Native Mass Spectrometry Captures the Conformational Plasticity of Proteins with Low-Complexity Domains.

Disordered regions are an important functional feature of many multidomain proteins. A prime example is proteins in membraneless organelles, which contain folded domains that engage in specific interactions and disordered low-complexity (LC) domains that mediate liquid-liquid phase separation. Studying these complex architectures remains challenging due to their conformational variability.

Post-spin Stretch Improves Mechanical Properties, Reduces Necking, and Reverts Effects of Aging in Biomimetic Artificial Spider Silk Fibers.

Recent biotechnological advancements in protein production and development of biomimetic spinning procedures make artificial spider silk a promising alternative to petroleum-based fibers. To enhance the competitiveness of artificial silk in terms of mechanical properties, refining the spinning techniques is imperative. One potential strategy involves the integration of post-spin stretching, known to improve fiber strength and stiffness while potentially offering additional advantages.

Liquid-liquid crystalline phase separation of spider silk proteins.

Liquid-liquid phase separation (LLPS) of proteins can be considered an intermediate solubility regime between disperse solutions and solid fibers. While LLPS has been described for several pathogenic amyloids, recent evidence suggests that it is similarly relevant for functional amyloids. Here, we review the evidence that links spider silk proteins (spidroins) and LLPS and its role in the spinning process.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 β-sheet crystals.

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 β-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.

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.

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.

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.

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.

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.

A Novel Approach for the Production of Aggregation-Prone Proteins Using the Spidroin-Derived NT* Tag.

Spiders have evolved proteins that can be kept in a highly concentrated soluble form in the silk gland yet rapidly assemble into stable silk fibers under certain environmental conditions. The transition between soluble and fibrillar states is partly regulated by the pH-sensitive N-terminal (NT) domain which has emerged as nature's own solubility-enhancing domain. NT has an inherent capacity to keep the silk proteins' partly hydrophobic and very aggregation-prone regions from premature fibrillation in spite of storage at enormous concentrations.

Treatment of Respiratory Distress Syndrome with Single Recombinant Polypeptides that Combine Features of SP-B and SP-C.

Treatment of respiratory distress syndrome (RDS) with surfactant replacement therapy in prematurely born infants was introduced more than 30 years ago; however, the surfactant preparations currently in clinical use are extracts from animal lungs. A synthetic surfactant that matches the currently used nature-derived surfactant preparations and can be produced in a cost-efficient manner would enable worldwide treatment of neonatal RDS and could also be tested against lung diseases in adults. The major challenge in developing fully functional synthetic surfactant preparations is to recapitulate the properties of the hydrophobic lung surfactant proteins B (SP-B) and SP-C.