Mesoscale Confinement in Bagworm Silk: A Hidden Structural Organization.

While silk fibers produced by silkworms and spiders are frequently described as a network of amorphous protein chains reinforced by crystalline ß-sheet nanodomains, the importance of higher-order, self-assembled structures has been recognized for advanced modeling of mechanical properties. General acceptance of hierarchical structural models is, however, currently limited by lack of experimental results. Indeed, X-ray scattering studies of spider's dragline-type fibers have been particularly limited by low crystallinities. Here we are reporting on probing the local structure of exceptionally crystalline bagworm silk fibers by X-ray nanobeam scattering. Probing the comparable thickness of cross sections with an X-ray nanobeam allows removing the strong scattering background from the outer sericin layer and reveals a hidden structural organization due to a radial gradient in diameters of mesoscale nanofibrillar bundles in the fibroin phase. Our results provide direct support for lateral interactions between nanofibrils.

Direct Recovery of the Rare Earth Elements Using a Silk Displaying a Metal-Recognizing Peptide.

Rare earth elements (RE) are indispensable metallic resources in the production of advanced materials; hence, a cost- and energy-effective recovery process is required to meet the rapidly increasing RE demand. Here, we propose an artificial RE recovery approach that uses a functional silk displaying a RE-recognizing peptide. Using the piggyBac system, we constructed a transgenic silkworm in which one or two copies of the gene coding for the RE-recognizing peptide (Lamp1) was fused with that of the fibroin L (FibL) protein. The purified FibL-Lamp1 fusion protein from the transgenic silkworm was able to recognize dysprosium (Dy3+), a RE, under physiological conditions. This method can also be used with silk from which sericin has been removed. Furthermore, the Dy-recovery ability of this silk was significantly improved by crushing the silk. Our simple approach is expected to facilitate the direct recovery of RE from an actual mixed solution of metal ions, such as seawater and industrial wastewater, under mild conditions without additional energy input.

Structure Water-Solubility Relationship in α-Helix-Rich Films Cast from Aqueous and 1,1,1,3,3,3-Hexafluoro-2-Propanol Solutions of S. c. ricini Silk Fibroin.

Silk fibroin (SF) produced by the domesticated wild silkworm, Samia cynthia ricini (S. c. ricini) is attracting increasing interest owing to its unique mechanical properties, biocompatibility, and abundance in nature. However, its utilization is limited, largely due to lack of appropriate processing strategies. Various strategies have been assessed to regenerate cocoon SF, as well as the use of aqueous liquid fibroin (LFaq) prepared by dissolution of silk dope obtained from the silk glands of mature silkworm larvae in water. However, films cast from these fibroin solutions in water or organic solvents are often water-soluble and require post-treatment to render them water-stable. Here, we present a strategy for fabrication of water-stable films from S. c. ricini silk gland fibroin (SGF) without post-treatment. Aqueous ethanol induced gelation of fibroin in the posterior silk glands (PSG), enabling its separation from the rest of the silk gland. When dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), the SGF-gel gave a solution from which a transparent, flexible, and water-insoluble film (SGFHFIP) was cast. Detailed structural characterization of the SGFHFIP as-cast film was carried out and compared to a conventional, water-soluble film cast from LFaq. FTIR and 13C solid-state NMR analyses revealed both cast films to be α-helix-rich. However, gelation of SGF induced by the 40%-EtOH-treatment resulted in an imperfect ß-sheet structure. As a result, the SGF-gel was soluble in HFIP, but some ß-sheet structural memory remains, and the SGFHFIP as-cast film obtained has some ß-sheet content which renders it water-resistant. These results reveal a structure water-solubility relationship in S. c. ricini SF films that may offer useful insights towards tunable fabrication of novel biomaterials. A plausible model of the mechanism that leads to the difference in water resistance of the two kinds of α-helix-rich films is proposed.

Aggregation State of Residual α-Helices and Their Influence on Physical Properties of S. c. ricini Native Fiber.

