Ricini Silk Fibroin Research Articles

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

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

Packing arrangement of 13C selectively labeled sequence model peptides of Samia cynthia ricini silk fibroin fibers studied by solid-state NMR.

Samia cynthia ricini (S. c. ricini) is one of the wild silkworms. Their silk fibroins have been paid attention as potentially valuable biomedical materials as well as Bombyx mori silk fibroins, but detailed information on the packing arrangement of the fibers is still not currently well understood at a molecular level. In this study, 34 mer model peptides, GGAGGGYGGDGG(A)12GGAGDGYGAG with different 13C labeled positions have been synthesized as a typical sequence of the primary structure of S. c. ricini silk fibroins made up of tandemly repeated sequences of polyalanine as the crystalline region and glycin-rich sequences as the non-crystalline region. The heterogeneous structure was obtained from the determination of the fraction of several conformations depending on the position of the Ala residue by 13C cross polarization/magic angle spinning NMR. The packing arrangement was studied by 13C dipolar assisted rotational resonance NMR and packing in a staggered arrangement rather than a rectangular arrangement of this peptide with an anti-parallel β-sheet structure was clarified, which is in good agreement with our previous report on the packing arrangement of (Ala)7 with an anti-parallel β-sheet structure.

Silk-silk blend materials

Silk fibroin materials can be used as various kinds of biomedical materials. Here, we report a comparative study of silk-silk blend materials using thermal analysis and infrared spectroscopy. Four groups of silk-silk blend films were fabricated from aqueous solutions by blending Chinese Bombyx mori (Mori) with Indian Antheraea mylitta (Tussah) silk fibroin (Mori-Tussah), Mori with Antheraea assama (Muga) silk fibroin (Mori-Muga), Mori with Philosamia ricini (Eri) silk fibroin (Mori-Eri), and Mori with Thailand mulberry (Thai) silk fibroin (Mori-Thai), respectively. These silk-silk blend systems exploit the beneficial material properties of both silks. Glass transition temperatures (T g), heat capacity increments at T g, and degradation temperatures (T d) of these water-based silk-silk blend films were measured by differential scanning calorimetry (DSC) and temperature-modulated DSC (TMDSC). It was found that those silk-silk film systems were well-blended without macrophase separation. And glass transition temperatures and degradation temperatures of those silk-silk blend films can be controlled by changing the mass ratio of different silks in the blend system. Fourier transform infrared spectrometer (FTIR) was used to characterize secondary structures of silk-silk blends. The contents of alpha-helix and random coils are tunable through changing the contents of Tussah, Muga, Eri, or Thai silk in the blend system. The study demonstrates that Mori silk are fully miscible with Tussah, Muga, Eri, and Thai silk at different mass ratios, and the features of Mori silk combined with the attributes of Tussah, Muga, Eri, and Thai silk offer a useful suite of materials for a variety of applications in the future.

Structural Determination of the Tandem Repeat Motif in Samia cynthia ricini Liquid Silk by Solution NMR

The wild silkworm Samia cynthia ricini produces silk fibroin containing polyalanine sequences. The structure of a typical tandem repeat sequence YGGDGG(A)12GGAG within S. c. ricini silk fibroin in liquid silk was determined by solution NMR. 13C, 15N, and 1H shifts were assigned from solution NMR spectra for the tandem repeat. TALOS-N was then used to predict the backbone dihedral angles from the chemical shifts. Dihedral angles revealed a well-structured α-helix for the polyalanine region and less stable capping motifs for the N- and C-terminal regions. Consistent with this, the amide proton temperature coefficients of the last glycine in the N-terminal region and the first two glycines in the C-terminal region show more positive values than that of a random coil structure. Thus, the α-helical structure of the polyalanine region of S. c. ricini liquid silk is stabilized by capping motifs.

Microscopic structural analysis of fractured silk fibers from Bombyx mori and Samia cynthia ricini using 13C CP/MAS NMR with a 1 mm microcoil MAS NMR probehead

Conformational changes have been studied in silk fibers from the domestic silkworm Bombyx mori and a wild silkworm Samia cynthia ricini as a result of fractured by stretching. About 300 samples consisting of only the fractured regions of [1- 13C]Ala or [1- 13C]Gly labeled silk fibers were collected and observed by 13C CP/MAS NMR spectra. The total amount of these fractured fibers is only about 1 mg and therefore we used a home-built 1 mm microcoil MAS NMR probehead. A very small increase in the fraction of random coil was noted for the alanine regions of both silk fibroins and for the glycine region of B. mori silk fibroin. However, there is no difference in the spectra before and after fractured for the glycine region of S. c. ricini silk fibroin. Thus, the influence of fracture occurs exclusively at the Ala region for S. c. ricini. The relationship between sequence, fracture and structure is discussed.

