintain activity and can diffuse through the gel. Interestingly, encapsulated ARPE-19 cells are viable and continuously produce the growth factor progranulin, which is detected in the cell culture medium over the study period of 31 days.

High-performance magnetic artificial silk fibers produced by a scalable and eco-friendly production method.

Flexible magnetic materials have great potential for biomedical and soft robotics applications, but they need to be mechanically robust. An extraordinary material from a mechanical point of view is spider silk. Recently, methods for producing artificial spider silk fibers in a scalable and all-aqueous-based process have been developed. If endowed with magnetic properties, such biomimetic artificial spider silk fibers would be excellent candidates for making magnetic actuators. In this study, we introduce magnetic artificial spider silk fibers, comprising magnetite nanoparticles coated with meso-2,3-dimercaptosuccinic acid. The composite fibers can be produced in large quantities, employing an environmentally friendly wet-spinning process. The nanoparticles were found to be uniformly dispersed in the protein matrix even at high concentrations (up to 20% w/w magnetite), and the fibers were superparamagnetic at room temperature. This enabled external magnetic field control of fiber movement, rendering the material suitable for actuation applications. Notably, the fibers exhibited superior mechanical properties and actuation stresses compared to conventional fiber-based magnetic actuators. Moreover, the fibers developed herein could be used to create macroscopic systems with self-recovery shapes, underscoring their potential in soft robotics applications. Supplementary information: The online version contains supplementary material available at 10.1007/s42114-024-00962-y.

Origin, structure, and composition of the spider major ampullate silk fiber revealed by genomics, proteomics, and single-cell and spatial transcriptomics.

Spiders produce nature's toughest fiber using renewable components at ambient temperatures and with water as solvent, making it highly interesting to replicate for the materials industry. Despite this, much remains to be understood about the bioprocessing and composition of spider silk fibers. Here, we identify 18 proteins that make up the spiders' strongest silk type, the major ampullate fiber. Single-cell RNA sequencing and spatial transcriptomics revealed that the secretory epithelium of the gland harbors six cell types. These cell types are confined to three distinct glandular zones that produce specific combinations of silk proteins. Image analysis of histological sections showed that the secretions from the three zones do not mix, and proteomics analysis revealed that these secretions form layers in the final fiber. Using a multi-omics approach, we provide substantial advancements in the understanding of the structure and function of the major ampullate silk gland as well as of the architecture and composition of the fiber it produces.

Structural interspecies variability of mammals' hairs.

Hairs are fundamental structures for mammals, serving crucial functions such as thermal insulation and hydrophobicity. In domestic animals, hair is also a valuable source of high-performance fibers for the textile industry, which has led to intensive study. However, there is limited comparative knowledge about the physical properties of hair across different wild mammalian species. In our lab, we are investigating the physical properties of hairs from a diverse range of wild mammalian species, laying the groundwork for an in-depth comparative study. These physical properties can be linked to the internal structures of the hairs. Using polarized light microscopy, we can visualize the internal structure of hairs, which are composed of a hollow channel (medulla) surrounded by a cortex and a keratin cuticle(1). By examining the brown hairs of three distinct mammals-the Patagonian mara, the brown bear, and the Amur tiger-we observe striking differences in their internal structures. We speculate that these structural differences correspond to varying physical properties, which we are currently investigating.

Controlling Drug Partitioning in Individual Protein Condensates through Laser-Induced Microscale Phase Transitions.

Gelation of protein condensates formed by liquid-liquid phase separation occurs in a wide range of biological contexts, from the assembly of biomaterials to the formation of fibrillar aggregates, and is therefore of interest for biomedical applications. Soluble-to-gel (sol-gel) transitions are controlled through macroscopic processes such as changes in temperature or buffer composition, resulting in bulk conversion of liquid droplets into microgels within minutes to hours. Using microscopy and mass spectrometry, we show that condensates of an engineered mini-spidroin (NT2repCTYF) undergo a spontaneous sol-gel transition resulting in the loss of exchange of proteins between the soluble and the condensed phase. This feature enables us to specifically trap a silk-domain-tagged target protein in the spidroin microgels. Surprisingly, laser pulses trigger near-instant gelation. By loading the condensates with fluorescent dyes or drugs, we can control the wavelength at which gelation is triggered. Fluorescence microscopy reveals that laser-induced gelation significantly further increases the partitioning of the fluorescent molecules into the condensates. In summary, our findings demonstrate direct control of phase transitions in individual condensates, opening new avenues for functional and structural characterization.

