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Proceedings of the Japan Academy. Series B, Physical and Biological Sciences logoLink to Proceedings of the Japan Academy. Series B, Physical and Biological Sciences
. 2026 Jan 9;102(1):40–56. doi: 10.2183/pjab.102.003

Spider silk-based structural proteins as tough, biodegradable, and sustainable polymers

Keiji NUMATA *1,*2,*3,†
PMCID: PMC12950840  PMID: 41526240

Abstract

Spider silk has garnered significant attention across diverse research fields because of its remarkable physical and biological properties, which have driven extensive efforts to develop materials and fabrication processes that replicate its unique characteristics. This review focuses on the structure of silk fibers and the mechanisms that underlie their formation, with an emphasis on the hierarchical organization that contributes to their outstanding performance. Additionally, the biodegradability of silk proteins and their degradation products are discussed, and their potential for sustainable material design are highlighted.

Keywords: spider silk, structural protein, liquid–liquid phase separation, fiber, toughness, biodegradability

Schematic diagram of the domain structure of major ampullate spidroin.

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1. Introduction

In general, polyamides exhibit desirable mechanical, thermal, and gas barrier properties that distinguish them from polyesters, which are commonly used in bioplastic materials. Polyamide 4 (nylon 4) and polyamide 11 (nylon 11) are generally considered bioplastics, as they are widely recognized as biobased polyamides. Nylon 4 is synthesized from gamma-aminobutyric acid, which is derived from the decarboxylation of glutamic acid, whereas nylon 11 is produced from castor oil.1)–4)) In addition to nylon and other biologically derived polyamides, increasing attention is being given to biobased polymers that are composed of amino acids, such as proteins. Natural proteins such as silk, keratin, and resilin are known for their unique mechanical properties, many of which are difficult to replicate with synthetic polymers.5),6)) However, the mechanisms that underlie these properties remain incompletely understood, and a molecular-level understanding of their hierarchical structures is still needed. Among these, spider silk—which is composed of spider silk proteins, also known as spidroins—is particularly notable for its exceptional toughness, which far surpasses that of synthetic polymers. This remarkable toughness makes spider silk a highly promising candidate for structural materials, which has in turn driven growing interest in spider silk across a broad range of materials science fields.

2. Spider silk

In recent years, spider silk has attracted considerable attention across various industries because of its exceptional mechanical properties, in turn spurring intensive research into the development of materials and processes that mimic its characteristics.5)–8)) Researchers who have studied silk for decades often remark that “research on artificial spider silk tends to surge in cycles”. However, with recent advances in biology and chemistry, the scope of research that is accessible to polymer scientists, including the author, has expanded significantly.9)) Furthermore, interdisciplinary collaborations have enabled entirely new approaches that differ from traditional methods. In this paper, from the perspective of polymer science, I review the process of spider silk formation and explore material design strategies that are inspired by its spinning mechanism.

Spider silk has unique properties and compositions that distinguish it from other types of silk,10)–12)) which contribute to its strong appeal as a biomaterial (Fig. 1). Spiders produce a variety of silk threads, with some species capable of generating up to seven distinct types, each of which serves different functions throughout their lifetime.13)–15)) These threads vary in terms of physical properties depending on their specific purpose.16)–18)) Among them, “dragline” silk is particularly notable for its exceptional toughness, which makes it a promising candidate for applications in structural materials and other advanced fields. In recent years, the biodegradability of structural proteins—such as spider silk—has also attracted attention, prompting interest in their use as environmentally friendly materials.19)–22)) Despite the growing expectations for these next-generation biomaterials, the precise spinning mechanism of spider silk remains unclear. This lack of understanding continues to be a major barrier to the efficient and scalable production of artificial spider silk.

Figure 1.

Figure 1.

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Examples of the natural spider silks. (a) Trichonephila clavata spider web at Kyoto, Japan. (b) Strings prepared from the natural Trichonephila clavata spider dragline.

2.1. Major ampullate spidroin (MaSp).

The dragline silk produced by Trichonephila clavata, a spider species that is widely distributed across Japan, is spun from a protein solution that passes through large, vase-shaped major ampullate glands (Fig. 2).23)) This silk is composed primarily of a complex mixture of proteins known as major ampullate spidroins (MaSp). The number of distinct MaSp types remains a topic of ongoing debate; however, at least two—MaSp1 and MaSp2—are widely recognized.24),25)) These proteins are considered to contribute differently to the mechanical properties of silk because of variations in their amino acid sequences.9)) Although MaSp proteins have been identified in a variety of spider species, the relationships between their molecular structure and resulting physical properties are not yet fully understood. In particular, the key determinants of mechanical performance, such as toughness, have not been conclusively demonstrated, which makes this an intriguing research area not only in biology but also in materials science and chemistry.