Formation of the α-helical conformation in the poly-l-alanine (PA) sequence regions, subsequent structural transition to ß-sheet during natural spinning, and presence of residual α-helices in Samia cynthia ricini (S. c. ricini) native silk fiber have been experimentally proven. However, the aggregation state of the residual α-helices, and their influence on the mechanical deformation behavior in native fiber remain unclear. Here we show that the α-helices form an ordered aggregation state with a hexagonal packing in the aqueous solution, some of which remain during natural spinning. X-ray scattering and differential scanning calorimetry (DSC) analyses revealed occurrence of a structural transition of the residual α-helices to the ß-sheet structure, accompanied by disappearance of the plateau region in the force-strain curve, due to heat-treatment at ~220 °C. On the basis of X-ray scattering before and after tensile stretching of S. c. ricini native silk, a direct connection between the plateau region and the α-helix to ß-sheet structural transition was confirmed. Our findings demonstrate the importance of the PA sequence regions in fiber structure formation and their influence on the tensile deformation behavior of S. c. ricini silk, features believed to be essentially similar in other saturniid silks. We strongly believe the residual ordered α-helices to be strategically and systematically designed by S. c. ricini silkworms to impart flexibility in native silk fiber. We anticipate that these knowledge forms a basis for fruitful strategies in the design and development of amino acid sequences for artificial silks with desired mechanical properties.

Conformation change of hornet silk proteins in the solid phase in response to external stimulation.

Hornet silks adopt a variety of morphology such as fibers, sponge, films, and gels depending on the preparation methods. We have studied the conformation change of hornet silk proteins (Vespa mandarina) as regenerated films, using chiroptical spectrophotometer universal chiroptical spectrophotometer 1, which can measure true circular dichroism spectra without artifact signals that are intrinsic to solid-state samples. The spectra showed that the proteins in films alter the conformation rapidly from the α-helix to the coiled coil and then to a ß-sheet structure in response to heat/moisture treatment, but the transformation stopped at the coiled coil state when the sample was soaked in EtOH/water solution. Water is required for the α-helix to the coiled coil transition, and extra energy is required for the further transition to a ß-sheet structure. This is the first successful circular dichroism study of fibril silk proteins to follow the conformation changes in the solid state. This work shows that proteins can undergo conformational changes easily even in the solid phase in response to external stimuli, and this can be traced by solid-phase-feasible chiroptical spectrophotometers. Application of unnatural stress to proteins gives valuable insights into their structure and characteristics.

Transformation of Coiled α-Helices into Cross-ß-Sheets Superstructure.

The fibrous silk produced by bees, wasps, ants, or hornets is known to form a four-strand α-helical coiled coil superstructure. We have succeeded in showing the formation of this coiled coil structure not only in natural fibers, but also in artificial films made of regenerated silk of the hornet Vespa simillima xanthoptera using wide- and small-angle X-ray scatterings and polarized Fourier transform infrared spectroscopy. On the basis of time-resolved simultaneous synchrotron X-ray scattering observations for in situ monitoring of the structural changes in regenerated silk material during tensile deformation, we have shown that the application of tensile force under appropriate conditions induces a transition from the coiled α-helices to a cross-ß-sheet superstructure. The four-stranded tertiary superstructure remains unchanged during this process. It has also been shown that the amorphous protein chains in the regenerated silk material are transformed into conventional ß-sheet arrangements with varying orientation.

Influence of pH, temperature, and concentration on stabilization of aqueous hornet silk solution and fabrication of salt-free materials.

We found that an aqueous solution of silk from cocoons produced by hornet larvae (hornet silk) can be obtained when the solution is adjusted to basic conditions of pH > 9.2. It is known that native hornet cocoons can be dissolved in concentrated aqueous solution of salts, such as lithium bromide (LiBr) and calcium chloride (CaCl2). Upon the removal of these salts from solution by dialysis, solidification, gelation, or sedimentation of hornet silk is known to occur. In the present study, under basic conditions, however, no such solidification occurred, even after salt removal. In this study, ammonia was used for alkalization of solution because it is volatilized during the casting process and pure hornet silk materials can be obtained after drying. The effects of the concentrations of hornet silk and ammonia, as well as dialysis temperature, on preventing gelation during dialysis were investigated. Dialysis conditions that limit the degradation of hornet silk by hydrolysis in alkali solution were identified. Moreover, casting conditions to prepare flexible and transparent hornet silk film from aqueous ammonia solution were optimized. Molecular structural analysis of hornet silk in aqueous ammonia solution and cast film indicated the formation of α-helix conformations.

