13C CP/MAS NMR study on structural heterogeneity in Bombyx mori silk fiber and their generation by stretching

It is important to resolve the structure of Bombyx mori silk fibroin before spinning (silk I) and after spinning (silk II), and the mechanism of the structural transition during fiber formation in developing new silk-like fiber. The silk I structure has been recently resolved by (13)C solid-state NMR as a "repeated beta-turn type II structure." Here, we used (13)C solid-state NMR to clarify the heterogeneous structure of the natural fiber from Bombyx mori silk fibroin in the silk II form. Interestingly, the (13)C CP/MAS NMR revealed a broad and asymmetric peak for the Ala Cbeta carbon. The relative proportions of the various heterogeneous components were determined from their relative peak intensities after line shape deconvolution. Namely, for 56% crystalline fraction (mainly repeated Ala-Gly-Ser-Gly-Ala-Gly sequences), 18% distorted beta-turn, 13% beta-sheet (parallel Ala residues), and 25% beta-sheet (alternating Ala residues). The remaining fraction of 44% amorphous Tyr-rich region, 22% in both distorted beta-turn and distorted beta-sheet. Such a heterogeneous structure including distorted beta-turn can be observed for the peptides (AG)(n) (n > 9 ). The structural change from silk I to silk II occurs exclusively for the sequence (Ala-Gly-Ser-Gly-Ala-Gly)(n) in B. mori silk fibroin. The generation of the heterogeneous structure can be studied by change in the Ala Cbeta peak of (13)C CP/MAS NMR spectra of the silk fibroin samples with different stretching ratios.     

家蚕丝蛋白重复片段替换人体α-突触核蛋白核心片段的研究

家蚕Bombyx mori丝丝心蛋白(silk fibroin)结晶域与人类帕金森综合症的致病蛋白α-突触核蛋白(α-synuclein, α-Syn)的积聚原理类似,都是由一段非常疏水的氨基酸组成的保守序列和一些无序卷曲,并在一定条件下发生整体的结构转换而发生纤维化所致。研究发现在这2种蛋白中存在的疏水片段是它们形成β折叠的关键。为了研究α-Syn蛋白的积聚核心在被别的积聚核心所替换后是否还可以正常纤维化,我们用PCR技术将家蚕丝丝心蛋白的核心片段替换α-Syn_1-74(α-Syn₇₄)的核心,组成一个重组蛋白α-Syn₇₄SFX,纯化后温育6天,用ThT荧光和原子力显微镜检测该重组蛋白的结构,结果表明α-Syn₇₄SFX未能发生纤维化。这说明具备能形成β片层的片段和无序卷曲这2个因素,并不能绝对使蛋白发生整体的结构转换。这对人工蚕丝的研究具有参考价值。     

X-ray evidence for a "super"-secondary structure in silk fibers

X-ray studies on degummed B. mori silk fibers and on hydrogels prepared under a variety of conditions reveal moderately small angle reflections. These reflections are often highly oriented and are correlated to silk II lattice reflections. A superstructure can explain these features. Silk fibroin hydrogels were monitored as they dried to form the silk II structure. The silk II wide angle and moderately small angle patterns obtained from dried hydrogels and silk fibers are identical. The "superstructure" reflections at moderately small angle (3-7 nm) were first to appear, followed by the "intersheet" spacing, and then the remainder of the silk II wide angle scattering pattern. Thus, any superstructure hypothesized for the hydrogels (and for Silk II in fibers) must be both stable in a highly hydrated environment and must convert to silk II with little large scale diffusion. A folded structure, similar to amyloids and cross-beta-sheets but with much longer beta-strand stems, is proposed for silk II in fibers.     

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."     

Possible implications of serine and tyrosine residues and intermolecular interactions on the appearance of silk I structure of Bombyx mori silk fibroin-derived synthetic peptides: high-resolution 13C cross-polarization/magic-angle spinning NMR study