Figure 2.

Figure 2.

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Overview of the major ampullate spider silk gland. (a) Image of the major ampullate gland from Trichonephila clavata. The arrows indicate the flow direction inside the gland. (b) A schematic diagram of a silk gland and the reported pH and ion gradients. In the spider major ampullate gland, chaotropic ions are replaced by kosmotropic ions, which induce phase separation, while a gradient of decreasing pH induces the dimerization of the NTD, which promotes fibril formation. Reproduced and modified from Lay et al. Polymer Journal 2025.70))

In MaSp, a type of silk protein, both the N-terminal domain (NTD) and the C-terminal domain (CTD) are believed to play key roles in intermolecular interactions that facilitate the stepwise assembly of silk fibers (Fig. 3). The NTD is thought to undergo dimerization in response to the decrease in pH that occurs during the spinning process,26)) whereas the CTD is believed to promote intermolecular bonding through the formation of disulfide bonds between cysteine residues.27)) The repetitive core sequence, which comprises alternating crystalline (beta-sheet-forming hard segments) and amorphous (noncrystalline soft segments) regions, lies between these terminal domains. This region constitutes the majority of the MaSp protein and is critical for driving crystallization and molecular alignment within the fiber.28),29))

Figure 3.

Figure 3.

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(a) Schematic diagram of the domain structure of major ampullate spidroin (MaSp). The N-terminal domain (NTD) and C-terminal domain (CTD) are present at both ends, along with a repetitive sequence of crystalline and amorphous sequences (repetitive domain).71)) (b) Schematic illustrations of spider silk spinning process from the secondary structure to bundle structures of microfibrils of spider silk.

For many years, scientists and researchers have sought to elucidate the hierarchical structure of spider silk and to develop artificial spider silk materials. Today, numerous ventures and start-ups worldwide are actively working to replicate or innovate spider silk-like materials, despite the incomplete scientific understanding of the complexity of this natural fiber.30)) However, no artificial material has yet matched the exceptional toughness of natural spider silk, which uniquely combines both strength and elongation. This shortfall largely stems from attempts to fabricate structural protein fibers via conventional synthetic fiber technologies without fully understanding the native spinning mechanism. Without leveraging the intrinsic biochemical properties of the protein, the necessary hierarchical structures that give rise to its remarkable physical characteristics cannot be properly formed.31)) By harnessing the inherent biochemical features of MaSp silk proteins, it should be possible to create tough structural protein materials that combine strength and extensibility, thereby enabling truly original developments distinct from existing research efforts. The author’s research group aims to clarify the self-organization behavior of MaSp—which is the main component of spider dragline silk—and, in particular, to elucidate the formation process of its hierarchical structure. Understanding the process of spider silk formation is critically important not only for creating artificial spider silk but also for realizing an eco-friendly manufacturing process that avoids the use of high temperatures, high pressures, and organic solvents.

Among the MaSp found in spider dragline silk, MaSp2 is more hydrophilic than MaSp1 is. The increased water sensitivity and solubility of MaSp2 is attributed to the periodic occurrence of diglutamine (two consecutive glutamine residues) and proline in its sequence.8),32)) To mimic the properties of MaSp2, a water-soluble silk protein was produced from genetically engineered Escherichia coli. To further investigate the roles of the NTD and CTD, variants of MaSp2 that lack the NTD and/or CTD were also prepared.31)) When potassium phosphate was added to MaSp2 under neutral pH conditions at concentrations above a certain threshold, white turbidity appeared in the solution. This turbidity was confirmed to result from liquid–liquid phase separation (LLPS), which was characterized by numerous heterogeneous spherical droplets that range from 0.1 to 10 micrometers in size undergoing dynamic fusion (Fig. 4).31)) Furthermore, under acidic conditions, pH-induced NTD dimerization led to the formation of a network of microscopic fibers (microfibrils), thus indicating its role in fiber assembly.

Figure 4.

Figure 4.

