Synthesis and characterization of chimeric silkworm silk

A synthetic gene encoding a chimeric silklike protein was constructed that combined a polyalanine encoding region (Ala)(18), a sequence slightly longer than the (Ala)(12-13) found in the silk fibroin from the wild silkworm Samia cynthia ricini, and a sequence encoding GVGAGYGAGAGYGVGAGYGAGVGYGAGAGY, found in the silk fibroin from the silkworm Bombyx mori. A tetramer of the chimeric repeat sequence encoding a approximately 29 kDa protein was expressed as a fusion protein in Escherichia coli. In comparison to S. c. ricini silk, the chimeric protein demonstrated improved solubility because it could be dissolved in 8 M urea. The purified protein assumed an alpha-helical structure based on solid-state (13)C CP/MAS NMR and was less prone to conformational transition to a beta-sheet, unlike native silk proteins from S. c. ricini. Model peptides representing the crystalline region of S. c. ricini silk fibroin, (Ala)(12) and (Ala)(18), formed beta-sheet structures. Therefore, the solubility and structural transitions of the chimeric protein were significantly altered through the formation of this chimeric silk. This experimental strategy to the study of silk structure and function can be used to develop an improved understanding of the contributions of protein domains in repetitive silkworm and spider silk sequences to structure development and structural transitions.     

New silk protein: modification of silk protein by gene engineering for production of biomaterials

The interest in silk fibroin morphology and structure have increased due to its attractiveness for bio-related applications. Silk fibers have been used as sutures for a long time in the surgical field, due to the biocompatibility of silk fibroin fibers with human living tissue. In addition, it has been demonstrated that silk can be used as a substrate for enzyme immobilization in biosensors. A more complete understanding of silk structure would provide the possibility to further exploit silk fibroin for a wide range of new uses, such as the production of oxygen-permeable membranes and biocompatible materials. Silk fibroin-based membranes could be utilized as soft tissue compatible polymers. Baculovirus-mediated transgenesis of the silkworm allows specific alterations in a target sequence. Homologous recombination of a foreign gene downstream from a powerful promoter, such as the fibroin promoter, would allow the constitutive production of a useful protein in the silkworm and the modification of the character of silk protein. A chimeric protein consisted of fibroin and green fluorescent protein was expressed under the control of fibroin in the posterior silk gland and the gene product was spun into the cocoon layer. This technique, gene targeting, will lead to the modification and enhancement of physicochemical properties of silk protein.     

New silk protein: modification of silk protein by gene engineering for production of biomaterials.

The interest in silk fibroin morphology and structure have increased due to its attractiveness for bio-related applications. Silk fibers have been used as sutures for a long time in the surgical field, due to the biocompatibility of silk fibroin fibers with human living tissue. In addition, it has been demonstrated that silk can be used as a substrate for enzyme immobilization in biosensors. A more complete understanding of silk structure would provide the possibility to further exploit silk fibroin for a wide range of new uses, such as the production of oxygen-permeable membranes and biocompatible materials. Silk fibroin-based membranes could be utilized as soft tissue compatible polymers. Baculovirus-mediated transgenesis of the silkworm allows specific alterations in a target sequence. Homologous recombination of a foreign gene downstream from a powerful promoter, such as the fibroin promoter, would allow the constitutive production of a useful protein in the silkworm and the modification of the character of silk protein. A chimeric protein consisted of fibroin and green fluorescent protein was expressed under the control of fibroin in the posterior silk gland and the gene product was spun into the cocoon layer. This technique, gene targeting, will lead to the modification and enhancement of physicochemical properties of silk protein.     

