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.     

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.     

Fibroin silk proteins from the nonmulberry silkworm Philosamia ricini are biochemically and immunochemically distinct from those of the mulberry silkworm Bombyx mori

Silk proteins were isolated from the cocoons of the nonmulberry silkworm, Philosamia ricini. Three polypeptides of 97, 66, and 45 kDa were identified. The 66-kDa molecule represented sericin, whereas the 97-kDa and the 45-kDa polypeptides linked together through a disulfide bond constituted the fibroin protein. Antibodies raised against the 97-kDa P. ricini fibroin heavy chain reacted specifically with this molecule and did not recognize fibroin heavy chain from another nonmulberry silkworm, Antheraea assama or from the mulberry silkworm, Bombyx mori, suggesting the presence of P. ricini species-specific determinants in this heavy chain. Antibodies generated against fibroin light chain of P. ricini also showed similar reactivity pattern. Immunoblot analysis with proteins isolated from the silk glands of P. ricini at different stages of larval development showed that the expression of fibroin heavy chain was developmentally and spatially regulated. The protein was most abundant in the 5th instar larva, and could be detected in the middle and the posterior but not the anterior silk glands. The amino acid composition of the 97-kDa fibroin protein showed abundance of glutamic acid and did not contain (Gly-Ala)(n) motifs, a characteristic feature of B. mori fibroin heavy chain. Our study reveals significant differences between the nonmulberry silkworm P. ricini and the mulberry silkworm B. mori in the biochemical composition and immunochemical characteristics of fibroin heavy chain. These differences might be responsible for the differences seen in the quality of silk produced by these two silkworms.     

Heterogeneous exchange behavior of Samia cynthia ricini silk fibroin during helix-coil transition studied with (13)C NMR

The structure and structural transition of the glycine residue adjacent to the N-terminal alanine residue of the poly(L-alanine), (Ala)(12-13), region in Samia cynthia ricini silk fibroin was studied using (13)C nuclear magnetic resonance (NMR). Most of the glycine carbonyl peaks in the (13)C solution NMR spectrum of [1-(13)C]glycine-silk fibroin could be assigned to the primary structure from the comparison of the (13)C chemical shifts of seven glycine-containing tripeptides. The slow exchange between helix and coil forms in the NMR time scale was observed with increasing temperature exclusively for the underlined glycine residue in the Gly-Gly-(Ala)(12-13) sequence during fast helix-coil transition of the (Ala)(12-13) region.     

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.     

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.     

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.     

Tightly winding structure of sequential model peptide for repeated helical region in Samia cynthia ricini silk fibroin studied with solid-state NMR

There are many kinds of silks from silkworms and spiders with different structures and properties, and thus, silks are suitable to study the structure-property relationship of fibrous proteins. Silk fibroin from a wild silkworm, Samia cynthia ricini, mainly consists of the repeated similar sequences by about 100 times where there are alternative appearances of the polyalanine (Ala)(12-13) region and the Gly-rich region. In this paper, a sequential model peptide, GGAGGGYGGDGG(A)(12)GGAGDGYGAG, which is a typical sequence of the silk fibroin, was synthesized, and the atomic-level conformations of Gly residues at the N- and C-terminal ends of the polyalanine region were determined as well as that of the central Ala residue using (13)C 2D spin diffusion solid-state nuclear magnetic resonance (NMR) under off-magic angle spinning. In the model peptide with alpha-helical conformation, the torsion angle of the central Ala residue, the 19th Ala, was determined to be (phi, psi) = (-60 degrees, -50 degrees ), which was a typical alpha-helical structure, but the torsion angles of two Gly residues, the 12th and 25th Gly residues, which are located at the N- and C-terminal ends of the polyalanine region, were determined to be (phi, psi) = (-70 degrees, -30 degrees ) and (phi, psi) = (-70 degrees, -20 degrees ), respectively. Thus, it was observed that the turns at both ends of polyalanine with alpha-helix conformation in the model peptide are tightly wound.     

