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.     

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.     

Copper in the silk formation process of Bombyx mori silkworm

Evidence is presented here that cupric ions play a part in the natural spinning of Bombyx mori silk. Proton induced X-ray emission studies revealed that the copper content increased from the posterior part to the anterior part of silk gland, and then further increased in the silk fiber. Spectrophotometric analysis demonstrated that cupric ions formed coordination complexes with silk fibroin chains while Raman spectroscopy indicated that they induced a conformation transition from random coil/helix to beta-sheet. Taken together these findings indicate that copper could play a role in the natural spinning process in silkworms.     

Plasticity in major ampullate silk production in relation to spider phylogeny and ecology

Spider major ampullate silk is a high-performance biomaterial that has received much attention. However, most studies ignore plasticity in silk properties. A better understanding of silk plasticity could clarify the relative importance of chemical composition versus processing of silk dope for silk properties. It could also provide insight into how control of silk properties relates to spider ecology and silk uses. We compared silk plasticity (defined as variation in the properties of silk spun by a spider under different conditions) between three spider clades in relation to their anatomy and silk biochemistry. We found that silk plasticity exists in RTA clade and orbicularian spiders, two clades that differ in their silk biochemistry. Orbiculariae seem less dependent on external spinning conditions. They probably use a valve in their spinning duct to control friction forces and speed during spinning. Our results suggest that plasticity results from different processing of the silk dope in the spinning duct. Orbicularian spiders seem to display better control of silk properties, perhaps in relation to their more complex spinning duct valve.     

Structural characteristics and properties of Bombyx mori silk fiber obtained by different artificial forcibly silking speeds

To study the spinning condition of natural biopolymer silk, the silk fibers were directly acquired from Bombyx mori silkworm, N140 x C140 by a simple artificial forcibly silking method at the speed of 60, 120, 180 and 240 cm min(-1), respectively and its microstructure and physical properties were evaluated. The fine silk fibers (about 8 microm) were obtained at faster spinning speed, 240 cm min(-1). The tensile properties of silk fibers were remarkably increased with raising the forcibly spinning speeds. The beta-sheet structure contents of silk fibers obtained at higher speed were considerably increased. The fibers obtained by different spinning speeds exhibited a fairly similar X-ray crystallinity, while the degree of molecular orientation increased with decreasing the fiber diameter. The fine silk fibers obtained at higher speed (240 cm min(-1)) exhibited a slightly higher thermal stability, as shown by the upward shift of differential scanning calorimetry (DSC) decomposition temperature.     

Comparative structure analysis of tyrosine and valine residues in unprocessed silk fibroin (silk I) and in the processed silk fiber (silk II) from Bombyx mori using solid-state (13)C,(15)N, and (2)H NMR

The solid-state (13)C CP-MAS NMR spectra of biosynthetically labeled [(13)C(alpha)]Tyr, [(13)C(beta)]Tyr, and [(13)C(alpha)]Val silk fibroin samples of Bombyx mori, in silk I (the solid-state structure before spinning) and silk II (the solid-state structure after spinning) forms, have been examined to gain insight into the conformational preferences of the semicrystalline regions. To establish the relationship between the primary structure of B. mori silk fibroin and the "local" structure, the conformation-dependent (13)C chemical shift contour plots for Tyr C(alpha), Tyr C(beta), and Val C(alpha) carbons were generated from the atomic coordinates of high-resolution crystal structures of 40 proteins and their characteristic (13)C isotropic NMR chemical shifts. From comparison of the observed Tyr C(alpha) and Tyr C(beta) chemical shifts with those predicted by the contour plots, there is strong evidence in favor of an antiparallel beta-sheet structure of the Tyr residues in the silk fibroin fibers. On the other hand, Tyr residues take a random coil conformation in the fibroin film with a silk I form. The Val residues are likely to assume a structure similar to those of Tyr residues in silk fiber and film. Solid-state (2)H NMR measurements of [3,3-(2)H(2)]Tyr-labeled B. mori silk fibroin indicate that the local mobility of the backbone and the C(alpha)-C(beta) bond is essentially "static" in both silk I and silk II forms. The orientation-dependent (i.e., parallel and perpendicular to the magnetic field) solid-state (15)N NMR spectra of biosynthetically labeled [(15)N]Tyr and [(15)N]Val silk fibers reveal the presence of highly oriented semicrystalline regions.     