Secondary structure in properdin of the complement cascade and related proteins: a study by Fourier transform infrared spectroscopy

Six structural repeat motifs of 58 amino acids are found in the sequence of both mouse and human properdins. Twelve more examples of the motif are available from the sequences of thrombospondin, the terminal complement components, and the thrombospondin-related anonymous protein. The averaged Robson and Chou-Fasman secondary structure predictions show that there are 57-66% turn and 19-38% beta-sheet structures in the typical repeat motif. The high amount of turn structure is consistent with Gly, Pro, Cys, and Ser being the four most abundant amino acid residues in properdin. Comparisons with sequences found in the circumsporozoite protein from several species of malaria parasites show that their sequences and secondary structures strongly coincide only in a 18-residue segment. Further secondary structure analysis utilized Fourier transform infrared spectroscopy of human properdin in 2H2O buffers. These show a broad amide I band that, after second-derivative and deconvolution calculations, is shown to be composed of several components. Two at 1633 and 1683 cm-1 are strong evidence for beta-sheet structure, although overlap from beta-turns can also contribute. The presence of beta-turn structure is indicated by absorptions at 1662-1675 and 1645 cm-1. The properdin structure contains substantial quantities of beta-sheet and beta-turn structures, which is consistent with the secondary structure predictions and amino acid compositions. The length of the repeat motif is estimated as 3.3-4.3 nm, and an estimated 14-22% of nonexchanged amide protons reside in properdin. This is suggestive of a high degree of solvent accessibility in the structure.     

Isolation of a clone encoding a second dragline silk fibroin. Nephila clavipes dragline silk is a two-protein fiber.

Spider dragline silk is a unique protein fiber possessing both high tensile strength and high elasticity. A partial cDNA clone for one dragline silk protein (Spidroin 1) was previously isolated. However, the predicted amino acid sequence could not account for the amino acid composition of dragline silk. We have isolated a partial cDNA clone for another dragline silk protein (Spidroin 2), demonstrating that dragline silk is composed of multiple proteins. The amino acid sequence exhibits an entirely different repetitive motif than Spidroin 1. Spidroin 2 is predicted to consist of linked beta-turns in proline-rich regions which alternate with beta-sheet regions composed of polyalanine segments. This structure for Spidroin 2 provides a model for dragline silk structure and function.     

The 1.7 A crystal structure of the apo form of the soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus reveals a novel internal conserved sequence repeat.

The crystal structure of a dimeric apo form of the soluble quinoprotein glucose dehydrogenase (s-GDH) from Acinetobacter calcoaceticus has been solved by multiple isomorphous replacement followed by density modification, and was subsequently refined at 1. 72 A resolution to a final crystallographic R-factor of 16.5% and free R-factor of 20.8% [corrected]. The s-GDH monomer has a beta-propeller fold consisting of six four-stranded anti-parallel beta-sheets aligned around a pseudo 6-fold symmetry axis. The enzyme binds three calcium ions per monomer, two of which are located in the dimer interface. The third is bound in the putative active site, where it may bind and functionalize the pyrroloquinoline quinone (PQQ) cofactor. A data base search unexpectedly showed that four uncharacterized protein sequences are homologous to s-GDH with many residues in the putative active site absolutely conserved. This indicates that these homologs may have a similar structure and that they may catalyze similar PQQ-dependent reactions.A structure-based sequence alignment of the six four-stranded beta-sheets in s-GDH's beta-propeller fold shows an internally conserved sequence repeat that gives rise to two distinct conserved structural motifs. The first structural motif is found at the corner of the short beta-turn between the inner two beta-strands of the beta-sheets, where an Asp side-chain points back into the beta-sheet to form a hydrogen-bond with the OH/NH of a Tyr/Trp side-chain in the same beta-sheet. The second motif involves an Arg/Lys side-chain in the C beta-strand of one beta-sheet, which forms a bidentate salt-bridge with an Asp/Glu in the CD loop of the next beta-sheet. These intra and inter-beta-sheet hydrogen-bonds are likely to contribute to the stability of the s-GDH beta-propeller fold.     