Bombyx mori silk fibroin molecule is known to exist in two distinct structural forms: silk I (unprocessed silk fibroin) and silk II (processed silk fibroin). Using synthetic peptides, we attempt to explore the structural role played by Ser and Tyr residues on the appearance of silk I structural form of the fibroin. Twelve selected peptides (1-12) incorporating Ser and Tyr residues in the (Ala-Gly)(n) copolypeptide, that is, the sequences mimicking the primary structure of B. mori silk fibroin molecule, have been investigated under the silk I state, employing high-resolution (13)C cross-polarization/magic-angle spinning (CP/MAS) NMR spectroscopy. To acquire the silk I structural form, all the peptides were dissolved in 9 M LiBr and then dialyzed extensively against water, as established previously for the synthetic (Ala-Gly)(15) copolypeptide and B. mori silk fibroin. The diagnostic line shape of the Ala C(beta) peaks and the conformation-dependent (13)C chemical shifts of Ala and Gly resonances are presented to analyze and characterize the structural features. The results indicate that the incorporation of one Ser and/or one Tyr residue(s) at selected position in the basic (Ala-Gly)(15) sequence tend to retain predominantly the silk I structure. Conversely, the repeat pentameric and octameric Ala-Gly-Ser-Gly-Ala-Gly sequences, for example, (Ala-Gly-Ser-Gly-Ala-Gly)(5) or (Ala-Gly-Ser-Gly-Ala-Gly)(8), preferred predominantly the silk II form. The peptide sequences incorporating Ser and Tyr residue(s) into repeat Ala-Gly-Ser-Gly-Ala-Gly sequences, however, adopted the silk II structure with certain content structural heterogeneity or randomness, more pronounced for specific peptides studied. Interestingly, the crystalline Cp fraction of B. mori silk fibroin, when mixed with (Ala-Gly-Ser-Gly-Ala-Gly)(5) sequence in a 5:1 molar ratio, dissolved in 9 M LiBr, and dialyzed against distilled water, favor the silk I form. The finding tends to suggest that the less stable silk I form in (Ala-Gly-Ser-Gly-Ala-Gly)(n) sequences is likely to be induced and facilitated via intermolecular interactions with the Cp fraction, which predominantly prefers the silk I form under similar conditions; however, the hydrogen-bond formation involving O(gamma)H groups of the Ser residues may have some implications.     

Characterizing the S‐layer structure and anti‐S‐layer antibody recognition on intact Tannerella forsythia cells by scanning probe microscopy and small angle X‐ray scattering

Tannerella forsythia is among the most potent triggers of periodontal diseases, and approaches to understand underlying mechanisms are currently intensively pursued. A ~22‐nm‐thick, 2D crystalline surface (S‐) layer that completely covers Tannerella forsythia cells is crucially involved in the bacterium–host cross‐talk. The S‐layer is composed of two intercalating glycoproteins (TfsA‐GP, TfsB‐GP) that are aligned into a periodic lattice. To characterize this unique S‐layer structure at the nanometer scale directly on intact T. forsythia cells, three complementary methods, i.e., small‐angle X‐ray scattering (SAXS), atomic force microscopy (AFM), and single‐molecular force spectroscopy (SMFS), were applied. SAXS served as a difference method using signals from wild‐type and S‐layer‐deficient cells for data evaluation, revealing two possible models for the assembly of the glycoproteins. Direct high‐resolution imaging of the outer surface of T. forsythia wild‐type cells by AFM revealed a p4 structure with a lattice constant of ~9.0 nm. In contrast, on mutant cells, no periodic lattice could be visualized. Additionally, SMFS was used to probe specific interaction forces between an anti‐TfsA antibody coupled to the AFM tip and the S‐layer as present on T. forsythia wild‐type and mutant cells, displaying TfsA‐GP alone. Unbinding forces between the antibody and wild‐type cells were greater than with mutant cells. This indicated that the TfsA‐GP is not so strongly attached to the mutant cell surface when the co‐assembling TfsB‐GP is missing. Altogether, the data gained from SAXS, AFM, and SMFS confirm the current model of the S‐layer architecture with two intercalating S‐layer glycoproteins and TfsA‐GP being mainly outwardly oriented. Copyright © 2013 John Wiley & Sons, Ltd.     

Structures of Bombyx mori and Samia cynthia ricini silk fibroins studied with solid-state NMR

There are many kinds of silks spun by silkworms and spiders, which are suitable to study the structure-property relationship for molecular design of fibers with high strength and high elasticity. In this review, we mainly focus on the structural determination of two well-known silk fibroin proteins that are from the domesticated silkworm, Bombyx mori, and the wild silkworm, Samia cynthia ricini, respectively. The structures of B. mori silk fibroin before and after spinning were determined by using an appropriate model peptide, (AG)(15), with several solid-state NMR methods; (13)C two-dimensional spin-diffusion solid-state NMR and rotational echo double resonance (REDOR) NMR techniques along with the quantitative use of the conformation-dependent (13)C CP/MAS chemical shifts. The structure of S. c. ricini silk fibroin before spinning was also determined by using a model peptide, GGAGGGYGGDGG(A)(12)GGAGDGYGAG, which is a typical repeated sequence of the silk fibroin, with the solid-state NMR methods. The transition from the structure of B. mori silk fibroin before spinning to the structure after spinning was studied with molecular dynamics calculation by taking into account several external forces applied to the silk fibroin in the silkworm.     

