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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.

Spider silk fiber rapidly assembles from spidroin protein in soluble state via an incompletely understood mechanism. Here, we present an integrated model for silk formation that incorporates the effects of multiple chemical and physical gradients on the different spidroin functional domains. Central to the process is liquid-liquid phase separation (LLPS) that occurs in response to multivalent anions such as phosphate, mediated by the carboxyl-terminal and repetitive domains. Acidification coupled with LLPS triggers the swift self-assembly of nanofibril networks, facilitated by dimerization of the amino-terminal domain, and leads to a liquid-to-solid phase transition. Mechanical stress applied to the fibril structures yields macroscopic fibers with hierarchical organization and enriched for β-sheet conformations. Studies using native silk gland material corroborate our findings on spidroin phase separation. Our results suggest an intriguing parallel between silk assembly and other LLPS-mediated mechanisms, such as found in intracellular membraneless organelles and protein aggregation disorders.

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.

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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.

The assembly of core histones onto eukaryotic DNA is modulated by several histone chaperone complexes, including Asf1, CAF-1, and HIRA. Asf1 is a unique histone chaperone that participates in both the replication-dependent and replication-independent pathways. Here we report the crystal structures of the apo-form of fission yeast Asf1/Cia1 ( Eukaryotic genomic DNA forms hierarchical nucleoprotein complex structures in the nucleus. The nucleosome core particle is the basal repeating unit of the complex, which is composed of ϳ147 bp of DNA wrapped around a core histone particle, comprising a tetramer of H3 and H4 and two dimers of H2A and H2B (1, 2). The precise and regular arraying of nucleosomes is supposed to be a key determinant for the formation of upper hierarchical structures, such as the 30-nm fibers and chromatin fibers. The assembly of the nucleosome is regulated by several groups of chromatin-associated factors involving histone chaperones, chromatin remodeling factors, and histone modification enzymes, which are tightly linked to the regulation of DNA metabolism (2).Histone chaperones are factors that bind to core histones and facilitate their deposition onto nucleosomes (3). Among the variety of histone chaperones, Asf1 4 is the most evolutionarily conserved in its primary structure (4 -8), and its function as a histone chaperone is conserved throughout the eukaryotes (5, 6, 9 -11). Asf1 associates with a variety of chromatin-associated factors, including the histone chaperones CAF-1 (12, 13) and HIR (10, 14, 15), and stimulates both the assembly and disassembly of chromatin (5,6,9,16,17). Consequently, Asf1 affects most DNA-mediated events, including gene expression (15,16,18,19) and silencing (4, 20 -22) as well as DNA repair (5, 13, 23, 24), replication (5) and recombination (25).Asf1 interacts with a heterodimer of histones H3/H4 through the C-terminal region of H3 (26, 27). Importantly, two human family members of Asf1 (ASF1A and ASF1B) are involved in both the major S-phase histone H3.1-and histone variant H3.3 complexes, whereas the Asf1-interactive histone chaperones CAF-1 and HIRA are detected only in the histone H3.1 and H3.3 complexes, respectively (28). Consistently, Asf1 facilitates both DNA replication-dependent and -independent histone depositions cooperatively with the CAF-1 and HIR complexes, respectively (5,28,29), indicating the central role of Asf1 in controlling the state of histone deposition in the nucleus. Recently, the complex structure of human Asf1 with the B-domain of HIRA was reported (30). In addition, biochemical studies suggested that human Cac2, the second largest subunit of CAF-1, interacts with Asf1 at the HIRA-binding region of Asf1 through its B-domain-like motif at the C terminus (30). Hence, human Asf1 is thought to interact mutually exclusively with the histone chaperones HIRA and CAF-1. However, structural evidence for the interaction between Asf1 and CAF-1 has yet to be obtained. In addition, it is not clear how Asf1 recognizes HIRA and CAF-1 for histone assem...