Conformational transition and liquid crystalline state of regenerated silk fibroin in water

The conformational transition of molecular chains of regenerated silk fibroin (SF) aqueous solution is systematically investigated by circular dichroism, Raman, IR, and UV-vis spectroscopies. It is found that an initial random coil conformation of the SF can be readily changed into an ordered beta-sheet structure by optimizing the solution conditions, such as the SF concentration, pH, temperature, or metal-ion content. Circular dichroic spectra quantitatively confirm a steadily decreased content of the random coil conformation but a significantly increased beta-sheet content after an ultrasonic or extruding treatment. Furthermore, the extrusion is more powerful to achieve high beta-sheet content than the ultrasonic. It is interesting that the polarized optical micrographs of the SF aqueous solution extruded by injection illustrate the formation and existence of liquid crystalline state. A study of extrusion in vitro could be used as a model system to understand the natural silk spinning process in silkworm.     

Solution structure of the carboxyl-terminal cysteine-rich domain of the VHv1.1 polydnaviral gene product: comparison with other cystine knot structural folds.

Polydnaviruses are an unusual group of insect viruses that have an obligate symbiotic association with certain parasitic wasps. These viruses are transmitted with the wasp egg during oviposition into lepidopteran insects, enabling the survival and development of the egg inside the host larvae. We report the three-dimensional structure of a novel polydnaviral cysteine-rich motif (cys-motif), identified as the carboxyl-terminal domain of a two cys-motif containing polydnaviral VHv1.1 gene product, abbreviated "C-term VHv1.1". This 65-residue domain was identified experimentally by limited proteolysis of the full-length protein and was subsequently cloned in a bacterial expression system for NMR studies. The C-term VHv1.1 3D structure was determined in solution by two-dimensional (1)H NMR spectroscopy. Calculation of the structure was based on a total of 300 upper distance restraints and 20 dihedral angle constraints, and resulted in an ensemble of 25 representative conformers with an average rmsd of 0.47 A from the mean structure for core backbone atoms. The protein core is made of a four beta-strand scaffold held together in a compact structure by three disulfide bonds, which form a cystine knot. The four beta-strands are arranged in an unusual configuration to form a triple-stranded beta-sheet and double-stranded beta-sheet. Comparison with other classes of cystine knots provides indication that C-term VHv1.1 represents a new and distinct cystine knot motif. This analysis provides a structural basis for interpretation of the genetic and amino acid sequence data classifying polydnavirus gene products as members of cysteine-rich protein families.     

Identification and characterization of a novel sericin gene expressed in the anterior middle silk gland of the silkworm Bombyx mori

Sericin is a group of proteins expressed in the middle silk gland that covers the surface of fibroin in the cocoon filament of Bombyx mori. Sericin consists of several serine-rich proteins with different molecular masses. Sericin A is one of the proteins and is produced in the anterior portion of the middle silk gland. To identify the gene coding for the protein, we determined the primary structures of its partial peptides, and the gene was searched using the silkworm genomic databases. Three contigs containing the corresponding nucleotide sequences were identified and categorized as one group. The gene structure covering the 5' flanking and the 3' end was determined by PCR fragments from genomic DNA, RT-PCR, and 5' and 3' RACE. The amino acid sequence deduced from the nucleotide sequence mainly consists of two serine-rich regions of 86-amino acid motif and 8-amino acid repeated sequence. The expression of the gene is limited to the anterior and middle parts of the middle silk gland. In addition, because the sericin gene appeared different from the sericin 1 and 2 genes reported earlier, we designated the newly discovered gene as sericin 3.     