Short and long range order of the morphology of silk from Latrodectus hesperus (Black Widow) as characterized by atomic force microscopy.

The surfaces of both stretched and unstretched silk threads from the cobweb weaver, Latrodectus hesperus (Black Widow) have been examined by atomic force microscopy (AFM). AFM images of cobweb scaffolding threads show both unordered and highly ordered regions. Two types of fibers within the threads were observed: thicker (approximately 300 nm in diameter) fibers oriented parallel to the thread axis and thinner (10-100 nm) fibrils oriented across the thread axis. While regions which lacked parallel fibers or fibrils were observed on threads at all strain values, the probability of observing fibers and/or fibrils increased with strain. High-resolution AFM images show that with increasing strain, both mean fiber and fibril diameters decrease and that fibrils align themselves more closely with the thread axis. The observation of fibers and fibrils within the cobweb threads has implications for current models of the secondary and tertiary structure and organization of spider silk.     

Structural analysis of Bombyx mori silk fibroin peptides with formic acid treatment using high-resolution solid-state 13C NMR spectroscopy

Bombyx mori silk fibroin fiber is a fibrous protein produced by the silkworm at room temperature and from an aqueous solution whose primary structure is highly repetitive. In this study we analyzed the structural characteristics of native peptides, derived from B. mori silk fibroin, with formic acid treatment using high-resolution solid-state 13C NMR. We establish that the Ser residue bearing a short polar side chain has the ability to stabilize the conformation formed in the model peptides due to its ability to form intermolecular hydrogen bonds involving its hydroxyl group as a donor and the carbonyl groups of other residues as acceptors. On the other hand, insertion of Tyr residue in the basic (AG)n and (AGSGAG)n sequence motifs usually exhibited disruptive effects on the preferred conformations. Moreover, the environmental effect was investigated by mixing the native Cp fraction with the model peptides, showing that there is no significant structural difference on the Ser-containing peptides, while structural transformation was observed on the peptides containing the GAAS unit. This may be attributed to the fact that the Cp fraction promotes the formation of an antiparallel beta-sheet in the Ala-Ala unit. Such periodically disrupted ordered structures in the semicrystalline region of B. mori silk fibroin may be critical not only for facilitating the conformational transformation from silk I to silk II structural form but also for having some correlation with the unique properties of the silk materials.     

Electrospinning Bombyx mori silk with poly(ethylene oxide)

Electrospinning for the formation of nanoscale diameter fibers has been explored for high-performance filters and biomaterial scaffolds for vascular grafts or wound dressings. Fibers with nanoscale diameters provide benefits due to high surface area. In the present study we explore electrospinning for protein-based biomaterials to fabricate scaffolds and membranes from regenerated silkworm silk, Bombyx mori, solutions. To improve processability of the protein solution, poly(ethylene oxide) (PEO) with molecular weight of 900,000 was blended with the silk fibroin. A variety of compositions of the silk/PEO aqueous blends were successfully electrospun. The morphology of the fibers was characterized using high-resolution scanning electron microscopy. Fiber diameters were uniform and less than 800 nm. The composition was estimated by X-ray photoelectron spectroscopy to characterize silk/PEO surface content. Aqueous-based electrospining of silk and silk/PEO blends provides potentially useful options for the fabrication of biomaterial scaffolds based on this unique fibrous protein.     