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.     

Effects of pH and calcium ions on the conformational transitions in silk fibroin using 2D Raman correlation spectroscopy and 13C solid-state NMR

Silk fibroin exists in a number of different states, such as silk I and silk II, with different properties largely defined by differences in secondary structure composition. Numerous attempts have been made to control the transitions from silk I to silk II in vitro to produce high-performance materials. Of all the factors influencing the structural compositions, pH and some metal ions play important roles. This paper focuses on the influence of pH and Ca(2+) ions on the conformational transition from silk I to silk II in regenerated (redissolved) Bombyx mori fibroin. One- and two-dimensional correlation Raman spectroscopy was used to describe qualitatively the transitions in secondary structure in silk I, silk II, and their intermediates as pH and Ca(2+) ion concentration were changed, while (13)C cross polarization magic angle spinning (CP/MAS) solid-state NMR was used to quantify these changes. We showed that conditions (low pH, pH 5.2; a defined range of Ca(2+) ion concentrations; gradual water removal) that mimic natural silk spinning promote the formations of beta-sheet and distorted beta-sheet characteristic of silk II or silk II-related intermediate. In contrast, higher pH (pH 6.9-8.0) and higher Ca(2+) ion concentrations maintain "random coil" conformations typical of silk I or silk I-related intermediate. These results help to explain why the natural silk spinning process is attended by a reduction in pH from 6.9 to 4.8 and a change in the Ca(2+) ion concentration in the gland lumen as fibroin passes from the posterior division through the secretory pathway to the anterior division.     

Structural characterization and artificial fiber formation of Bombyx mori silk fibroin in hexafluoro-iso-propanol solvent system

High-resolution solution (13)C-NMR and CD studies of Bombyx mori silk fibroin revealed the presence of an ordered secondary structure 3(10)-helix, in hexafluoro-iso-propanol (HFIP). The solid-state structure of the silk fibroin film prepared by drying it gently from the HFIP solution still keep the structure, 3(10)-helix, which was studied with high-resolution solid state (13)C-NMR. The structural transition from the 3(10)-helix to silk II structure, heterogeneous structure including antiparallel beta-sheet, occurred during the artificial spinning from the HFIP solution. The wide-angle x-ray diffraction and differential scanning calorimetry thermograms of the artificial spinning fiber after postspinning treatments were observed together with the stress-strain curves. The results emphasize that the molecular structures, controlled morphology, and mechanical properties of the protein-based synthetic polymers can be modulated for enhancing biocompatibility.     

Structure of Bombyx mori silk fibroin before spinning in solid state studied with wide angle x-ray scattering and (13)C cross-polarization/magic angle spinning NMR

The structure of a crystalline form of Bombyx mori silk fibroin, commonly found before the spinning process (known as silk I), has been proposed as a repeated beta-turn type II-like structure by combining data obtained from solid-state two dimensional spin-diffusion nuclear magnetic resonance and rotational-echo double-resonance (T. Asakura et al., J Mol Biol, in press). In this paper, the WAXS pattern of alanine-glycine alternating copolypeptide, (Ala-Gly)(15) with silk I form which was used for a silk I model of B. mori silk fibroin was observed. The pattern calculated with the silk I model proposed by us is well reproduced the observed one, indicating the validity of the proposed silk I model. In addition, two peptides of the other repeated sequences which contain Tyr or Val residues in the silk fibroin,23 were synthesized; (Ala-Gly-Tyr-Gly-Ala-Gly)(5) and (X-Gly)(15) where X is Tyr for the 7th, 15th and 23th residues, and Val for the 11th residue and Ala for other residues. There are no sharp peaks in the WAXS patterns, and therefore both samples are in the non-crystalline state. This is in agreement with the (13)C CP/MAS NMR result, where the conformation is mainly random coil.     