Heparin-binding growth-associated molecule contains two heparin-binding beta -sheet domains that are homologous to the thrombospondin type I repeat

Heparin-binding growth-associated molecule (HB-GAM) is an extracellular matrix-associated protein implicated in the development and plasticity of neuronal connections of brain. Binding to cell surface heparan sulfate is indispensable for the biological activity of HB-GAM. In the present paper we have studied the structure of recombinant HB-GAM using heteronuclear NMR. These studies show that HB-GAM contains two beta-sheet domains connected by a flexible linker. Both of these domains contain three antiparallel beta-strands. In addition to this domain structure, HB-GAM contains the N- and C-terminal lysine-rich sequences that lack a detectable structure and appear to form random coils. Studies using CD and NMR spectroscopy suggest that HB-GAM undergoes a conformational change upon binding to heparin, and that the binding occurs primarily to the beta-sheet domains of the protein. Search of sequence data bases shows that the beta-sheet domains of HB-GAM are homologous to the thrombospondin type I repeat (TSR). Sequence comparisions show that the beta-sheet structures found previously in midkine, a protein homologous with HB-GAM, also correspond to the TSR motif. We suggest that the TSR sequence motif found in various extracellular proteins defines a beta-sheet structure similar to that found in HB-GAM and midkine. In addition to the apparent structural similarity, a similarity in biological functions is suggested by the occurrence of the TSR sequence motif in a wide variety of proteins that mediate cell-to-extracellular matrix and cell-to-cell interactions, in which the TSR domain mediates specific cell surface binding.     

Comparison of fibroin cDNAs from webspinning insects: insight into silk formation and function

Embiopterans (webspinning insects) are renowned for their prolific use of silk. These organisms spin silk to construct elaborate networks of tubes in which they live, forage, and reproduce. The silken galleries are essential for protecting these soft-bodied insects from predators and other environmental hazards. Despite the ecological importance of embiopteran silk, very little is known about its constituent proteins. Here, we characterize the silk protein cDNAs from four embiopteran species to better understand the function and evolution of these adaptive molecules. We show that webspinner fibroins (silk proteins) are highly repetitive in sequence and possess several conserved characteristics, despite differences in habitat preferences across species. The most striking similarities are in the codon usage biases of the fibroin genes, particularly in the repetitive regions, as well as sequence conservation of the carboxyl-terminal regions of the fibroins. Based on analyses of the silk genes, we propose hypotheses regarding codon bias and its effect on the translation and replication of these unusual genes. Furthermore, we discuss the significance of specific fibroin motifs to the mechanical and structural characteristics of silk fibers. Lastly, we report that the conservation of webspinner fibroin carboxyl-terminal regions suggests that fiber formation may occur through a mechanism analogous to that found in Lepidoptera. From these results, insight is gained into the tempo and mode of evolution that has shaped embiopteran fibroins.     

The role of irregular unit,GAAS, on the secondary structure of Bombyx mori silk fibroin studied with 13C CP/MAS NMR and wide-angle X-ray scattering

Bombyx mori silk fibroin is a fibrous protein whose fiber is extremely strong and tough, although it is produced by the silkworm at room temperature and from an aqueous solution. The primary structure is mainly Ala-Gly alternative copolypeptide, but Gly-Ala-Ala-Ser units appear frequently and periodically. Thus, this study aims at elucidating the role of such Gly-Ala-Ala-Ser units on the secondary structure. The sequential model peptides containing Gly-Ala-Ala-Ser units selected from the primary structure of B. mori silk fibroin were synthesized, and their secondary structure was studied with (13)C CP/MAS NMR and wide-angle X-ray scattering. The (13)C isotope labeling of the peptides and the (13)C conformation-dependent chemical shifts were used for the purpose. The Ala-Ala units take antiparallel beta-sheet structure locally, and the introduction of one Ala-Ala unit in (Ala-Gly)(15) chain promotes dramatical structural changes from silk I (repeated beta-turn type II structure) to silk II (antiparallel beta-sheet structure). Thus, the presence of Ala-Ala units in B. mori silk fibroin chain will be one of the inducing factors of the structural transition for silk fiber formation. The role of Tyr residue in the peptide chain was also studied and clarified to induce "locally nonordered structure."     