Regionalization of cell types in silk glands of Larinioides sclopetarius suggest that spider silk fibers are complex layered structures.

In order to produce artificial silk fibers with properties that match the native spider silk we likely need to closely mimic the spinning process as well as fiber architecture and composition. To increase our understanding of the structure and function of the different silk glands of the orb weaver Larinioides sclopetarius, we used resin sections for detailed morphology, paraffin embedded sections for a variety of different histological stainings, and a histochemical method for localization of carbonic anhydrase activity. Our results show that all silk glands, except the tubuliform glands, are composed of two or more columnar epithelial cell types, some of which have not been described previously. We observed distinct regionalization of the cell types indicating sequential addition of secretory products during silk formation. This means that the major ampullate, minor ampullate, aciniform type II, and piriform silk fibers most likely are layered and that each layer has a specific composition. Furthermore, a substance that stains positive for polysaccharides may be added to the silk in all glands except in the type I aciniform glands. Active carbonic anhydrase was found in all silk glands and/or ducts except in the type I aciniform and tubuliform glands, with the strongest staining in aggregate glands and their ductal nodules. Carbonic anhydrase plays an important role in the generation of a pH gradient in the major ampullate glands, and our results suggest that some other glands may also harbor pH gradients.

Mass Spectrometry of RNA-Binding Proteins during Liquid-Liquid Phase Separation Reveals Distinct Assembly Mechanisms and Droplet Architectures.

Liquid-liquid phase separation (LLPS) of heterogeneous ribonucleoproteins (hnRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility mass spectrometry, and molecular dynamics simulations. We used an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with liquid-liquid phase separation. We find that FUS monomers undergo an unfolded-to-globular transition, whereas TDP-43 oligomerizes into partially disordered dimers and trimers. hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over LLPS. The divergent assembly mechanisms revealed by ion mobility mass spectrometry of soluble protein species that exist under LLPS conditions suggest structurally distinct complexes inside liquid droplets that may impact RNA processing and translation depending on biological context.

Liquid-Liquid Phase Separation Primes Spider Silk Proteins for Fiber Formation via a Conditional Sticker Domain.

Many protein condensates can convert to fibrillar aggregates, but the underlying mechanisms are unclear. Liquid-liquid phase separation (LLPS) of spider silk proteins, spidroins, suggests a regulatory switch between both states. Here, we combine microscopy and native mass spectrometry to investigate the influence of protein sequence, ions, and regulatory domains on spidroin LLPS. We find that salting out-effects drive LLPS via low-affinity stickers in the repeat domains. Interestingly, conditions that enable LLPS simultaneously cause dissociation of the dimeric C-terminal domain (CTD), priming it for aggregation. Since the CTD enhances LLPS of spidroins but is also required for their conversion into amyloid-like fibers, we expand the stickers and spacers-model of phase separation with the concept of folded domains as conditional stickers that represent regulatory units.

Biological Materials Processing: Time-Tested Tricks for Sustainable Fiber Fabrication.

There is an urgent need to improve the sustainability of the materials we produce and use. Here, we explore what humans can learn from nature about how to sustainably fabricate polymeric fibers with excellent material properties by reviewing the physical and chemical aspects of materials processing distilled from diverse model systems, including spider silk, mussel byssus, velvet worm slime, hagfish slime, and mistletoe viscin. We identify common and divergent strategies, highlighting the potential for bioinspired design and technology transfer. Despite the diversity of the biopolymeric fibers surveyed, we identify several common strategies across multiple systems, including: (1) use of stimuli-responsive biomolecular building blocks, (2) use of concentrated fluid precursor phases (e.g., coacervates and liquid crystals) stored under controlled chemical conditions, and (3) use of chemical (pH, salt concentration, redox chemistry) and physical (mechanical shear, extensional flow) stimuli to trigger the transition from fluid precursor to solid material. Importantly, because these materials largely form and function outside of the body of the organisms, these principles can more easily be transferred for bioinspired design in synthetic systems. We end the review by discussing ongoing efforts and challenges to mimic biological model systems, with a particular focus on artificial spider silks and mussel-inspired materials.

Influence of experimental methods on the mechanical properties of silk fibers: A systematic literature review and future road map.