Spider silk fibre extrusion: combined wide- and small-angle X-ray microdiffraction experiments

The major and minor ampullate silks from live Nephila senegalensis (Tetragnathidae) and the major ampullate silk from Euprostenops spp. (Pisauridae) spiders were investigated in situ by X-ray diffraction during forced silking. Wide- (WAXS) and small-angle (SAXS) scattering patterns were obtained at the same time. WAXS data show that the thread at the exit of the spigots already contains beta-sheet poly(alanine) crystallites. SAXS data suggest the presence of microfibrils with an axial repeating period of approximately 8 nm for both Nephila and Euprostenops. Minor ampullate (MI) Nephila silk, however, does not show this axial repeat which is probably due to a higher amount of crystal forming poly(alanine). A microfibrillar morphology, connected by a network of random polymer chains, can explain the presence of highly oriented crystallites, an oriented halo and a diffuse background in the WAXS patterns. At high reeling speeds, bound water is co-extruded with the fibre. It can be squeezed out of the fibre by friction at a needle. Under natural conditions it is the spider's tarsal claws which might serve to squeeze out the water to improve the mechanical properties of the thread during dragline production.     

Microvoids in Bombyx mori silk. An electron microscope study.

The size and distribution of microvoids in Bombyx mori silk were examined by transmission electron microscopy of silver sulphide 'stained' filaments. Silver sulphide deposited in voids and accessible regions of molecular structure appears as dense particles in thin transverse and longitudinal sections of silk filaments. Small particles (about 8 nm or less in diameter) occur around or adjacent to the periphery of the filaments. Larger particles (around 10-15 nm in diameter) occur in the form of dendritic arrays in the core region of the filaments. The leading edges of the dendritic arrays are oriented towards the fibre periphery. The particles (microvoids) appear to be either spherical or rod-like in shape and are aligned parallel to the long axis of the filament. A skin/core structure is proposed.     

Occurrence of Lectin in the Silkgland of the Silkworm, Bombyx mori

Monoclonal antibodies were prepared against the 350 kDa lectin purified from larval hemolymph of the silkworm, Bombyx mori . The antibodies inhibited the hemagglutinating activity (HA activity) and bound specifically to the hemolymph 350 kDa lectin on Western blotting analysis. Immunohistological observations revealed the occurrence of lectin in the cuticular intima of the anterior silk gland, but not the middle or posterior silk glands of fifth instar larvae of Bombyx mori . Extracts from the anterior silk glands showed HA activity and exhibited the same biochemical characteristics as those of the 350 kDa lectin in the hemolymph. These results suggested that lectin-like molecules in epithelial tissues may be important in histolysis during molting and metamorphosis.     

双向电泳法在家蚕蛋白质分离中的应用

家蚕Bombyxmori(L.)既是重要的经济昆虫,又是鳞翅目昆虫研究的典型模式生物。开展家蚕蛋白质组研究,将有助于阐明家蚕绢丝蛋白的分泌机理,也是研究鳞翅目昆虫及其他生物生命本质的需要。双向电泳是蛋白质分离的关键技术。为探讨适宜家蚕蛋白质组研究的双向电泳条件,以家蚕丝腺、丝腺内容物、蚕卵和血液为材料,在不同条件下进行双向电泳,并对分离的蛋白点进行质谱分析。结果表明:通过改进的蛋白质裂解液辅以超声破碎制备的蛋白质,双向电泳后能够得到较好的2-DE图,也能满足进行MALDI-TOFMS分析的需要。因此本研究方法适用于家蚕不同组织中蛋白质的提取和双向电泳。     

Synthesis and characterization of PCL-b-PEO-b-PCL-based nanostructured and porous hydrogels

Hydrogels with nanoscale structure were synthesized using amphiphilic poly(epsilon-caprolactone)-poly(ethylene oxide)-poly(epsilon-caprolactone) (PCL-b-PEO-b-PCL) triblock copolymers. Small-angle X-ray scattering (SAXS) studies show that the block copolymers form 30-40 nm structures in aqueous solution and that these patterns are retained, with some increase in length scale, following electron beam cross-linking. Lamellar nanostructures were observed by SAXS and atomic force microscopy (AFM), with SAXS indicating cylindrical structure as the block lengths become more different in length. It is demonstrated through Fourier transform infrared spectroscopy (FTIR), mass loss, and differential scanning calorimetry (DSC) that the PCL can be completely removed by hydrolysis in NaOH(aq) to form porous PEO hydrogels. These hydrogels retain active functional groups following PCL removal that serve as sites for further chemical modification.     