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Self-assembly behavior of MaSp2 in response to pH. Fluorescence microscopy image of the liquid–liquid phase separation formed by MaSp2. Fluorescence microscopy image of MaSp2 under neutral pH conditions after the addition of potassium phosphate to MaSp2, showing liquid–liquid phase separation with numerous heterogeneous spherical droplets that range from 0.1 to 10 µm in size. Fluorescence microscopy images of reticulated microfibrils. MaSp2 was acidified to form reticulated microfibrils. The scale bars represent 10 µm. Reproduced from Malay et al. Science Advances 2020.31))

As a control experiment, MaSp2 variants lacking the NTD, CTD, or both were subjected to the same conditions used to induce self-assembly. The results revealed that the CTD is essential for LLPS, whereas the NTD is required for the formation of a fibrous network. Fluorescence recovery after photobleaching (FRAP), rheological analysis, and Raman spectroscopy demonstrated that under neutral to mildly acidic conditions, the presence of the CTD and the repetitive core sequence promotes intermolecular interactions that drive LLPS.31)) Although not discussed in detail here, the data suggest that the repetitive sequence is actually required for both LLPS and fibrillization. A more accurate interpretation is that LLPS requires both the CTD and the repetitive sequence, whereas fibrillization depends on the NTD and the repetitive sequence.

In the case of MaSp1—which is a more hydrophobic spidroin than MaSp2—the LLPS phase diagram exhibits notable differences. Biomimetic self-assembly of MaSp1 across multiple length scales, with a transition from a largely disordered soluble state to hierarchically organized macroscopic fibers, was reported recently.33)) Interestingly, compared with MaSp2, MaSp1 was significantly more sensitive to kosmotropic anions, such as phosphate, as indicated by its lower threshold concentrations required for LLPS induction. This greater sensitivity is likely due to strong cation–π interactions, primarily between arginine and tyrosine residues, which are known to be key drivers of biomolecular condensation—especially in systems that contain multivalent, repetitive sequences.34),35)) Notably, the consensus repeat of MaSp1 includes one tyrosine residue and one arginine residue, whereas MaSp2 includes a tyrosine residue but lacks an arginine residue. This distinction highlights an intriguing sequence–function relationship that warrants further investigation.33)) Moreover, the variability in phase separation behavior between MaSp1 and MaSp2—and potentially other MaSp subtypes—raises the possibility that, within the silk gland, different spidroin subtypes may form quasidiscrete, preorganized ensembles or clusters prior to fiber assembly.36))

2.2. Posttranslational modification of silk.

MaSp, the primary component of spider silk, is a prototypical biopolymer that exhibits exceptional tensile properties because of its complex hierarchical assembly. Although recent sequencing advances have provided extensive insights, relatively little is known about posttranslational modifications (PTMs) in MaSp. The authors’ group recently investigated the PTM landscape of dragline silk from Trichonephila clavata via a combination of mass spectrometry and solid-state nuclear magnetic resonance (NMR).37)) The results revealed a diverse array of potential modifications—including hydroxyproline, phosphorylation, and dityrosine crosslinks38))—across different spidroin constituents. Notably, the MaSp3 repetitive region exhibited numerous PTMs, whereas the MaSp1 and MaSp2 variants displayed distinct phosphorylation patterns in their terminal domains (Fig. 5). Phosphorylation sites in the NTD were located predominantly at the dimer interface, which suggests a modulatory role in its pH-dependent dimerization; this hypothesis is supported by studies in which phosphomimetic NTD mutants were used. The possible functions of phosphoserine in limiting β-sheet formation and of hydroxyproline in disrupting β-turns have also been discussed. The prevalence of PTMs that involve various MaSp proteins in spider dragline silk and their structural domains were first described in the group’s study. This characterization provides the first insights into the structural effects of these PTMs and their potential regulatory roles, particularly in the NTD. However, the authors acknowledge limitations, especially that the current methods do not enable the quantitative assessment of PTM abundance, which is an issue that future specialized approaches may address. Overall, this work highlights a significant gap in our understanding of the spider silk proteome, which is an essential component of the broader silk material, and highlights the potential for developing more sophisticated artificial silks that incorporate the added functionality and complexity derived from these PTMs.

Figure 5.

Figure 5.

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Comprehensive map of PTMs observed in spider MA silk.37)) The observed PTM positions with respect to the full-length sequences of the predominant MaSp and SpiCE components of dragline silk from T. clavata. A detailed breakdown of the fragments used to generate the map can be found in the supplementary material. The numbered residues correspond to the phosphorylation of Ser (S, aqua), Thr (T, purple) or Tyr (Y, orange) and the hydroxylation of Pro (P, blue). Each protein is drawn to scale, with the scale bar corresponding to 300 residues. Reproduced from Craig et al. MRS Bulletin 2024.37))