Structural analysis of silk with 13C NMR chemical shift contour plots.

The polymorphic structures of silk fibroins in the solid state were examined on the basis of a quantitative relationship between the 13C chemical shift and local structure in proteins. To determine this relationship, 13C chemical shift contour plots for C alpha and C beta carbons of Ala and Ser residues, and the C alpha chemical shift plot for Gly residues were prepared using atomic co-ordinates from the Protein Data Bank and 13C NMR chemical shift data in aqueous solution reported for 40 proteins. The 13C CP/MAS NMR chemical shifts of Ala, Ser and Gly residues of Bombyx mori silk fibroin in silk I and silk II forms were used along with 13C CP/MAS NMR chemical shifts of Ala residues of Samia cynthia ricini silk fibroin in beta-sheet and alpha-helix forms for the structure analyses of silk fibroins. The allowed regions in the 13C chemical shift contour plots for C alpha and C beta carbons of Ala and Ser residues for the structures in silk fibroins, i.e. Silk II, Silk I and alpha-helix, were determined using their 13C isotropic NMR chemical shifts in the solid state. There are two area of the phi,psi map which satisfy the observed Silk I chemical shift data for both the C alpha and C beta carbons of Ala and Ser residues in the 13C chemical shift contour plots.     

Synthesis and characterization of multiblock copolymers based on spider dragline silk proteins

Spider dragline silk with its superlative tensile properties provides an ideal system to study the relationship between morphology and mechanical properties of a structural protein. Accordingly, we synthesized two hybrid multiblock copolymers by condensing poly(alanine) [(Ala)(5)] blocks of the structural proteins (spidroin MaSp1 and MaSp2) of spider dragline silk with different oligomers of isoprene (2200 and 5000 Da) having reactive end groups. The synthetic multiblock polymer displayed similar secondary structure to that of natural spidroin, the peptide segment forming a beta-sheet structure. These multiblock polymers showed a significant solubility in the component solvents. Moreover, the copolymer which contains the short polyisoprene segment would aggregate into a micellar-like structure, as observed by TEM.     

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.     

Structure of model peptides based on Nephila clavipes dragline silk spidroin (MaSp1) studied by 13C cross polarization/magic angle spinning NMR

To obtain detailed structural information for spider dragline spidroin (MaSp1), we prepared three versions of the consensus peptide GGLGGQGAGAAAAAAGGAGQGGYGGLGSQGAGR labeled with 13C at six different sites. The 13C CP/MAS NMR spectra were observed after treating the peptides with different reagents known to alter silk protein conformations. The conformation-dependent 13C NMR chemical shifts and peak deconvolution were used to determine the local structure and the fractional compositions of the conformations, respectively. After trifluoroacetic acid (solvent)/diethyl ether (coagulant) treatment, the N-terminal region of poly-Ala (PLA) sequence, Ala8 and Ala10, adopted predominantly the alpha-helix with a substantial amount of beta-sheet. The central region, Ala15, Ala18, and Leu26, and C-terminal region, Ala31, of the peptide were dominated by either 3(1)-helix or alpha-helix. There was no indication of beta-sheet, although peak broadening indicates that the torsion angle distribution is relatively large. After 9 M LiBr/dialysis treatment, three kinds of conformation, beta-sheet, random coil, and 3(1)-helix, appeared, in almost equal amounts of beta-sheet and random coil conformations for Ala8 and Ala10 residues and distorted 3(1)-helix at the central region of the peptide. In contrast, after formic acid/methanol and 8 M urea/acetonitrile treatments, all of the local structure tends to beta-sheet, although small amounts of random coil are also observed. The peak pattern of the Ala Cbeta carbon after 8 M urea/acetonitrile treatment is similar to the corresponding patterns of silk fiber from Bombyx mori and Samia cynthia ricini. We also synthesized a longer 13C-labeled peptide containing two PLA blocks and three Gly-rich blocks. After 8 M urea/acetonitrile treatment, the conformation pattern was closely similar to that of the shorter peptide.     