Influence of Philosamia ricini Silk Fibroin Components on Morphology, Secondary Structure and Thermal Properties of Chitosan Biopolymer Film

This study aimed to prepare Eri (Philosamia ricini) Silk Fibroin (SF)/chitosan (CS) blend films by a solvent evaporation method and to compare the blend films with both native SF and CS films. Influence of SF ratios on the morphology, secondary structure and thermal decomposition of the CS blend films were investigated. The native SF and CS films were uniform and homogeneous without phase separation. For the blend films, the uniform can be found less than 60% of SF composition. All of SF/CS blend films showed both SF and CS characteristics. FT-IR results showed that the blend films composed of both random coil and beta-sheet with predominant of beta-sheet form. Interaction of intermolecular between SF and CS have occurred which were measured by thermogravimetric thermograms. Increasing of SF contents was leading to the increase of beta-sheet structures which were enhanced the thermal stability of the CS blend films.

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

AbstractFor Abstract see ChemInform Abstract in Full Text.

Spectroscopic Characterization of Heterogeneous Structure of Samia cynthia ricini Silk Fibroin Induced by Stretching and Molecular Dynamics Simulation

The 13C CP/MAS NMR, X-ray diffraction, and Raman spectroscopies were used for monitoring the structural transition of Samia cynthia ricini (S. c. ricini) silk fibroin induced by stretching. Here the silk fibroin was obtained from the aqueous solution stored in the silk gland. All of these spectroscopic data indicate that the structural transition from α-helix to β-sheet occurs with increasing the stretching ratio, especially between the stretching ratios, ×4 and ×6. The 13C chemical shifts of Ala Cβ peak in the 13C CP/MAS NMR spectrum change significantly depending on α-helix, random coil, and two kinds of β-sheet structure, which make it possible to clarify the local structure and structural transition. Actually, the fraction of individual structure in the silk fibroin samples was determined from decomposition of the Ala Cβ peak by assuming a Gaussian line shape. To examine the conformational change of Ala residues of the polyalanine region in S. c. ricini silk fibroin observed with these spectroscopic methods, MD simulations for four peptide molecules, AGGAGG(A)12GGAGAG, with α-helix conformation were performed in the presence of water molecules under different tensile strengths, and then additional MM calculations were performed after removal of water molecules. The change in the conformational character of Ala residues induced by stretching is explicable by these calculations.

Structure and structural changes of the silk fibroin from Samia cynthia ricini using nuclear magnetic resonance spectroscopy.

The structure of silk fibroin from a wild silkworm, S. c. ricini, the amino acid sequence of which consists of repeated poly-Ala and Gly-rich regions, was examined by using solution and solid-state NMR methods. The structural transition of the silk fibroin in aqueous solution was monitored by using 13C solution NMR spectroscopy as a function of temperature. The fast exchange with respect to the chemical shift between the helix and coil conformations was observed in the poly-Ala region and the slow conformational change from alpha-helix to random coil was observed for the Gly residue adjacent to the N-terminal Ala residue of the poly-Ala region. The torsion angles of several Ala and Gly residues in the model peptide, GGAGGGYGGDGG(A)12GGA-GDGYGAG, were determined by the conformation-dependent 13C chemical shifts, rotational echo double resonance (REDOR) and 2D spin-diffusion NMR methods. The solid-state NMR analysis leads to the precise silk structure before spinning, where the poly-Ala sequence takes a typical alpha-helix pattern with a tightly winded helical structure at both terminal regions of the poly-Ala sequence. This is expected to stabilize the alpha-helical structure of the poly-Ala region in S. c. ricini silk fibroin from the silkworm.

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.

Production and characterization of a silk-like hybrid protein, based on the polyalanine region of Samia cynthia ricini silk fibroin and a cell adhesive region derived from fibronectin

There are a variety of silkworms and silk fibroins produced by them. Silks have many inherent suitable properties for biomaterials. In this paper, a novel silk-like hybrid protein, [DGG(A) 12GGAASTGRGDSPAAS] 5, which consists of polyalanine region of silk fibroin from a wild silkworm, Samia cynthia ricini, and cell adhesive region including Arg-Gly-Asp (RGD) sequence, derived from fibronectin, was designed and produced. The genes encoding the hybrid protein were constructed and expressed in Escherichia coli. The main conformation of the polyalanine region, that is, either α-helix or β-sheet, could be easily controlled by treatment with different acidic solvents, trifluoroacetic acid or formic acid, respectively. This structural change was monitored with 13C CP/MAS NMR. Higher cell adhesive and growth activities of the hybrid protein compared with those of collagen were obtained.

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.

Structure of Silks Determined with Solid-State NMR and Development of New Silk-like Materials

The solid-state structures of Bombyx mori silk fibroin before and after spinning were determined with several solid-state NMR. A repeated β-turn type II structure was proposed for the former model and a heterogeneous structure which consists of two kinds of β-sheet structure with different inter-chain arrangement and distorted β-turn for the latter. New silk-like materials with several repeated sequences selected from B. mori and Samia cynthia ricini silk fibroins, and spider dragline silk were designed and produced using genetic engineering methods. The silk-like materials containing the cell-adhesive sequence, Arg-Gly-Asp, from fibronectin were produced similarly and their high cell-adhesive abilities were observed.