2.3. Hierarchical structures.

Natural spider dragline silk demonstrates a hierarchical structure that is composed of bundled microfibrils (Fig. 6a, b).39)) When fibers were prepared by applying shear stress to the mesh-like microfibrils formed via LLPS, a similarly well-organized hierarchical structure of bundled microfibrils was observed (Fig. 6c–h).31)) Raman spectroscopy revealed beta-sheet formation during the subsequent dehydration process rather than during shear-induced fiber formation.31)) The solid-state NMR studies of the other MaSp1 reported that the beta-sheet structures of the alanine-rich regions consisted of a mixture of rectangular and staggered arrangements, whereas the glycine-rich regions exhibit a mixed structure composed of random coils and beta-turns.40)) Consistent with the structure of natural spider silk, the LLPS-derived thread samples exhibited bundled microfibrils (Fig. 6c, d) with oriented beta-sheet structures, and scanning electron microscopy (SEM) and optical microscopy of the fractured surfaces clearly revealed individual microfibril fractures (Fig. 6e, f). In contrast, fibers that were produced without LLPS did not display reticulate microfibrils or the hierarchical structural characteristics of natural spider silk (Fig. 6g, h). These results demonstrate that through MaSp self-assembly via LLPS, we successfully reproduced—for the first time—a hierarchical structure in which microfibrils are bundled into fibers.31))

Figure 6.

Figure 6.

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Micrographs of spider silk threads. (a) SEM image of spider dragline72)) and (b) FE-SEM image of the surface of the trailing thread of a jumping spider after enzyme treatment. The microfibril bundle structure can be observed.39)) (c, d) SEM and (e, f) optical microscopy images of the surface (c, e) and fracture surface (d, f) of the artificial spider silk threads prepared via LLPS.31)) (g, h) Optical microscopy and SEM images of artificial spider silk threads prepared without the LLPS process.31)) Reproduced from Yazawa et al. Macromolecular Bioscience 2019,72)) Sogawa et al. Frontier in Bioengineering and Biotechnology, 2020,39)) Malay et al. Science Advances 2020.31))

As described above, various experiments in which MaSp2—a highly water-soluble and pH-responsive silk protein41))—has been used have successfully reproduced a spinning mechanism that closely resembles that of natural spider silk, particularly when implemented with a microfluidic system.42)) The key finding was that silk proteins that are capable of self-assembly in response to pH changes and specific ionic conditions undergo LLPS. Specifically, LLPS is triggered by interactions between the CTD and the central repetitive sequence, while the NTD undergoes dimerization in response to a decrease in pH. Subsequently, the repetitive sequence forms beta-sheet structures in response to shear stress and dehydration, thus revealing a multistep assembly mechanism. In addition to the roles of the terminal domains, the role of the central repetitive sequence in the early stages of the spinning process has also been investigated. Nuclear magnetic resonance (NMR) and circular dichroism (CD) analyses have shown that glycine-rich amorphous regions adopt a polyproline II helix conformation when at least two repeated units are present in solution.28)) Although the precise biological role of this partial helical structure remains unclear, it is thought to help prevent nonspecific aggregation of highly concentrated silk proteins just before spinning. Furthermore, our studies have shown that pH changes that occur during the spinning process specifically affect the N- and C-terminal domains but not the repetitive sequence itself.43),44))

3. Diversity of silk proteins

All spiders use silk for a wide range of critical functions, including foraging, locomotion, nesting, mating, egg protection, and communication.45)) Different types of threads are employed for these diverse purposes, and each type is produced by specific silk glands located in the abdomen.46)) For instance, orb-weaving spiders can produce up to seven distinct types of silk, each of which is named after the gland that synthesizes it. Major ampullate silk, which is the toughest type, is used for draglines and as the structural frame of orb webs. Minor ampullate silk serves as a scaffold during web construction. Piriform silk functions as an adhesive and anchors the web frame to wood or other substrates. The capture thread of orb webs consists of a flagelliform silk core that is coated with aggregate glue. Aciniform silk is used for prey wrapping and occasionally for web decoration, whereas tubuliform (or cylindrical) silk is responsible for forming the egg sac. While spiders are often associated with orb webs and are successful predators, orb-weaving spiders of the superfamily Araneoidea constitute only approximately 25% of all known spider species. More ancestral clades, such as those in the Mygalomorphae order, are predominantly ground-dwelling spiders that construct sheet and maze webs for prey capture. In contrast, a more modern clade—the RTA clade—encompasses approximately 50% of all spider species. These are largely wandering hunters that have abandoned silk-based prey capture strategies.47)) This remarkable diversity in silk use illustrates how spiders have adapted, specialized, and optimized silk production according to their ecological needs. The extraordinary plasticity and versatility of spider silk and its proteins make them an ideal model for studying the relationships between sequence and physical properties. Understanding the underlying design principles of silk could enable the application of its wide-ranging mechanical characteristics in biomaterials.

