Conformation transition kinetics of Bombyx mori silk protein

Time-resolved FTIR analysis was used to monitor the conformation transition induced by treating regenerated Bombyx mori silk fibroin films and solutions with different concentrations of ethanol. The resulting curves showing the kinetics of the transition for both films and fibroin solutions were influenced by the ethanol concentration. In addition, for silk fibroin solutions the protein concentration also had an effect on the kinetics. At low ethanol concentrations (for example, less than 40% v/v in the case of film), films and fibroin solutions showed a phase in which beta-sheets slowly formed at a rate dependent on the ethanol concentration. Reducing the concentration of the fibroin in solutions also slowed the formation of beta-sheets. These observations suggest that this phase represents a nucleation step. Such a nucleation phase was not seen in the conformation transition at ethanol concentrations > 40% in films or > 50% in silk fibroin solutions. Our results indicate that the ethanol-induced conformation transition of silk fibroin in films and solutions is a three-phase process. The first phase is the initiation of beta-sheet structure (nucleation), the second is a fast phase of beta-sheet growth while the third phase represents a slow perfection of previously formed beta-sheet structure. The nucleation step can be very fast or relatively slow, depending on factors that influence protein chain mobility and intermolecular hydrogen bond formation. The findings give support to the previous evidence that natural silk spinning in silkworms is nucleation-dependent, and that silkworms (like spiders) use concentrated silk protein solutions, and careful control of the pH value and metallic ion content of the processing environment to speed up the nucleation step to produce a rapid conformation transition to convert the water soluble spinning dope to a tough solid silk fiber.     

X-ray structural study of noncrystalline regenerated Bombyx mori silk fibroin

X-ray diffraction measurements of regenerated Bombyx mori silk fibroin were carried out to determine its structural characteristic from an analysis of differential radial distribution functions (DRDFs). The temperature dependence of X-ray diffraction patterns from noncrystalline and crystal structures of regenerated silk fibroin was investigated using a high temperature furnace. Time resolved X-ray diffraction profiles were also obtained to construct kinematical models of structural changes caused by the addition of water. DRDFs, calculated from the experimental data, were compared with the DRDFs simulated on the basis of the Monte Carlo method. In order to model the noncrystalline structures, structural units were assumed to be parts of the crystalline structure of silk and those with appropriate structural defects reported previously. From the comparison of experimental and simulated DRDFs, it was determined that noncrystalline regenerated silk consisted of locally ordered atomic sheets similar to the atomic arrangement in the silk I crystal (Type-I sheets), and the final state of the structural change was noncrystalline, consisting of small crystallites, the structure of which is similar to that of silk II (Type-II crystallites). Time resolved DRDFs were also qualitatively interpreted by both the ordering of Type-I sheets and structural changes from Type-I to Type-II. The formation of the small Type-II crystallites obtained in this study was consistent with the nucleation of silk II by birefringence measurements of silk glands and the spinneret of Bombyx mori silkworm reported previously. X-ray diffraction should be a useful technique to understand the structural characteristics of noncrystalline organic materials.     

Optical spectroscopy to investigate the structure of regenerated Bombyx mori silk fibroin in solution

Fluorescence and circular dichroism spectroscopy were used to monitor the conformational transition of regenerated Bombyx mori silk fibroin (RSF) in aqueous solutions under different conditions. According to the analysis of fluorescence spectra using anilinonaphthalene-8-sulfonic acid magnesium salt (ANS) as an external probe, the destruction of the hydrophobic core prior to the secondary structure change suggests that this collapse may initiate the conformational transition from random coil to beta-sheet for RSF. The temperature dependence of the structural changes of RSF, detected by both fluorescence spectroscopy and circular dichroism, shows a reversible process upon heating and recooling, with the midpoint around 45 degrees C. The results also indicate that most of the tryptophan (Trp) residues contained in silk fibroin are concentrated on the surface of the unfolded protein. However, they will change their location in the highly ordered structure (e.g., becoming more homogeneous) with the conformational transition of silk fibroin. Moreover, our studies also suggest that the presence of water plays a crucial role during the structure changes of fibroin.     

Effect of shearing on formation of silk fibers from regenerated Bombyx mori silk fibroin aqueous solution

In this paper, the spinnable regenerated silk fibroin aqueous solution with high concentration was prepared and the regenerated silk fibers were obtained from the aqueous solution by two different spinning processes at ambient temperature. The orientation of these fibers was characterized by polarizing microscope. Their secondary structure was investigated by Raman spectroscopy and related mechanical properties were also measured. These data showed that shearing is an important step for increasing orientation and silk II (β-sheet) structure, and the mechanical properties of the regenerated silk fibers can also be improved by shearing.     

Materials fabrication from Bombyx mori silk fibroin

Silk fibroin, derived from Bombyx mori cocoons, is a widely used and studied protein polymer for biomaterial applications. Silk fibroin has remarkable mechanical properties when formed into different materials, demonstrates biocompatibility, has controllable degradation rates from hours to years and can be chemically modified to alter surface properties or to immobilize growth factors. A variety of aqueous or organic solvent-processing methods can be used to generate silk biomaterials for a range of applications. In this protocol, we include methods to extract silk from B. mori cocoons to fabricate hydrogels, tubes, sponges, composites, fibers, microspheres and thin films. These materials can be used directly as biomaterials for implants, as scaffolding in tissue engineering and in vitro disease models, as well as for drug delivery.     

