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The development of artificial spider silk with properties similar to native silk has been a challenging task in materials science. In this study, we use a microfluidic device to create continuous fibers based on recombinant MaSp2 spidroin. The strategy incorporates ion-induced liquid-liquid phase separation, pH-driven fibrillation, and shear-dependent induction of β-sheet formation. We find that a threshold shear stress of approximately 72 Pa is required for fiber formation, and that β-sheet formation is dependent on the presence of polyalanine blocks in the repetitive sequence. The MaSp2 fiber formed has a β-sheet content (29.2%) comparable to that of native dragline with a shear stress requirement of 111 Pa. Interestingly, the polyalanine blocks have limited influence on the occurrence of liquid-liquid phase separation and hierarchical structure. These results offer insights into the shear-induced crystallization and sequence-structure relationship of spider silk and have significant implications for the rational design of artificially spun fibers., (© 2024. The Author(s).)

Spider dragline silk has attracted great interest due to its outstanding mechanical properties, which exceed those of man-made synthetic materials. Dragline silk, which is composed of at least major ampullate spider silk protein 1 and 2 (MaSp1 and MaSp2), contains a long repetitive domain flanked by N-terminal and C-terminal domains (NTD and CTD). Despite the small size of the CTD, this domain plays a crucial role as a molecular switch that regulates and directs spider silk self-assembly. In this study, we report the 1 H, 13 C, and 15 N chemical shift assignments of the Latrodectus hesperus MaSp2 CTD in dimeric form at pH 7. Our solution NMR data demonstrated that this protein contains five helix regions connected by a flexible linker., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Spider dragline silk is a remarkably tough biomaterial and composed primarily of spidroins MaSp1 and MaSp2. During fiber self-assembly, the spidroin N-terminal domains (NTDs) undergo rapid dimerization in response to a pH gradient. However, obtaining a detailed understanding of this mechanism has been hampered by a lack of direct evidence regarding the protonation states of key ionic residues. Here, we elucidated the solution structures of MaSp1 and MaSp2 NTDs from Trichonephila clavipes and determined the experimental p K a values of conserved residues involved in dimerization using NMR. Surprisingly, we found that the Asp40 located on an acidic cluster protonates at an unusually high pH (∼6.5-7.1), suggesting the first step in the pH response. Then, protonation of Glu119 and Glu79 follows, with p K a s above their intrinsic values, contributing toward stable dimer formation. We propose that exploiting the atypical p K a values is a strategy to achieve tight spatiotemporal control of spider silk self-assembly.

The tiny spider makes dragline silk fibers with unbeatable toughness, all under the most innocuous conditions. Scientists have persistently tried to emulate its natural silk spinning process using recombinant proteins with a view toward creating a new wave of smart materials, yet most efforts have fallen short of attaining the native fiber's excellent mechanical properties. One reason for these shortcomings may be that artificial spider silk systems tend to be overly simplified and may not sufficiently take into account the true complexity of the underlying protein sequences and of the multidimensional aspects of the natural self-assembly process that give rise to the hierarchically structured fibers. Here, we discuss recent findings regarding the material constituents of spider dragline silk, including novel spidroin subtypes, nonspidroin proteins, and possible involvement of post-translational modifications, which together suggest a complexity that transcends the two-component MaSp1/MaSp2 system. We subsequently consider insights into the spidroin domain functions, structures, and overall mechanisms for the rapid transition from disordered soluble protein into a highly organized fiber, including the possibility of viewing spider silk self-assembly through a framework relevant to biomolecular condensates. Finally, we consider the concept of "biomimetics" as it applies to artificial spider silk production with a focus on key practical aspects of design and evaluation that may hopefully inform efforts to more closely reproduce the remarkable structure and function of the native silk fiber using artificial methods.