In a previous study, an international collaborative group that included the author established a comprehensive dataset that encompasses the genotypes and phenotypes—including mechanotypes—of spider silks and identified the key design elements that are responsible for their extraordinary mechanical and physical performance (Fig. 7a). Although silk proteins have evolved convergently across various lineages,48)) they share common features such as sequence motifs,49)) amino acid compositions,50)) and a characteristic trade-off between tensile strength and elasticity, which is governed by the ratio of amorphous to crystalline regions.51)) These findings offer a valuable framework for the future analysis of silk proteins and other structural proteins as biomaterials. The application of similar data-driven approaches to protein materials with properties beyond toughness—such as elastomers and adhesive proteins—could further accelerate our understanding of the genetic design principles that underlie functional biomaterials. In particular, computational modeling and simulations that can predict molecular interactions among multiple components, such as various MaSp-type spidroins and spider-silk-constituting element (SpiCE) proteins, represent promising directions for future research.52))

Figure 7.

Figure 7.

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(a) Overview of the taxonomic distribution of spidroins and the physical properties of dragline silks. Left, phylogenetic tree of spider families constructed from the transcriptome data obtained from 1,000 spiders in this work. The Araneoidea superfamily and the RTA clade are highlighted in red and blue, respectively. The family names in red represent those without previous reports of spidroins in the NCBI Protein database. Families marked with orange circles represent those without previous transcriptome data. The total number of species that were sequenced in this work for each family, as well as species-level decompositions of unreported spidroin and transcriptomes, are shown to the right of the family names. As the table shows, the vast majority of the species that were reported in this work were previously unreported in terms of their spidroin sequences or transcriptomes. Middle, heatmap of the conservation level of spidroin types within the spider families. For example, MaSp3 of the family Araneidae has a value of approximately 0.5, which is indicated by the color code shown in the bottom-left corner and indicates that the silks produced by approximately 50% of the 191 species studied in this work contain MaSp3. The orb-weaving spiders in the superfamily Araneoidea (highlighted in pink) possessed greater diversity of spidroin types, while the RTA clade (highlighted in light blue) lost the capture web silk components Flag and AgSp. MaSp sequence subtypes are not well differentiated in the RTA clade, where MiSp, AmSp, and MaSp are more conserved than MaSp1 and MaSp2 are. Right, distribution of physical properties among the spider families. Mirrored by the diversity of spidroins, the silks produced by orb-weaving Araneoidea spiders tend to perform better than those produced by other clades do. (b) Comparison of phylogenetic trees of silkworm-derived silk.73)) Amino acid sequences and various physical properties are also compared.53)) Reproduced from Arakawa et al. Science Advances 2022,73)) Malay et al. Scientific Reports 2016.53))

Silk proteins are produced not only by spiders but also by other species, such as silkworms. While both types of silk share beta-sheet structures as their crystalline component, their amino acid sequences and resulting physical properties differ significantly. Spider silk is composed of multiple protein components, yet many of the low-molecular-weight proteins that are present in small amounts remain unidentified. On the other hand, silkworm-derived silk is the most extensively studied polymeric material. In recent years, silk has been re-evaluated as a versatile macromolecular material. A consortium of Asian countries—particularly Japan—has reported a wide range of physical properties derived from silkworm cocoon silk (Fig. 7b).53)) Differences in mechanical and thermal characteristics can be attributed to differences in amino acid sequences and the hierarchical structures that arise from them. Furthermore, some silk proteins contain arginine-glycine-aspartic acid (RGD) sequences,54)) which promote cell adhesion, thus making them promising candidates for use in biomaterials.

The author’s group previously succeeded in elucidating the amino acid sequence of silkworm cocoon silk and compared its physical properties with those of chemically synthesized model peptides, thereby providing valuable insights for material design. For example, the copolymerization ratio of alanine and glycine—amino acids that make up the crystalline regions of silk—affects the resulting secondary structure, which can adopt a polyglycine II, polyglycine I, or beta-sheet conformation. These structural variations also lead to corresponding changes in thermal degradation behavior (Fig. 8).55)) This correlation has been confirmed not only in synthetic model peptides but also in natural silk fibers that were produced by silkworms.53)) These results suggest that differences in the arrangement of partial polypeptide motifs within proteins significantly influence material properties. In silk in particular, both thermal and mechanical characteristics appear to be uniquely determined by the organization of the crystalline domains. These findings provide important insights for designing the thermophysical properties of silk materials at the molecular level.

Figure 8.

Figure 8.