Evidence of α-helical coiled coils and ß-sheets in hornet silk.

α-Helical coiled coil and ß-sheet complexes are essential structural building elements of silk proteins produced by different species of the Hymenoptera. Beside X-ray scattering at wide and small angles we applied cryo-electron diffraction and microscopy to demonstrate the presence and the details of such structures in silk of the giant hornet Vespa mandarinia japonica. Our studies on the assembly of the fibrous silk proteins and their internal organization in relation to the primary chain structure suggest a 172 Å pitch supercoil consisting of four intertwined alanine-rich α-helical strands. The axial periodicity may adopt even multiples of the pitch value. Coiled coil motifs form the largest portion of the hornet silk structure and are aligned nearly parallel to the cocoon fiber axis in the same way as the membrane-like parts of the cocoon are molecularly orientated in the spinning direction. Supercoils were found to be associated with ß-crystals, predominantly localized in the l-serine-rich chain sequences terminating each of the four predominant silk proteins. Such ß-sheet blocks are considered resulting from transformation of random coil molecular sequences due to the action of elongational forces during the spinning process.

Effects of RGD-fused silk fibroin in a solution format on fibroblast proliferation and collagen production.

BACKGROUND: Collagen production in fibroblasts is important for skin tissue repair. Cell-adhesive Arg-Gly-Asp (RGD) peptides immobilized on scaffolds stimulate fibroblast collagen production, but RGD peptides in solution exhibit opposite effects. Transgenic silkworm technology enables the design of fusion positions for RGD peptides in silk fibroin molecules. The effect of RGD-fused silk fibroin in solution on fibroblast cell activity remains unclear. OBJECTIVE: To clarify the effects of RGD peptides fused to silk fibroin heavy (H)-chain or light (L)-chain on fibroblast proliferation and collagen production when RGD-fused silk fibroin proteins were added to the culture medium. METHODS: Silk fibers with RGD-fused H-chains (H-RGD) or L-chains (L-RGD) were degummed, dissolved, and dialyzed to prepare H-RGD or L-RGD aqueous solutions, respectively. These solutions were added to the fibroblast medium, and their proliferation and collagen production were quantified. RESULTS: Both L- and H-RGD stimulated fibroblast proliferation at a similar level, even in a solution format, but L-RGD promoted fibroblast collagen production significantly, indicating the synergistic effect of the native H-chain and RGD-fused L-chain. CONCLUSION: RGD-fused silk fibroin in solution stimulated fibroblast proliferation and collagen production, depending on the fusion position of the peptides.

Immobilization of Arg-Gly-Asp peptides on silk fibroin via Gly-Ala-Gly-Ala-Gly-Ser sequences.

A simple method by which the functional peptide of Gly-Arg-Gly-Asp-Ser (GRGDS) is immobilized on the surface of silk fibroin (SF) films via Gly-Ala-Gly-Ala-Gly-Ser (GAGAGS) sequences is proposed. GAGAGS, a repeating amino acid sequence in the crystal region of Bombyx mori SF, performs a key role in interacting with and immobilizing SF molecules. Immobilization by this proposed method involves no chemical reaction, thereby preserving the original properties of the SF molecule. The density of GRGDS peptides existing on SF film was found to be higher in the GAGAGS-bound type than in the non-GAGAGS-bound type. Furthermore, results showed that the amount of immobilized (GAGAGS)GRGDS peptide increased as the ß-sheet crystallization was promoted in the SF film. Fibroblasts, which adhered to the surface of the SF film, showed more extensibility because of the (GAGAGS)GRGDS immobilization, which suggests that the cell adhesion activity of RGD is functioning effectively.

Natural templates for coiled-coil biomaterials from praying mantis egg cases.