The genes for silk fibroin in Bombyx mori

The genes for the protein silk fibroin were quantitated by hybridizaton of purified fibroin messenger RNA with the DNA from several tissues of the silk-worm Bombyx mori.     

Studies on silk fibroin of the silkworm Bombyx mori

A procedure has been developed to obtain native fibroin in a pure state from the reservoir part of the silk gland. The purified protein has a sedimentation coefficient of 10 S as determined on sucrose density gradients and the amino acid composition is similar to that reported for fibroin from the cocoons. The effects of various solvents has been studied; lithium thiocyanate was found to be the solvent of choice. By in vivo labeling of fibroin with [³H]glycine and [¹⁴C]alanine it was demonstrated that fibroin synthesized in the posterior part of the gland and that stored in the reservoir part are identical.     

Wet spinning of Bombyx mori silk fibroin dissolved in N-methyl morpholine N-oxide and properties of regenerated fibres

Silk fibroin (SF) was dissolved in N-methyl morpholine N-oxide (NMMO) at a polymer concentration of 13% (w/w); thermal and rheological solution properties were characterized. The melting/crystallization behaviour of NMMO was influenced by SF presence. Melting of NMMO hydrate decreased to 71 degrees C and a cold crystallization peak appeared at 35 degrees C on heating. None crystallization occurred on cooling. Quenching at a temperature of 50 degrees C or higher did not induce any crystallization on heating. Viscosity of SF-NMMO solutions decreased as a function of temperature. At 75 degrees C, viscosity remained constant for 360 min. SF-NMMO dope was spun by using a lab-scale wet spinning line. The extruded filament was coagulated in an ethanol bath. Regenerated SF fibres were collected at different draw ratios and their morphological, physical, and mechanical properties were characterized. Fibre diameters ranged from 133 to 19mum, cross-section was regularly circular, and surface was generally smooth, with a very fine granular aspect. Birefringence increased with increasing the draw ratio, especially when take up and post-spinning draw were coupled. FT-IR spectra and DSC thermograms confirmed that SF fibres crystallized into Silk II structure. The IR crystallinity index did not change as a function of drawing. Regenerated SF fibres undrawn or drawn only during the coagulation step showed the mechanical behaviour typical of a brittle material. However, when both take up and post-spinning draw were applied, fibres displayed a ductile-stable behaviour. Typical values of the mechanical parameters of regenerated SF fibres were: E=8.7 GPa, sigma(b)=120 MPa and epsilon(b)=35%.     

Fractionation of the chymotryptic precipitate of Bombyx mori silk fibroin

1. A solution of Bombyx mori silk fibroin was digested with chymotrypsin. Amino acid analyses of the chymotryptic precipitate showed in addition to the main constituents Gly, Ala, Ser and Tyr, very small amounts of Lys, His, Arg, Asp, Thr, Glu, Pro, Cys, Val, Met, Ile, Leu, Phe and Trp. 2. A stable solution of the chymotryptic precipitate in 6m-urea was obtained by dialysing a solution in 50% (w/v) lithium thiocyanate against 6m-urea. 3. The dinitrophenylated chymotryptic precipitate in 6m-urea was fractionated by gel filtration and by ion-exchange chromatography. On Dowex 1 (X2), a main fraction I_d and three further fractions with different amino acid compositions and molecular weights were obtained. 4. Specific rearrangement and fission of the bonds involving the serine nitrogen atoms of fraction I_d and fractionation of the resulting mixture by gel filtration yielded five fractions. Two of these fractions had the compositions DNP-Ser-(Gly₆,Ala₄,Ser) and DNP-Ser-(Gly₄,Ala₂ or Ala₃,Ser) and are presumably double repeating units according to the proposed formula of Lucas, Shaw & Smith (1957), namely [Ser-Gly-(Ala-Gly)_n]₂, for n values of 2 and 1 respectively.     

Conformation transition in silk protein films monitored by time-resolved Fourier transform infrared spectroscopy: effect of potassium ions on Nephila spidroin films

We used time-resolved Fourier transform infrared spectroscopy (FTIR) to follow a conformation transition in Nephila spidroin film from random coil and/or helical structures to beta-sheet induced by the addition of KCl from 0.01 to 1.0 mol/L in D(2)O. Time series difference spectra showed parallel increases in absorption at 1620 and 1691 cm(-)(1), indicating formation of beta-sheet, together with a coincident loss of intensity of approximately 1650 cm(-)(1), indicating decrease of random coil and/or helical structures. Increase in KCl concentration produced an increased rate of the conformation transition that may attributable to weakening of hydrogen bonds within spidroin macromolecules. The conformation transition was a biphasic process with [KCl] > or = 0.3 mol/L but monophasic with [KCl] < or = 0.1 mol/L. This suggests that, at high KCl concentrations, segments of the molecular chain are adjusted first and then the whole molecule undergoes rearrangement. We discuss the possible significance of these findings to an understanding of the way that spiders spin silk.     