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Comparison of the amino acid sequence, thermophysical properties, and secondary structure of the silk protein crystalline sequence.53),55)) Reproduced from Ageitos et al. Biomacromolecules, 2016.55))

4. Silk as a high-toughness material

The exceptional toughness of spider dragline silk is believed to originate from its molecular and microfibrillar network structures. Similarly, the network architecture of silk proteins or silk microfibers is a key factor that contributes to the toughness of silk-based biomaterials. In addition, the hydration state—which influences both crystallization and plasticization—plays a crucial role in determining the mechanical behavior of silk. Typically, in polymeric materials, increased crystallinity leads to reduced elongation. However, in silk, the presence of excess water can increase both crystallinity and elongation simultaneously.56),57)) This counterintuitive effect is likely because of the plasticizing action of water, which increases molecular mobility and promotes chain reorientation and crystallization. In amorphous regions, water also improves the flexibility of molecular chains. Therefore, both the hydration state and the higher-order structure of silk proteins are critical considerations in the design and application of silk-based materials.

To investigate the effects of network structure and hydration state on the mechanical properties of silk materials, studies have examined silk-based chemical gels, physical gels, and chemical–physical gels—each with distinct network architectures (Fig. 9).21)) Chemical gels are prepared by adding horseradish peroxidase and hydrogen peroxide to an aqueous silk solution, thereby inducing covalent cross-linking between tyrosine residues. Physical gels are formed by treating aqueous silk solutions with ethanol, which promotes beta-sheet formation and phase separation. Chemical–physical gels are obtained by immersing chemical gels in ethanol, thereby combining covalent cross-linking with beta-sheet–induced structural reinforcement. Among the three gel types, chemical–physical gels have the highest compressive strength. In contrast, physical gels tend to be hard and brittle, whereas chemical gels are soft and mechanically weak. The greater mechanical performance of chemical–physical gels is attributed to ethanol-induced beta-sheet formation in the chemically cross-linked network. This process reduces the molecular weight between cross-linking points and increases crystallinity, thereby resulting in a stiffer and more robust material.

Figure 9.

Figure 9.

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Silk gels with different network structures. (a) Silk chemical gel, (b) physical gel, and (c) chemical–physical gel. (d–f) SEM images of the interior of the silk gels. The scale bar represents 20 µm. (g–i) Schematic diagrams of the network structures of the chemical gel, physical gel, and chemical–physical gel. The red dots represent chemical cross-linking points, and the blue structure indicates sheet-like crystals. Reproduced from Numata et al. Biomacromolecules, 2017.21))

The properties of silk and its related proteins are sensitive to water content and humidity.56),57)) Silk materials with varying moisture contents were prepared by subjecting each gel to different relative humidity (RH) conditions, thereby controlling their hydration states (Fig. 10).21)) The compressive strength of the resulting silk materials increased as the moisture content decreased, with the highest strength observed at 23% RH. This trend suggests that the formation of beta-sheet structures, which is increased under low-humidity conditions, contributes significantly to the increased mechanical stress resistance. These findings indicate that both the network structure and the hydration state of the silk are critical factors that influence the mechanical properties of silk materials, including compressive strength and toughness.

Figure 10.

Figure 10.

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Silk materials that were prepared from chemical–physical gels and the corresponding results of compression tests. (a) Chemical–physical gel prepared from aqueous silk solution. (b–h) Silk materials obtained when the chemical physical gel was prepared at RHs of 97% (b), 75% (c), 58% (d), 43% (e), 23% (f), 11% (g) and 6% (h). (i) Compression test results for these eight samples. Reproduced from Numata et al. Biomacromolecules, 2017.21))

5. Biodegradability and degradation products of silk

In a white paper from Chemical Sciences and Society Summit (CS3), London, 2019 (“Science to enable sustainable plastics” URL: https://www.rsc.org/news-events/articles/2020/jun/science-to-enable-sustainable-plastics/), most of the biodegradable plastics and bio-based polymers were introduced as a key material for sustainable society development. Notably, structural proteins such as spider silk were introduced as environmentally degradable and sustainable polymers. The utilization of biodegradable plastics, particularly those designed for on-demand biodegradation in which degradation is initiated post-use, remains limited. Achieving precise control over polymer degradation is of critical importance, not only to secure the functional properties of degradable polymers but also to enhance the long-term stability of polymeric materials. At present, the suppression of deterioration and premature decomposition, as well as the maintenance of polymer stability over several decades, represent formidable challenges that impede their deployment as primary structural materials in the manner that metals are currently employed. Nonetheless, these challenges associated with long-term stability and controlled degradability may be addressed through a comprehensive, multilevel understanding of polymer decomposition processes.