Spider silk fibers are of scientific and industrial interest because of their extraordinary mechanical properties. These properties are normally determined by tensile tests, but the values obtained are dependent on the morphology of the fibers, the test conditions, and the methods by which stress and strain are calculated. Because of this, results from many studies are not directly comparable, which has led to widespread misconceptions in the field. Here, we critically review most of the reports from the past 50 years on spider silk mechanical performance and use artificial spider silk and native silks as models to highlight the effect that different experimental setups have on the fibers' mechanical properties. The results clearly illustrate the importance of carefully evaluating the tensile test methods when comparing the results from different studies. Finally, we suggest a protocol for how to perform tensile tests on silk and biobased fibers.

Engineered Spider Silk Proteins for Biomimetic Spinning of Fibers with Toughness Equal to Dragline Silks.

Spider silk is the toughest fiber found in nature, and bulk production of artificial spider silk that matches its mechanical properties remains elusive. Development of miniature spider silk proteins (mini-spidroins) has made large-scale fiber production economically feasible, but the fibers' mechanical properties are inferior to native silk. The spider silk fiber's tensile strength is conferred by poly-alanine stretches that are zipped together by tight side chain packing in ß-sheet crystals. Spidroins are secreted so they must be void of long stretches of hydrophobic residues, since such segments get inserted into the endoplasmic reticulum membrane. At the same time, hydrophobic residues have high ß-strand propensity and can mediate tight inter-ß-sheet interactions, features that are attractive for generation of strong artificial silks. Protein production in prokaryotes can circumvent biological laws that spiders, being eukaryotic organisms, must obey, and the authors thus design mini-spidroins that are predicted to more avidly form stronger ß-sheets than the wildtype protein. Biomimetic spinning of the engineered mini-spidroins indeed results in fibers with increased tensile strength and two fiber types display toughness equal to native dragline silks. Bioreactor expression and purification result in a protein yield of ≈9 g L-1 which is in line with requirements for economically feasible bulk scale production.

Abilities of the BRICHOS domain to prevent neurotoxicity and fibril formation are dependent on a highly conserved Asp residue.

Proteins can self-assemble into amyloid fibrils or amorphous aggregates and thereby cause disease. Molecular chaperones can prevent both these types of protein aggregation, but to what extent the respective mechanisms are overlapping is not fully understood. The BRICHOS domain constitutes a disease-associated chaperone family, with activities against amyloid neurotoxicity, fibril formation, and amorphous protein aggregation. Here, we show that the activities of BRICHOS against amyloid-induced neurotoxicity and fibril formation, respectively, are oppositely dependent on a conserved aspartate residue, while the ability to suppress amorphous protein aggregation is unchanged by Asp to Asn mutations. The Asp is evolutionarily highly conserved in >3000 analysed BRICHOS domains but is replaced by Asn in some BRICHOS families. The conserved Asp in its ionized state promotes structural flexibility and has a pK a value between pH 6.0 and 7.0, suggesting that chaperone effects can be differently affected by physiological pH variations.

Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform.

Recombinant spider silk proteins (spidroins) have multiple potential applications in development of novel biomaterials, but their multimodal and aggregation-prone nature have complicated production and straightforward applications. Here, we report that recombinant miniature spidroins, and importantly also the N-terminal domain (NT) on its own, rapidly form self-supporting and transparent hydrogels at 37 °C. The gelation is caused by NT α-helix to ß-sheet conversion and formation of amyloid-like fibrils, and fusion proteins composed of NT and green fluorescent protein or purine nucleoside phosphorylase form hydrogels with intact functions of the fusion moieties. Our findings demonstrate that recombinant NT and fusion proteins give high expression yields and bestow attractive properties to hydrogels, e.g., transparency, cross-linker free gelation and straightforward immobilization of active proteins at high density.

Solution Structure of Tubuliform Spidroin N-Terminal Domain and Implications for pH Dependent Dimerization.