Whereas there is growing interest in producing biomaterials containing coiled-coils, relatively few studies have made use of naturally occurring fibrous proteins. In this study, we have characterized fibrous proteins used by mother praying mantises to produce an extensive covering for their eggs called an ootheca and demonstrate the production of artificial ootheca using recombinantly produced proteins. Examination of natural oothecae by infrared spectroscopy and solid-state nuclear magnetic resonance revealed the material to consist of proteins organized predominately as coiled-coils. Two structural proteins, Mantis Fibroin 1 and Mantis Fibroin 2, were identified in ootheca from each of three species. Between species, the primary sequences of both proteins had diverged considerably, but other features were tightly conserved, including low molecular weight, high abundance of Ala, Glu, Lys, and Ser, and a triblock-like architecture with extensive central coiled-coil domain. Mantis fibroin hydrophobic cores had an unusual composition containing high levels of alanine and aromatic residues. Recombinantly produced mantis fibroins folded into coiled-coils in solution and could be fabricated into solid materials with high coiled-coil content. The structural features of mantis fibroins and their straightforward recombinant production make them promising templates for the production of coiled-coil biomimetics materials.

Silk Fibroin Conjugated with Heparin Promotes Epithelialization and Wound Healing.

Silk fibroin (SF) has attracted attention as a base biomaterial that could be suitable in many applications because of its shape and structure. Highly functional SF has been developed to promote tissue regeneration with heparin conjugation. However, the hydrophobic three-dimensional structure of SF makes it difficult to bind to high-molecular-weight and hydrophilic compounds such as heparin. In this study, sufficient heparin modification was achieved using tyrosine residues as reaction points to improve cellular response. As it was considered that there was a trade-off between the improvement of water wettability and cell responsiveness induced by heparin modification, influences on the structure, and mechanical properties, the structure and physical properties of the SF conjugated with heparin were extensively evaluated. Results showed that increased amounts of heparin modification raised heparin content and water wettability on film surfaces even though SF formation was not inhibited. In addition, the proliferation of endothelial cells and fibroblasts were enhanced when a surface with sufficient heparin assumed its potential in assisting wound healing. This research emphasizes the importance of material design focusing on the crystal structure inherent in SF in the development of functionalized SF materials.

A study of ladder-like silk foothold for the locomotion of bagworms.

While walking on horizontal substrates, caterpillars skilfully engage all their legs, including three pairs of thoracic legs and a maximum of five pairs of prolegs, to move in a flexible wave-like motion. Such locomotory behaviours, represented by 'crawling' and 'inching' motions, have widely inspired the development of locomotion systems in soft robotics. However, bagworms are unable to use their prolegs for walking because these are always accommodated in a portable bag; thus, they are unable to walk using such general locomotory behaviours. Indeed, how they walk with only three pairs of thoracic legs is unknown at present. In this study, we show that bagworms construct a ladder-like foothold using their silk to walk without using prolegs. This enables them to walk not only on horizontal floor surfaces but also on wall and ceiling surfaces, even those with slippery or smooth surfaces. They construct the foothold by spinning a continuous silk thread in a zigzag manner and controlling the discharge of adhesive to attach the folded parts of the silk to a substrate. Discovery of this elaborate silk utilisation technique offers fresh insights into the diversity of silk use in lepidopteran larvae and provides potential designs for robot locomotion systems.

Transcriptomic analysis of the bagworm moth silk gland reveals a number of silk genes conserved within Lepidoptera.

Lepidopteran insects produce cocoons with unique properties. The cocoons are made of silk produced in the larval tissue silk gland and our understanding of the silk genes is still very limited. Here, we investigated silk genes in the bagworm moth Eumeta variegata, a species that has recently been found to produce extraordinarily strong and tough silk. Using short-read transcriptomic analysis, we identified a partial sequence of the fibroin heavy chain gene and its product was found to have a C-terminal structure that is conserved within nonsaturniid species. This is in accordance with the presence of fibroin light chain/fibrohexamerin genes and it is suggested that the bagworm moth is producing silk composed of fibroin ternary complex. This indicates that the fibroin structure has been evolutionarily conserved longer than previously thought. Other than fibroins we identified candidates for sericin genes, expressed strongly in the middle region of the silk gland and encoding serine-rich proteins, and other silk genes, that are structurally conserved with other lepidopteran homologues. The bagworm moth is thus considered to be producing conventional lepidopteran type of silk. We further found a number of genes expressed in a specific region of the silk gland and some genes showed conserved expression with Bombyx mori counterparts. This is the first study allowing comprehensive silk gene identification and expression analysis in the lepidopteran Psychidae family and should contribute to the understanding of silk gene evolution as well as to the development of novel types of silk.