Silk, as both a structural protein and a natural polymer, is inherently biodegradable, rendering it a promising alternative to synthetic, petroleum-derived polymers. In previous studies, the biodegradability of silk resins was investigated using biochemical oxygen demand (BOD) assays, confirming their capacity to undergo degradation in natural environments (Fig. 11). In addition, scanning electron microscopy (SEM) characterization with the silk films revealed the formation of surface roughness and cracks in the coated films after exposure to seawater, demonstrating degradation of the material over time.22)) In contrast to synthetic petroleum-derived polymers and biodegradable polyesters, natural structural proteins and artificially designed silk materials such as fibers, resins, sponges, films, nano/microparticles, gels, coatings, and composites with the other materials can degrade in various natural environments,21),22),58),59)) and the biodegradation products aren’t significantly cytotoxic to model organisms, e.g., Daphnia magna.60)–62)) In this way, these biodegradation products are scientifically different and unique compared with those of biodegradable polyesters and other materials.62))

Figure 11.

Figure 11.

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Biodegradation and environmental effects of model silk fragments, namely, polymers and oligomers of Ala and various nylon units. (a) BOD tests of poly(AlaNylXAla) and polyAla in seawater at 25 ℃.60)) (b) Daphnia magna, which was used for the environmental toxicity test. (c) Kaplan–Meier survival curves for evaluating the effects of poly(AlaNylXAla) and polyAla on the aquatic model animal Daphnia magna at 20 ℃. Control refers to the background values without any test samples.60)) Reproduced from Gudeangadi et al. Polymer Chemistry, 2020.60))

Silk proteins and other structural proteins are biodegradable in the natural environment, which makes sense, as silk is a natural biological resource. However, the solid structure and chemical structure of proteins clearly influence their degradation rate. One example is keratin, which is a persistent protein that possesses many disulfide bonds.7)) This finding suggests that controlling the biodegradability of protein materials through modifications of their amino acid sequences and chemical structures is possible. The degradation behavior of silk protein in activated sludge was evaluated by measuring the biochemical oxygen demand (BOD) in a resin-like solidified state, and the results showed that differences in network structure and hydration state had no significant effect on biodegradability, whereas the network density, which was represented by the silk concentration, had a significant effect on biodegradability (Fig. 12).2),22))

Figure 12.

Figure 12.

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Comparison of degradability in terms of biochemical oxygen demand (BOD).2),22)) The degradability of polyhydroxybutyrate and nylon 4 films, which are representative bioplastics, in water is compared with that of silkworm-derived silk resin in activated sludge.

The antiparallel beta-pleated sheet represents a fundamental secondary structure in proteins and constitutes a major structural element of silk fibers produced by silkworms and spiders, where it contributes to protein stabilization through physical cross-links. Notably, unlike beta-amyloid aggregates formed under pathological conditions, beta-sheet structures in silk are fully degradable and nontoxic in biological systems. Elucidating the mechanisms underlying their enzymatic degradation is therefore essential for understanding the distinct behaviors of these stable yet structurally divergent assemblies in biology. A previous study reported the enzymatic degradation pathway of antiparallel beta-pleated sheets in Bombyx mori silk, demonstrating a sequential process in which silk crystals are digested into fibrils and subsequently into nanofibrils (Fig. 13).19)) This work provided the first mechanistic model describing the degradation of silk beta-sheet crystals and further suggested that the resulting natural degradation products exhibit no significant cytotoxicity toward cells. Moreover, the nanofibrils generated during degradation function as nucleators of crystalline regions, a newly identified and significant feature that can be leveraged in the rational design of silk-based biomaterials with tunable biodegradability and mechanical properties.

Figure 13.

Figure 13.

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Silk beta-sheet crystals before enzymatic degradation. (A) Typical AFM amplitude image of silk crystals before enzymatic degradation. The white broken line denotes the nanofibrils that compose the crystal. (B) Line profile data for the crystals indicated by the white solid line in (A). Enzymatic degradation processes of silk beta-sheet crystals. AFM height images of silk crystals during enzymatic degradation by protease XIV for 24 h (C). (D) Line profile data of the crystals indicated by the white line in (C). Each scale bar represents 1 µm. Reproduced from Numata et al. Biomaterials 2010.19))