Preparation of non-woven nanofibers of Bombyx mori silk, Samia cynthia ricini silk and recombinant hybrid silk with electrospinning method

Electrospinning is a good method to obtain nanoscale fibers from polymer solutions. In this paper, we successfully prepared non-woven nanofibers of Bombyx mori and Samia cynthia ricini silk fibroins, and of the recombinant hybrid fiber involving the crystalline domain of B. mori silk and non-crystalline domain of S. c. ricini silk from hexafluoroacetone (HFA) solution using electrospinning method. 13C cross polarization/magic angle spinning NMR spectroscopy was used to monitor the structural change of silk fibroins together with the detection of the residual HFA during the process of the fiber formation. Scanning electronic microscope was used to determine the diameters and their distributions of the fibers.

Heterogeneous exchange behavior of Samia cynthia ricini silk fibroin during helix–coil transition studied with 13C 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 13C nuclear magnetic resonance (NMR). Most of the glycine carbonyl peaks in the 13C solution NMR spectrum of [1- 13C]glycine-silk fibroin could be assigned to the primary structure from the comparison of the 13C 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.

Dynamics of the Tyrosine Side Chain in Bombyx mori and Samia cynthia ricini Silk Fibroin Studied by Solid State 2H NMR

Solid-state deuterium NMR (2H NMR) was used to study the dynamics of the tyrosine (Tyr) residue in silk fibroin from Bombyx mori (B. mori) and Samia cynthia ricini (S. c. ricini). Specifically deuterated cocoon silk was obtained by feeding silk worms with Tyr, labeled either at the Cβ carbon ([3,3-2H2]Tyr) or at the aromatic ring ([3‘,5‘-2H2]Tyr). The 2H NMR spectra of the [3,3-2H2]Tyr-labeled silk fibroins showed typical rigid powder patterns, indicating that there is essentially no motion about the Cα−Cβ bond axis, both in B. mori and S. c. ricini. In contrast, the 2H NMR spectra of the [3‘,5‘-2H2]Tyr-labeled silk fibroins consisted of two dynamic components each: a rigid powder contribution, plus a motionally averaged contribution. Hence, some of the Tyr side chains are mobile at the phenolic ring. This motion was characterized to be a π-flip as is typical of aromatic rings. The corresponding 2H NMR line-shape of the B. mori sample could be successfully simulated by attributing 20% of the signal to a motionally averaged component with a fast rate (106 Hz) and the remaining 80% to a much slower component (<103 Hz). Likewise, the simulation of S. c. ricini silk fibroin indicated that 60% of the rings are engaged in fast motional averaging (107 Hz), while 40% undergo slow motion (104 Hz). Thus, the fraction of the fast component is considerably higher for S. c. ricini silk fibroin than for B. mori, which must be a consequence of their different amino acid sequences. It appears that the side-chain mobility depends on the local packing density around the Tyr residue. We conclude that the Gly-Ala repeats in B. mori silk fibroin are relatively tightly packed. In contrast, a large part of the Gly-rich regions in S. c. ricini are comparatively loosely packed.

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 Cα and Cβ carbons of Ala and Ser residues, and the Cα 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 β-sheet and α-helix forms for the structure analyses of silk fibroins. The allowed regions in the 13C chemical shift contour plots for Cα and Cβ carbons of Ala and Ser residues for the structures in silk fibroins, i.e. Silk II, Silk I and α-helix, were determined using their 13C isotropic NMR chemical shifts in the solid state. There are two area of the φ,Ψ map which satisfy the observed Silk I chemical shift data for both the Cα and Cβ carbons of Ala and Ser residues in the 13C chemical shift contour plots.

NMR study of silk I structure ofBombyx mori silk fibroin with15N- and13C-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.

Nuclear magnetic resonance study on the relationship between the helicity and L‐alanine content of L‐alanine/β‐alanine random copolypeptides in connection with silk fibroin conformation

AbstractThe helicities of six kinds of L‐alanine/β‐alanine random copolypeptides having different L‐alanine contents were determined using 1H NMR. Next, the theoretical helicities of these copolypeptides were calculated on the basis of a statistical thermodynamic analysis of the helixcoil transition of the random copolypeptide — using Monte Carlo methods — as a function of the L‐alanine content. The statistical weight parameters used in the model were determined from a comparison of the observed and calculated results. The experimental and theoretical results are in agreement with the fact that Bombyx mori silk fibroin with 30,0 mol‐% L‐alanine content assumes exclusively a random‐coil conformation and that Philosamia cynthia ricini silk fibroin with 48,8 mol‐% L‐alanine content locally assumes an α‐helical conformation in the sequence of the L‐alanine residues, both in aqueous solution.