Ecdysteroid as a mediator in the regulation of silk protein synthesis and its influence on silkworm (Bom., Lepidoptera) genome

Abstract:  Ecdysteroid is one of the two most important insect hormones, which controls growth and developmental activities of insect. In silkworm, Bombyx mori L., basically ecdysteroid induces molting and metamorphosis in larvae and exogenous administration hastens the silk synthetic activity and cocoon spinning process but it strictly depends on the time of administration. In the present study, we administered phytogenous ecdysteroid to the 48-h-old 5th instar silkworm, B. mori at a single dose of 2  μ g per larva to study its effects on the larval growth, cocoon and silk variables. The possible role of ecdysteroid in altering the developmental sequence of silkworm and thereby its effect on cocoon and silk production at molecular level have been tried to get elucidated. The genomic DNA was isolated from the posterior silk gland on day 5 and 7 of the 5th instar from the ecdysteroid treated and the control larvae and was randomly amplified with arbitrary primers. The result presented notable variation in the amplified product suggesting the participation of ecdysteroid in regulating the silk gene. The feeding period of treated larvae was unaffected while the cocoon characters exhibited considerable improvement. The filament traits also were improved notably in the treated larvae. The participation of ecdysteroid in the silk biosynthetic process with its physiological and molecular implications was discussed.     

Phase behavior and hydration of silk fibroin

The osmotic stress method was applied to study the thermodynamics of supramolecular self-assembly phenomena in crystallizable segments of Bombyx mori silkworm silk fibroin. By controlling compositions and phases of silk fibroin solution, the method provided a means for the direct investigation of microscopic and thermodynamic details of these intermolecular interactions in aqueous media. It is apparent that as osmotic pressure increases, silk fibroin molecules are crowded together to form silk I structure and then with further increase in osmotic pressure become an antiparallel beta-sheet structure, silk II. A partial ternary phase diagram of water-silk fibroin-LiBr was constructed based on the results. The results provide quantitative evidence that the silk I structure must contain water of hydration. The enhanced control over structure and phase behavior using osmotic stress, as embodied in the phase diagram, could potentially be utilized to design a new route for water-based wet spinning of regenerated silk fibroin.     

Insights into the regulatory characteristics of silkworm fibroin gene promoters using a modified Gal4/UAS system

Transgenic Research - The silkworm Bombyx mori is a valuable insect that synthesizes bulk amounts of fibroin protein in its posterior silk gland (PSG) and weaves these proteins into silk cocoons....     

High-Toughness Silk Produced by a Transgenic Silkworm Expressing Spider (Araneus ventricosus) Dragline Silk Protein

Spider dragline silk is a natural fiber that has excellent tensile properties; however, it is difficult to produce artificially as a long, strong fiber. Here, the spider (Araneus ventricosus) dragline protein gene was cloned and a transgenic silkworm was generated, that expressed the fusion protein of the fibroin heavy chain and spider dragline protein in cocoon silk. The spider silk protein content ranged from 0.37 to 0.61% w/w (1.4–2.4 mol%) native silkworm fibroin. Using a good silk-producing strain, C515, as the transgenic silkworm can make the raw silk from its cocoons for the first time. The tensile characteristics (toughness) of the raw silk improved by 53% after the introduction of spider dragline silk protein; the improvement depended on the quantity of the expressed spider dragline protein. To demonstrate the commercial feasibility for machine reeling, weaving, and sewing, we used the transgenic spider silk to weave a vest and scarf; this was the first application of spider silk fibers from transgenic silkworms.     