Drawing-induced changes in morphology and mechanical properties of hornet silk gel films.

Complete amino acid sequences of the four major proteins (Vssilk 1-4) of silk (hornet silk) obtained from yellow hornet ( Vespa simillima , Vespinae, Vespidae) cocoons have been determined. The native structure of the hornet silk (HS), in which Vssilk 1-4 have an alpha-helix domain with coiled-coil alpha-helices and a beta-sheet domain, is restored when hornet silk gel films (HSGFs) are formed by pressing and drying HS hydrogel. Necking occurs when dry HSGFs are drawn; however, wet HSGFs can be uniaxially drawn with a draw ratio (DR) of 2. Drawing helps obtain high-performance films with a maximum tensile strength and tensile modulus of 170 MPa and 5.5 GPa, respectively. Drawing-induced changes in the orientation and conformation of the coiled-coil structure are investigated.

Genome-wide SNP marker discovery and phylogenetic analysis of mulberry varieties using double-digest restriction site-associated DNA sequencing.

There has been long taxonomic debate on mulberry species (genus Morus) because the classification of mulberry species has relied on morphological characteristics. Although attempts for classifying mulberry species using molecular markers have been performed, phylogenetic relationships among diploid mulberry species remain unclear. In this study, we aim to investigate the genetic relationship between 54 diploid mulberry varieties belonging to seven different Morus species (M. alba, M. indica, M. bombycis, M. acidosa, M. latifolia, M. kagayamae, and M. rotundiloba) and one unspecified Morus species ('Enbu') using genome-wide SNP discovery and phylogenetic analysis via double-digest restriction site-associated DNA sequencing (ddRAD-seq). Genome-wide 2229 homozygous SNPs of 54 mulberry varieties in the eight species were identified by ddRAD-seq. Results of the phylogenetic analysis identified only three clear monophyletic clades in two Japanese native species, M. acidosa and M. kagayamae, which are found on different geographically isolated islands and a Thai species, M. rotundiloba, whereas the other species were non-monophyletic. Varieties of M. bombycis, another Japanese native species, were roughly classified into three groups. Of these, two M. bombycis groups were monophyletic with M. acidosa and M. kagayamae, respectively, while another M. bombycis group was not monophyletic. Varieties of M. indica, an Indian native species, were classified into two different monophyletic clades. Of these, one clade was clearly monophyletic with an indigenous variety in Kenya, 'Enbu', while another clade was monophyletic with M. rotundiloba and one M. latifolia variety. There were no clear monophyletic clades within M. alba and M. latifolia varieties, which could be a result of several hybridization events after their introductions from China to Japan. Our results suggested that it was difficult to clearly classify the hybridized mulberry varieties even with genome-wide DNA markers. In addition to phylogenetic analysis, we also evaluated morphological characteristics of mulberry leaves for each variety. The results of morphological evaluation indicated that leaf tip ratio may correlate to genetic difference among the two M. bombycis groups in monophyletic clades and another M. bombycis group in non-monophyletic clades. These results suggested that leaf tip ratio might be used for evaluating hybridization of M. bombycis varieties. Over all, our results may provide new insights into taxonomic debate of mulberry species.

Variable-temperature 13C solid-state NMR study of the molecular structure of honeybee wax and silk.