An Australian webspinner species makes the finest known insect silk fibers

Aposthonia gurneyi, an Australian webspinner species, is a primitive insect that constructs and lives in a silken tunnel which screens it from the attentions of predators. The insect spins silk threads from many tiny spines on its forelegs to weave a filmy sheet. We found that the webspinner silk fibers have a mean diameter of only 65nm, an order of magnitude smaller than any previously reported insect silk. The purpose of such fine silk may be to reduce the metabolic cost of building the extensive tunnels. At the molecular level, the A. gurneyi silk has a predominantly beta-sheet protein structure. The most abundant clone in a cDNA library produced from the webspinner silk glands encoded a protein with extensive glycine-serine repeat regions. The GSGSGS repeat motif of the A. gurneyi silk protein is similar to the well-known GAGAGS repeat motif found in the heavy fibroin of silkworm silk, which also has beta-sheet structure. As the webspinner silk gene is unrelated to the silk gene of the phylogenetically distant silkworm, this is a striking example of convergent evolution.     

Characterization by Raman microspectroscopy of the strain-induced conformational transition in fibroin fibers from the silkworm Samia cynthia ricini

Raman microspectroscopy has been used to quantitatively study the effect of a mechanical deformation on the conformation and orientation of Samia cynthia ricini (S. c. ricini) silk fibroin. Samples were obtained from the aqueous solution stored in the silk gland and stretched at draw ratios (lambda) ranging from 0 to 11. Using an appropriate band decomposition procedure, polarized and orientation-insensitive spectra have been analyzed to determine order parameters and the content of secondary structures, respectively. The data unambiguously show that, in response to mechanical deformation, S. c. ricini fibroin undergoes a cooperative alpha-helix to beta-sheet conformational transition above a critical draw ratio of 4. The alpha-helix content decreases from 33 to 13% when lambda increases from 0 to 11, while the amount of beta-sheets increases from 15 to 37%. In comparison, cocoon silk is devoid of alpha-helical structure and always contains a larger amount of beta-sheets. Although the presence of isosbestic points in different spectral regions reveals that the conformational change induced by mechanical deformation is a two-state process, our results suggest that part of the glycine residues might be incorporated into beta-poly(alanine) structures. The beta-sheets are initially isotropically distributed and orient along the fiber axis as lambda increases, but do not reach the high level of orientation found in the cocoon fiber. The increase in the orientation level of the beta-sheets is found to be concomitant with the alpha --> beta conformational conversion, whereas alpha-helices do not orient under the applied strain but are rather readily converted into beta-sheets. The components assigned to turns exhibit a small orientation perpendicular to the fiber axis in stretched samples, showing that, overall, the polypeptide chains are aligned along the stretching direction. Our results suggest that, in nature, factors other than stretching contribute to the optimization of the amount of beta-sheets and the high degree of orientation found in natural cocoon silk.     

NMR study of silk I structure of Bombyx mori silk fibroin with 15N- and 13C-NMR chemical shift contour plots