Conformational transitions in model silk peptides

Protein structural transitions and beta-sheet formation are a common problem both in vivo and in vitro and are of critical relevance in disparate areas such as protein processing and beta-amyloid and prion behavior. Silks provide a "databank" of well-characterized polymorphic sequences, acting as a window onto structural transitions. Peptides with conformationally polymorphic silk-like sequences, expected to exhibit an intractable beta-sheet form, were characterized using Fourier transform infrared spectroscopy, circular dichroism, and electron diffraction. Polymorphs resembling the silk I, silk II (beta-sheet), and silk III (threefold polyglycine II-like helix) crystal structures were identified for the peptide fibroin C (GAGAGS repetitive sequence). Two peptides based on silk amorphous sequences, fibroin A (GAGAGY) and fibroin V (GDVGGAGATGGS), crystallized as silk I under most conditions. Methanol treatment of fibroin A resulted in a gradual transition from silk I to silk II, with an intermediate state involving a high proportion of beta-turns. Attenuated total reflectance Fourier transform infrared spectroscopy has been used to observe conformational changes as the peptides adsorb from solution onto a hydrophobic surface. Fibroin C has a beta-strand structure in solution but adopts a silk I-like structure upon adsorption, which when dried on the ZnSe crystal contains silk III crystallites.     

Conformational energy studies of beta-sheets of model silk fibroin peptides. I. Sheets of poly(Ala-Gly) chains

A new model structure is proposed for the silk I form of the crystalline domains of Bombyx mori silk fibroin and the corresponding crystal form of poly(L-Ala-Gly). It was deduced from conformational energy computations on stacked sheet structures of poly(L-Ala-Gly). The novel sheet structure contains interstrand hydrogen bonds but is composed of anti-parallel polypeptide chains whose conformation differs from that of the antiparallel beta-sheets that constitute the silk II structure. The strands of the new sheet have a two-residue repeat, in which the Ala residues adopt a right-handed and the Gly residues a left-handed sheet-like conformation. The computed unit cell is orthorhombic, with cell dimensions a = 8.94 A, b = 6.46 A, and c = 11.26 A. The model accounts for most spacings in the observed fiber x-ray diffraction patterns of silk I and of the silk-I-like form of poly(L-Ala-Gly), and it is consistent with nmr and ir spectroscopic data. As a test of the computations, the well-established beta-sheet structure of silk II and the corresponding form of poly(L-Ala-Gly) have been reproduced. The computed energies for the two forms of poly(L-Ala-Gly) indicate that the silk-II-like form is more stable, by about 1.0 kcal/mol per residue. The main difference between the two structures is the orientation of the Ala side chains of neighboring strands in each sheet. In the Pauling-Corey beta-sheet and in the silk II form, referred to as an "in-register" structure, the Ala side chains of every strand point to the same side of a sheet. In the silk I structure, referred to as "out-of-register," the side chains of Ala residues in adjacent strands point to opposite sides of the sheet.     

Comparative Proteome Analysis Reveals that Cuticular Proteins Analogous to Peritrophin‐Motif Proteins are Involved in the Regeneration of Chitin Layer in the Silk Gland of Bombyx mori at the Molting Stage

The silk gland of silkworm produces silk proteins during larval development. Many studies have long focused on the silk gland of the fifth instar larvae, but few have investigated this gland at other larval stages. In the present study, the silk gland proteomes of the fourth instar and fourth molt are analyzed using liquid chromatography–tandem mass spectrometry. In total, 2654 proteins are identified from the silk gland. A high abundance of ribosomal proteins and RR‐motif chitin‐binding proteins is identified during day 2 of the fourth instar (IV‐2) larval developmental stage, and the expression of cuticular proteins analogous to peritrophin (CPAP)‐motif chitin‐binding proteins is higher during the fourth molt (IV‐M). In all, nine enzymes are found to be involved in the chitin regeneration pathway in the silk gland. Among them, two chitinase and two chitin deacetylases are identified as CPAP‐motif proteins. Furthermore, the expression of CPAP3‐G, the most abundant CPAP‐motif cuticular protein in the silk gland during the IV‐M stage, is investigated using western blot and immunofluorescence analyses; CPAP3‐G shows a reverse changing trend with chitin in the silk gland. The findings of this study suggest that CPAP‐motif chitin‐binding proteins are involved in the degradation of the chitin layer in the silk gland. The data have been deposited to the ProteomeXchange with identifier PXD008677.