Furthermore, new approaches for exploiting silk-based beta-sheet structures within complex designed protein biomaterial systems will benefit from this understanding as a basis for controlling degradation rates in vitro and in vivo. Beta-sheet silk crystals were shown to undergo enzymatic degradation by protease XIV, initially producing nanofibrils and subsequently nanofilaments approximately 2 nm in diameter and 160 nm in length. In contrast, the termini and edges of the beta-sheet crystals were degraded more slowly by alpha-chymotrypsin, yielding soluble silk molecules with random-coil conformations. Notably, the degradation products generated by protease XIV exhibited greater cytotoxicity than either the intact silk crystals or the natural degradation products obtained with alpha-chymotrypsin, a result attributed to the rapid formation of soluble beta-sheet fragments derived from higher-molecular-weight intermediates. These findings indicate that silk beta-sheet crystals and their natural degradation products are unlikely to induce cytotoxicity in vivo owing to their intrinsically slow degradation kinetics. Collectively, these newly elucidated mechanistic features provide a foundation for the rational design of silk-based biomaterials with predictable biodegradability, minimal cytotoxicity, and tailored mechanical properties.19)) Furthermore, comparison of the degradation pathways of antiparallel beta-sheet silk structures with those of amyloid aggregates yields novel insights into the determinants of biological stability and cytotoxicity in amyloid-beta assemblies.63),64))

6. Future perspectives

As mentioned at the beginning of this review, structural proteins such as silk and elastin serve as essential building blocks in nature. However, their practical application as bulk-scale structural materials for human use remains unrealized.8)) The challenges that are associated with using polypeptides in this capacity span the entire construction pipeline—from synthesis and purification to processing. In particular, replicating the remarkable physical properties of natural spider silk remains a major scientific and technological challenge. A deeper understanding of these natural phenomena at the molecular level is therefore not only of academic interest but also key to reproducing and utilizing spider silk in practical applications. Moreover, the development of materials that are derived from natural sources and produced through non-petroleum-based processes holds great promise for building a sustainable society and mitigating environmental issues, such as microplastic pollution, by expanding the use of biodegradable substances. Achieving sustainable production of protein- and polypeptide-based materials is a critical hurdle. Recently, the photosynthetic biosynthesis of proteins and polypeptides has emerged as a promising avenue,65)–69)) but further research is needed to elucidate the underlying molecular mechanisms and to advance synthetic biology approaches. Elucidating the molecular mechanisms of structural proteins and applying these insights to material design is expected to be more widely adopted as a relevant strategy in the future. Finally, the author sincerely hopes that more researchers will enter this field and contribute to the development of innovative synthetic methods and molecular designs for structural proteins.

Acknowledgments

This work was financially supported by the Cabinet Office, the Impulsing Paradigm Change through Disruptive Technologies (ImPACT) Program, Japan Science and Technology (JST) ERATO (Grant No. JPMJER1602), Grant-in-Aid for Transformative Research Areas (B) (Grant No. JP20H05735), JST COI-Next (Grant Number JPMJPF2114), and the MEXT Program: Data Creation and Utilization-Type Material Research and Development Project (Grant Number JPMXP1122714694).

Abbreviations

BOD

biochemical oxygen demand

CD

circular dichroism

CTD

C-terminal domain

LLPS

liquid–liquid phase separation

MaSp

major ampullate spidroin

NMR

nuclear magnetic resonance

NTD

N-terminal domain

PTM

posttranslational modification

RH

relative humidity

SEM

scanning electron microscopy

SpiCE

spider-silk-constituting elements

Profile

Keiji Numata earned his Ph.D. (2007) with a thesis centered on enzymatic degradation and synthesis with hydrolases of biopolymers under the supervision of Prof. Yoshiharu Doi, Tokyo Institute of Technology. He worked as a JSPS Postdoctoral Fellow for Research Abroad at Tufts University (U.S.A.) where he studied biosynthesis of silk-based polymers via bacterial pathways. He moved to RIKEN in 2010 to start up a laboratory to investigate biosynthesis and material design of structural proteins and poly(amino acid). He has been a Research Director for JST-ERATO Numata Organelle Reaction Cluster Project (2016–2023), Research Director for JST-COI-NEXT (2022–), Research Director for MEXT Program: Data Creation and Utilization-Type Material Research and Development Project (2022–). In 2020, he moved to Department of Material Chemistry, Kyoto University, as a full professor. He received Nagase Prize from the frontier salon foundation (2022), the 2020 ACS Macro Letters/Biomacromolecules/Macromolecules Young Investigator Award (American Chemical Society, 2020), The Young Scientists’ Prize for Minister of MEXT (2018), JSPS Prize (2023), Fellow of the Royal Society of Chemistry (FRSC) and so on. He was appointed as an associate editor of Polymer Journal (2018–2020) and is currently an associate editor of ACS Biomaterials Science and Engineering (2020–).graphic file with name pjab-102-040-p001.gif

Conflict of interest

The author declares no conflicts of interest.

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