To elucidate the native-state crystal structure of beeswax from the Japanese bee, Apis cerana japonica, we determined the relationship between temperature and the 13C solid-state nuclear magnetic resonance (NMR) chemical shift of methylene carbon of beeswax, with comparison to n-alkanes and polyethylene in the orthorhombic, monoclinic, or triclinic crystal form. Variable-temperature 13C solid-state NMR observations of n-alkanes and polyethylene revealed that the chemical shifts of methylene carbon in the orthorhombic crystal form increased linearly with increasing temperature, that of the triclinic form decreased, and that of the monoclinic form was unaltered. These relations were compared with results of variable-temperature 13C solid-state NMR observation of beeswax. Results clarified that the two crystal forms comprising the beeswax in the native state are orthorhombic and monoclinic. The variable-temperature 13C solid-state NMR observations were also applied to interpret the differential scanning calorimetry (DSC) curve of beeswax. They were used to clarify the structural changes of beeswax for widely various temperatures. For beeswax secreted by the Japanese bee, the transition from the orthorhombic form to the rotator phase occurred at 36 degrees C, that is from the crystalline to the intermediate state at 45 degrees C. Moreover, the variable-temperature 13C solid-state NMR spectrum of honeybee silk in the native state was observed. Results demonstrated that the secondary structures of honeybee silk proteins in the native state comprised coexisting alpha-helix and beta-sheet conformations and that the amount of alpha-helices was greater. The alpha-helix content of honeybee silk was compared with that of hornet silk produced by Vespa larvae.

A study of the extraordinarily strong and tough silk produced by bagworms.

Global ecological damage has heightened the demand for silk as 'a structural material made from sustainable resources'. Scientists have earnestly searched for stronger and tougher silks. Bagworm silk might be a promising candidate considering its superior capacity to dangle a heavy weight, summed up by the weights of the larva and its house. However, detailed mechanical and structural studies on bagworm silks have been lacking. Herein, we show the superior potential of the silk produced by Japan's largest bagworm, Eumeta variegata. This bagworm silk is extraordinarily strong and tough, and its tensile deformation behaviour is quite elastic. The outstanding mechanical property is the result of a highly ordered hierarchical structure, which remains unchanged until fracture. Our findings demonstrate how the hierarchical structure of silk proteins plays an important role in the mechanical property of silk fibres.

Transdermal delivery of 40-nm silk fibroin nanoparticles.

Transdermal administration of drugs improves their bioavailability and is capable of systemic and local treatment. To improve the skin permeability of drugs, nano-sized systems have attracted attention as drug carriers for transdermal drug delivery system. We considered that silk fibroin composed of a crystalline region with many hydrophobic amino acids and an amorphous region with many hydrophilic amino acids was useful as a carrier for transdermal administration of a drug because of the balance between hydrophilicity and hydrophobicity. In this study, silk fibroin nanoparticles with mean volume diameters of 42.3 nm were successfully prepared, and storage stability was confirmed by storing the nanoparticle suspension at 4, 32, and 37 °C for a week. At any storage temperature, the mean volume diameter and standard deviation were stable. The polydispersity indexes were 0.19-0.23, and no specific trends were observed. Then, to investigate the transdermal delivery route of the silk fibroin nanoparticles, skin permeability in vivo was evaluated using mice. Six hours after administration, fluorescent substances were observed in the dermis in addition to the stratum corneum, hair follicles and the epidermis around them. This result indicated that fibroin nanoparticles with the mean volume diameter of 40-nm penetrated the stratum corneum and was delivered deep into the skin. Therefore, it was suggested that small nanoparticles prepared using silk fibroin are useful for drug delivery to the dermis.

Preparation of gel film from Bombyx mori silk sericin and its characterization as a wound dressing.

Sericin is a highly hydrophilic protein family acting as the glue in Bombyx mori silk. In order to apply sericin as a wound dressing, a novel sericin film named gel film was prepared by a simple process without using any chemical modifications: sericin solution was gelled with ethanol into a sheet shape and then dried. Infrared analysis revealed that the sericin gel film contained water-stable beta-sheet networks formed in the gelation step. This structural feature rendered the gel film morphologically stable against swelling and gave it good handling properties in the wet state. The sericin gel film rapidly absorbed water, equilibrating at a water content of about 80%, and exhibited elastic deformation up to a strain of about 25% in the wet state. A culture of mouse fibroblasts on the gel film indicated that it had low cell adhesion properties and no cytotoxicity. These characteristics of sericin gel film suggest its potential as a wound dressing.