The metastable state silk I structures of Bombyx mori silk fibroin in the solid state were studied on the basis of ¹⁵N- and ¹³C-nmr chemical shifts of Ala, Ser, and Gly residues. The ¹⁵N cross-polarization magic angle spinning (CP/MAS) nmr spectra of the precipitated fraction after chymotrypsin hydrolysis of B. mori silk fibroin with the silk I and silk II forms were measured to determine the ¹⁵N chemical shifts of Gly, Ala, and Ser residues. For comparison, ¹⁵N CP/MAS nmr chemical shifts of Ala were measured for [¹⁵N] Ala Philosamia cynthia ricini silk fibroin with antiparallel β-sheet and α-helix forms. The ¹³C CP/MAS nmr chemical shifts of Ala, Ser, and Gly residues of B. mori silk fibroin with the silk I and silk II forms, as well as ¹³C CP/MAS nmr chemical shifts of Ala residue of P. c. ricini silk fibroin with β-sheet and α-helix forms, are used for the examination of the silk I structure. Both silk I and α-helix peaks are shifted to a lower field than silk II (β-sheet) for the Cα carbons of the Ala residues, while both Cβ carbon peaks are shifted to higher field. However, the silk I peak of the ¹⁵N nucleus of the Ala residue is shifted to lower field than the silk II peak, but the α-helix peak is shifted to high field. Thus, the difference in the structure between the silk I and α-helix is reflected in a different manner between the ¹³C and ¹⁵N chemical shifts. The Cα and Cβ chemical shift contour plots for Ala and Ser residues, and the Cα plot for the Gly residue, were prepared from the Protein Data Bank data obtained for 12 proteins and used for discussing the silk I structure quantitatively from the conformation-dependent chemical shifts. The plots reported by Le and Oldfield for ¹⁵N chemical shifts were also used for the purpose. All these chemical shift data support Fossey's model (Ala: ϕ = −80°, φ = 150°, Gly: ϕ = −150°, φ = 80°) and do not support Lotz and Keith's model (Ala: ϕ = −104.6°, φ = 112.2°, Gly: ϕ = 79.8°, φ = 49.7° or Ala: ϕ = −124.5°, φ = 88.2°, Gly: ϕ = −49.8°, φ = −76.1°) as the silk I structure. © 1997 John Wiley & Sons, Inc.     

Purification and biochemical characterization of a 70 kDa sericin from tropical tasar silkworm, Antheraea mylitta

Sericin isolated from the cocoon of the tropical tasar silkmoth Antheraea mylitta showed three major bands, with the lowest 70 kDa. This band was purified by anion exchange chromatography. Immunoblotting with concanavalin-A suggests a glycoprotein and CD analysis of secondary structure includes beta-sheet. Amino acid analysis shows that the protein is enriched in glycine and serine while the mole percentages of these two amino acids are different from sericin of mulberry silkworm. An anti A. mylitta sericin antibody was able to cross-react with sericin from A. assamensis but not the sericin of Bombyx mori and Philosamia ricini. Immunoblot analysis with proteins isolated from middle silk gland of A. mylitta at different developmental stages of larva showed that the 70 kDa sericin is developmentally regulated. These data extend the range of biochemical features found in this unusual family of proteins and may help in developing an improved understanding of their role in forming environmentally stable fibroin fiber-sericin composite structures (cocoons).     

Structure and properties of silk hydrogels

Control of silk fibroin concentration in aqueous solutions via osmotic stress was studied to assess relationships to gel formation and structural, morphological, and functional (mechanical) changes associated with this process. Environmental factors potentially important in the in vivo processing of aqueous silk fibroin were also studied to determine their contributions to this process. Gelation of silk fibroin aqueous solutions was affected by temperature, Ca(2+), pH, and poly(ethylene oxide) (PEO). Gelation time decreased with increase in protein concentration, decrease in pH, increase in temperature, addition of Ca(2+), and addition of PEO. No change of gelation time was observed with the addition of K(+). Upon gelation, a random coil structure of the silk fibroin was transformed into a beta-sheet structure. Hydrogels with fibroin concentrations >4 wt % exhibited network and spongelike structures on the basis of scanning electron microscopy. Pore sizes of the freeze-dried hydrogels were smaller as the silk fibroin concentration or gelation temperature was increased. Freeze-dried hydrogels formed in the presence of Ca(2+) exhibited larger pores as the concentration of this ion was increased. Mechanical compressive strength and modulus of the hydrogels increased with increase in protein concentration and gelation temperature. The results of these studies provide insight into the sol-gel transitions that silk fibroin undergoes in glands during aqueous processing while also providing important insight in the in vitro processing of these proteins into useful new materials.