The spidroin N-terminal domain (NT) is responsible for high solubility and pH-dependent assembly of spider silk proteins during storage and fiber formation, respectively. It forms a monomeric five-helix bundle at neutral pH and dimerizes at lowered pH, thereby firmly interconnecting the spidroins. Mechanistic studies with the NTs from major ampullate, minor ampullate, and flagelliform spidroins (MaSp, MiSp, and FlSp) have shown that the pH dependency is conserved between different silk types, although the residues that mediate this process can differ. Here we study the tubuliform spidroin (TuSp) NT from Argiope argentata, which lacks several well conserved residues involved in the dimerization of other NTs. We solve its structure at low pH revealing an antiparallel dimer of two five-α-helix bundles, which contrasts with a previously determined Nephila antipodiana TuSp NT monomer structure. Further, we study a set of mutants and find that the residues participating in the protonation events during dimerization are different from MaSp and MiSp NT. Charge reversal of one of these residues (R117 in TuSp) results in significantly altered electrostatic interactions between monomer subunits. Altogether, the structure and mutant studies suggest that TuSp NT monomers assemble by elimination of intramolecular repulsive charge interactions, which could lead to slight tilting of α-helices.

The dimerization mechanism of the N-terminal domain of spider silk proteins is conserved despite extensive sequence divergence.

The N-terminal (NT) domain of spider silk proteins (spidroins) is crucial for their storage at high concentrations and also regulates silk assembly. NTs from the major ampullate spidroin (MaSp) and the minor ampullate spidroin are monomeric at neutral pH and confer solubility to spidroins, whereas at lower pH, they dimerize to interconnect spidroins in a fiber. This dimerization is known to result from modulation of electrostatic interactions by protonation of well-conserved glutamates, although it is undetermined if this mechanism applies to other spidroin types as well. Here, we determine the solution and crystal structures of the flagelliform spidroin NT, which shares only 35% identity with MaSp NT, and investigate the mechanisms of its dimerization. We show that flagelliform spidroin NT is structurally similar to MaSp NT and that the electrostatic intermolecular interaction between Asp 40 and Lys 65 residues is conserved. However, the protonation events involve a different set of residues than in MaSp, indicating that an overall mechanism of pH-dependent dimerization is conserved but can be mediated by different pathways in different silk types.

A "spindle and thread" mechanism unblocks p53 translation by modulating N-terminal disorder.

Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of "life on the edge of solubility." Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT∗ domain. We conclude that interactions with NT∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT∗. In summary, we demonstrate that inducing co-translational folding via a molecular "spindle and thread" mechanism unblocks protein translation in vitro.

Artificial and natural silk materials have high mechanical property variability regardless of sample size.

Silk fibres attract great interest in materials science for their biological and mechanical properties. Hitherto, the mechanical properties of the silk fibres have been explored mainly by tensile tests, which provide information on their strength, Young's modulus, strain at break and toughness modulus. Several hypotheses have been based on these data, but the intrinsic and often overlooked variability of natural and artificial silk fibres makes it challenging to identify trends and correlations. In this work, we determined the mechanical properties of Bombyx mori cocoon and degummed silk, native spider silk, and artificial spider silk, and compared them with classical commercial carbon fibres using large sample sizes (from 10 to 100 fibres, in total 200 specimens per fibre type). The results confirm a substantial variability of the mechanical properties of silk fibres compared to commercial carbon fibres, as the relative standard deviation for strength and strain at break is 10-50%. Moreover, the variability does not decrease significantly when the number of tested fibres is increased, which was surprising considering the low variability frequently reported for silk fibres in the literature. Based on this, we prove that tensile testing of 10 fibres per type is representative of a silk fibre population. Finally, we show that the ideal shape of the stress-strain curve for spider silk, characterized by a pronounced exponential stiffening regime, occurs in only 25% of all tested spider silk fibres.

An Image-Analysis-Based Method for the Prediction of Recombinant Protein Fiber Tensile Strength.

Silk fibers derived from the cocoon of silk moths and the wide range of silks produced by spiders exhibit an array of features, such as extraordinary tensile strength, elasticity, and adhesive properties. The functional features and mechanical properties can be derived from the structural composition and organization of the silk fibers. Artificial recombinant protein fibers based on engineered spider silk proteins have been successfully made previously and represent a promising way towards the large-scale production of fibers with predesigned features. However, for the production and use of protein fibers, there is a need for reliable objective quality control procedures that could be automated and that do not destroy the fibers in the process. Furthermore, there is still a lack of understanding the specifics of how the structural composition and organization relate to the ultimate function of silk-like fibers. In this study, we develop a new method for the categorization of protein fibers that enabled a highly accurate prediction of fiber tensile strength. Based on the use of a common light microscope equipped with polarizers together with image analysis for the precise determination of fiber morphology and optical properties, this represents an easy-to-use, objective non-destructive quality control process for protein fiber manufacturing and provides further insights into the link between the supramolecular organization and mechanical functionality of protein fibers.