Spider silk is a protein-based material whose toughness suggests possible novel applications. A particularly fascinating example of silk toughness is provided by Darwin's bark spider ( Caerostris darwini ) found in Madagascar. This spider produces extraordinarily tough silk, with an average toughness of 350 MJ m -1 and over 50% extensibility, and can build river-bridging webs with a size of 2.8 m 2 . Recent studies have suggested that specific spidroins expressed in C. darwini are responsible for the mechanical properties of its silk. Therefore, a more comprehensive investigation of spidroin sequences, silk thread protein contents and phylogenetic conservation among closely related species is required. Here, we conducted genomic, transcriptomic and proteomic analyses of C. darwini and its close relative Caerostris extrusa . A variety of spidroins and low-molecular-weight proteins were found in the dragline silk of these species; all of the genes encoding these proteins were conserved in both genomes, but their genes were more expressed in C. darwini . The potential to produce very tough silk is common in the genus Caerostris , and our results may suggest the existence of plasticity allowing silk mechanical properties to be changed by optimizing related gene expression in response to the environment.

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 300 mM NaCl. Our NMR data demonstrate that secondary structure of monomeric form of NTD MaSp2 consists of five helix regions.

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 CO 2 and N 2 , thus making this photosynthetic microbial cell factory a promising green and sustainable production platform for proteins and biopolymers, including spider silks.

The extraordinary mechanical properties of spider dragline silk are dependent on the highly repetitive sequences of the component proteins, major ampullate spidroin 1 and 2 (MaSp2 and MaSp2). MaSp sequences are dominated by repetitive modules composed of short amino acid motifs; however, the patterns of motif conservation through evolution and their relevance to silk characteristics are not well understood. We performed a systematic analysis of MaSp sequences encompassing infraorder Araneomorphae based on the conservation of explicitly defined motifs, with the aim of elucidating the essential elements of MaSp1 and MaSp2. The results show that the GGY motif is nearly ubiquitous in the two types of MaSp, while MaSp2 is invariably associated with GP and di-glutamine (QQ) motifs. Further analysis revealed an extended MaSp2 consensus sequence in family Araneidae, with implications for the classification of the archetypal spidroins ADF3 and ADF4. Additionally, the analysis of RNA-seq data showed the expression of a set of distinct MaSp-like variants in genus Tetragnatha. Finally, an apparent association was uncovered between web architecture and the abundance of GP, QQ, and GGY motifs in MaSp2, which suggests a co-expansion of these motifs in response to the evolution of spiders' prey capture strategy.

The spider silk spinning process converts spidroins from an aqueous form to a tough fiber. This spinning process has been investigated by numerous researchers, and micelles or liquid crystals of spidroins have been reported to form silk fibers, which are bundles of silk microfibrils. However, the formation process of silk microfibrils has not been clarified previously. Here, we report that silk microfibrils are generated through the formation, homogenization, and linkage of liquid crystalline granules without micelle-like structures. Heterogeneous granules on the submicron to micron scale were observed in the storage sac, whereas homogeneous granules with diameters of approximately 100 nm were aligned along the tapering duct. In the spun fibers, the homogeneous granules were connected along the fiber axis. This is the first clear description of the formation of granule-based microfibrils in the spinning process, which is the key conversion process leading to the unique hierarchical structure of spider dragline.

Silk has attracted widespread attention due to its superlative material properties and promising applications. However, the determinants behind the variations in material properties among different types of silk are not well understood. We analysed the physical properties of silk samples from a variety of silkmoth cocoons, including domesticated Bombyx mori varieties and several species from Saturniidae. Tensile deformation tests, thermal analyses, and investigations on crystalline structure and orientation of the fibres were performed. The results showed that saturniid silks produce more highly-defined structural transitions compared to B. mori, as seen in the yielding and strain hardening events during tensile deformation and in the changes observed during thermal analyses. These observations were analysed in terms of the constituent fibroin sequences, which in B. mori are predicted to produce heterogeneous structures, whereas the strictly modular repeats of the saturniid sequences are hypothesized to produce structures that respond in a concerted manner. Within saturniid fibroins, thermal stability was found to correlate with the abundance of poly-alanine residues, whereas differences in fibre extensibility can be related to varying ratios of GGX motifs versus bulky hydrophobic residues in the amorphous phase.