Vibrational 13C-cross-polarization/magic angle spinning NMR spectroscopic and thermal characterization of poly(alanine-glycine) as model for silk I Bombyx mori fibroin

This study focuses on the conformational characterization of poly(alanine-glycine) II (pAG II) as a model for a Bombyx mori fibroin silk I structure. Raman, IR, and 13C-cross-polarization/magic angle spinning NMR spectra of pAG II are discussed in comparison with those of the crystalline fraction of B. mori silk fibroin (chymotryptic precipitate, Cp) with a silk I (silk I-Cp) structure. The spectral data give evidence that silk I-Cp and the synthetic copolypeptide pAG II have similar conformations. Moreover, the spectral findings reveal that silk I-Cp is more crystalline than pAG II; consequently, the latter contains a larger amount of the random coil conformation. Differential scanning calorimetry measurements confirm this result. N-Deuteration experiments on pAG II allow us to attribute the Raman component at 1320 cm(-1) to the amide III mode of a beta-turn type II conformation, thus confirming the results of those who propose a repeated beta-turn type II structure for silk I. The analysis of the Raman spectra in the nuNH region confirms that the silk I structure is characterized by the presence of different types of H-bonding arrangements, in agreement with the above model.     

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.     

The natural silk spinning process. A nucleation-dependent aggregation mechanism?

The spinning mechanism of natural silk has been an open issue. In this study, both the conformation transition from random coil to beta sheet and the beta sheet aggregation growth of silk fibroin are identified in the B. mori regenerated silk fibroin aqueous solution by circular dichroism (CD) spectroscopy. A nucleation-dependent aggregation mechanism, similar to that found in prion protein, amyloid beta (Abeta) protein, and alpha-synuclein protein with the conformation transition from a soluble protein to a neurotoxic, insoluble beta sheet containing aggregate, is a novel suggestion for the silk spinning process. We present evidence that two steps are involved in this mechanism: (a) nucleation, a rate-limiting step involving the conversion of the soluble random coil to insoluble beta sheet and subsequently a series of thermodynamically unfavorable association of beta sheet unit, i.e. the formation of a nucleus or seed; (b) once the nucleus forms, further growth of the beta sheet unit becomes thermodynamically favorable, resulting a rapid extension of beta sheet aggregation. The aggregation growth follows a first order kinetic process with respect to the random coil fibroin concentration. The increase of temperature accelerates the beta sheet aggregation growth if the beta sheet seed is introduced into the random coil fibroin solution. This work enhances our understanding of the natural silk spinning process in vivo.     

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.     

Proteomic analysis of silk gland programmed cell death during metamorphosis of the silkworm Bombyx mori

The silk gland of the silkworm Bombyx mori undergoes programmed cell death (PCD) during pupal metamorphosis. On the basis of their morphological changes and the occurrence of a DNA ladder, the tissue cells were categorized into three groups: intact, committed, and dying. To identify the proteins involved in this process, we conducted a comparative proteomic analysis. Protein expression changes among the three different cell types were examined by two-dimensional gel electrophoresis. Among approximately 1000 reproducibly detected protein spots on each gel, 43 were down-regulated and 34 were up-regulated in PCD process. Mass spectrometry identified 17 differentially expressed proteins, including some well-studied proteins as well as some novel PCD related proteins, such as caspases, proteasome subunit, elongation factor, heat shock protein, and hypothetical proteins. Our results suggest that these proteins may participate in the silk gland PCD process of B. mori and, thus, provide new insights for this mechanism.