Design, expression and solid-state NMR characterization of silk-like materials constructed from sequences of spider silk, Samia cynthia ricini and Bombyx mori silk fibroins

Silk has a long history of use in medicine as sutures. To address the requirements of a mechanically robust and biocompatible material, basic research to clarify the role of repeated sequences in silk fibroin in its structures and properties seems important as well as the development of a processing technique suitable for the preparation of fibers with excellent mechanical properties. In this study, three silk-like protein analogs were constructed from two regions selected from among the crystalline region of Bombyx mori silk fibroin, (GAGSGA)(2), the crystalline region of Samia cynthia ricini silk fibroin, (Ala)(12), the crystalline region of spider dragline silk fibroin, (Ala)(6), and the Gly-rich region of spider silk fibroin, (GGA)(4). The silk-like protein analog constructed from the crystalline regions of the spider dragline silk and B. mori silk fibroins, (A(6)SCS)(8), that constructed from the crystalline regions of the S. c.ricini and B. mori silk fibroins, (A(12)SGS)(4), that constructed from and the crystalline region of S. c.ricini silk fibroin and the glycine-rich region of spider dragline silk fibroin, (A(12)SGS)(4),were expressed their molecular weights being about 36.0 kDa, 17.0 kDa and 17.5 kDa, respectively in E. coli by means of genetic engineering technologies. (A(12)SCS)(4) and (A(12)SGS)(4 )undergo a structural transition from alpha-helix to beta-sheet on a change in the solvent treatment from trifluoroacetic acid (TFA) to formic acid (FA). However, (A(6)SCS)(8) takes on the beta-sheet structure predominantly on TFA treatment and FA treatment. Structural analysis was performed on model peptides selected from spider dragline and S. c.ricini silks by means of (13)C CP/MAS NMR.     

DNA polymerase-delta from the silk glands of Bombyx mori.

The silk gland of Bombyx mori is a terminally differentiated tissue in which DNA replication continues without cell or nuclear division during larval development. DNA polymerase-delta activity increases in the posterior and middle silk glands during the development period, reaching maximal levels in the middle of the fifth instar larvae. The enzyme has been purified to homogeneity by a series of column chromatographic and affinity purification steps. It is a multimer comprising of three heterogeneous subunits, M(r) 170,000, 70,000, and 42,000. An auxiliary protein from B. mori silk glands, analogous to the proliferating cell nuclear antigen, enhances the processivity of the enzyme and stimulates catalytic activity by 3-fold. This auxiliary protein has also been purified to homogeneity. It is a dimer comprised of a single type M(r) 40,000 subunit. Polymerase-delta possesses an intrinsic 3'----5' exonuclease activity which participates in proofreading by mismatch repair during DNA synthesis and is devoid of any primase activity. DNA polymerase-delta activity could be further distinguished from polymerase-alpha from the same tissue based on its sensitivity to various inhibitors and polyclonal antibodies to the individual enzymes. Like DNA polymerase-alpha, polymerase-delta is also tightly associated with the nuclear matrix. The polymerase alpha-primase complex could be readily separated from polymerase-delta (exonuclease) in the purification protocol adopted. DNA polymerase-delta from B. mori silk glands resembles the mammalian delta-polymerases. Considering that both DNA polymerase-delta and -alpha are present in nearly equal amounts in this highly replicative tissue and their close association with the nuclear matrix, the involvement of both the enzymes in the chromosomal endoreplication process in B. mori is strongly implicated.     

家蚕Wnt信号通路下游关键基因Pangolin转录剪接体X3的克隆和分析

【目的】克隆家蚕Bombyx mori Wnt信号通路下游关键基因Pangolin isoforms A/H/I/S转录剪接体X3 (Pangolin X3),分析其序列和表达特征。【方法】从NCBI数据库检索家蚕Pangolin X3,根据其编码序列(coding sequence, CDS)设计引物,利用PCR从家蚕幼虫中肠和血淋巴中进行克隆并测序验证。利用SilkDB 3.0, SMART,多序列比对和系统发育树分析Pangolin X3的序列特征。利用qRT-PCR分析Pangolin X3在家蚕5龄第3天幼虫不同组织(头、血淋巴、体壁、性腺、中肠、前部丝腺、中部丝腺、后部丝腺、脂肪体和马氏管)中的相对表达水平。【结果】从家蚕幼虫中肠和血淋巴克隆了Pangolin X3(GenBank登录号：XM＿038020921)的CDS,其开放阅读框长1 560 bp,编码519个氨基酸残基,预测分子量为55.86 kD,预测等电点为7.53。Pangolin X3蛋白含有保守的β-catenin结合位点和HMG结构域,其氨基酸序列在不同的昆虫中比较保守,特别是与DNA结合的HMG结构域...