Ultrastructure of fibroin in the silk gland of larval Bombyx mori

The fibroin molecules stored in Golgi vacuoles in the posterior silk gland cells of 72-h-old, fifth instar larvae of Bombyx mori L. were observed electron-microscopically. The fibers which float in the Golgi vacuoles often have their ends attached to the limiting membrane. The fibers are helical bundles about 130 Å in diameter composed of 5–7 threads, each 20–30 Å thick.     

Localization of the genes for silk fibroin in silk gland cells of Bombyx mori.

Radioactive iodinated silk fibroin messenger RNA and ribosomal RNA have been used as probes to localize their genes in tissue sections of Bombyx mori by in situ hybridization. From filter hybridization experiments it is inferred that the majority of the grains produced by in situ hybridization with fibroin mRNA represents specific hybridization to fibroin genes. Sections of the posterior silk gland where silk is synthesized have been compared with those of the middle gland which does not synthesize fibroin. Glands have been analyzed from the second through the fifth (last) larval instar during feeding and moulting periods. During later stages when the gland cells increase their DNA content by polyploidization, serial sections were required to follow the distribution of grains through entire nuclei. At all stages, both ribosomal DNA and fibroin genes are distributed randomly throughout the nuclei without a preferred relationship to any nuclear structure.     

In vitro production of Bombyx mori silk fibroin by organ culture of the posterior silk glands; isotope labeling and fluorination of the silk fibroin

An in vitro silk fibroin production system has been developed by culture of posterior silk glands from Bombyx mori. A large amount of the silk fibroin was produced continuously and effectively with a rotation culture procedure. Modified Grace's insect medium was used, and oxygen bubbling in the medium was performed. In addition, half of the medium was replaced with fresh medium every 6 h. The production yield of silk fibroin produced after 100 h culture was 81 mg/g wet weight of posterior silk gland. This culture system was used successfully for efficient (15)N isotope labeling of silk fibroin, which is required for (15)N solid state nuclear magnetic resonance (NMR) analysis of silk fibroin. Moreover, the introduction of fluorinated amino acids into silk fibroin was also carried out using this culture system. (c) 1993 John Wiley & Sons, Inc.     

The chemical structure and the crystalline structures of Bombyx mori silk fibroin.

Some recent data (i.e. published in the last ten years) on the chemical and crystalline structures of B. mori silk are reviewed. The main emphasis is put on the crystallizable portion of silk fibroin, including its chemical constitution and its molecular conformation (at the crystallographic unit-cell level) in the two crystalline modifications : the beta pleated sheet and the silk I structures. The structural aspects are based on a discussion of X-ray and electron diffraction data, and on conformational energy analyses of a model (Ala-Gly)n polypeptide of silk fibroin.     

Biomaterial films of Bombyx mori silk fibroin with poly(ethylene oxide)

Phase separation into controllable patterned microstructures was observed for Bombyx mori silkworm silk and poly(ethylene oxide) (PEO) (900000 g/mol) blends cast from solution. The evolution of the microstructures with increasing PEO volume fraction is strikingly similar to the progression of phases and microstructures observed with surfactants. The chemically patterned materials obtained provide engineerable biomaterial surfaces with predictable microscale features which can be used to create topographically patterned or chemically functionalized biomaterials. Solution blending was used to incorporate water-soluble PEO into silk to enhance elasticity and hydrophilicity. The sizes of the globule fibroin phase ranged from 2.1 +/- 0.5 to 18.2 +/- 2.1 microm depending on the ratio of silk/PEO. Optical microscopy and SEM analysis confirmed the micro-phase separation between PEO and silk. Surface properties were determined by XPS and contact angle. Methanol can be used to control the conformational transition of silk fibroin to the insoluble beta-sheet state. Subsequentially, the PEO can be easily extracted from the films with water to generate silk matrixes with definable porosity and enhanced surface roughness. These blend films formed from two biocompatible polymers provide potential new biomaterials for tissue engineering scaffolds.     

Isolation and identification of the messenger RNA for silk fibroin from Bombyx mori

The messenger RNA for the protein silk fibroin has been isolated from the posterior silk gland of Bombyx mori and identified by partial sequence analysis. The sequence of mRNA could be predicted because the protein has a simple repetitious primary structure in which glycine residues comprise 45% of all residues and alternate predominantly with alanine and serine.     

The biosynthesis of fibroin. I. The intracellular form of silk fibroin of Bombyx mori L

After in vivo labeling with [³H]glycine the synthesis and transport of fibroin has been studied by radioautography and cell fractionation.Radioactivity appearing in the cytoplasm is rapidly transferred to the lumen where it accumulates in the so-called silk layer before reaching the central core of secreted fibroin. By sucrose density gradients it was demonstrated that the radioactivity appears immediately in the fibroin fraction, no precursors being observed.A simple fractionation procedure, based on the utilization of detergent, gives three fractions tentatively interpreted as synthesis, transport, and accumulation compartments in accordance with their kinetics of labeling.