Conformational Transition Of Silk Fibroin Research Articles

Regenerated Silk Fibroin Nanofibers: Water Vapor‐Induced Structural Changes and Their Effects on the Behavior of Normal Human Cells

Nanofibrous non-woven matrices were prepared by electrospinning a regenerated silk fibroin (SF) solution, and the structural changes of SF nanofibers treated with water vapor were investigated using time-resolved IR and (13)C CP/MAS NMR spectroscopy. Conformational transitions of SF from random coil to beta-sheet structures were induced by water vapor treatment and were strongly dependent on the treatment time and temperature. Water vapor treatment provided a useful means of stabilizing the SF nanofiber matrices, resulting in the formation of matrices with a decreased solubility in water and increased mechanical strength. The adhesion and spreading of both normal human keratinocytes and fibroblasts onto the SF nanofiber matrices were also investigated, and the water vapor-treated SF nanofiber matrices showed good cellular compatibility, in comparison with traditional methanol-treated ones. This approach to controlling the conformational changes of SF nanofibers by water vapor treatment may be useful in the design and tailoring of novel materials for biomedical applications, including wound dressings and scaffolds for tissue engineering.

X‐ray photoelectron spectroscopic and Raman analysis of silk fibroin–Cu(II) films

There is evidence to suggest that Cu(II) is involved in the natural spinning process of a silkworm helping to convert the concentrated silk fibroin (SF) solution (or dope) into tough insoluble threads. To investigate the interaction between SF and Cu(II), a series of regenerated SF (RSF) films with different mass ratios of Cu(II) to SF were prepared. X-ray photoelectron spectroscopy (XPS) was employed to study the chemical interaction between Cu(II) and SF in these Cu(II)-RSF films. A significant change in the binding energy of Cu 2p(3/2) demonstrated that the chemical state of Cu(II) in the Cu(II)-RSF films was affected by the interaction between Cu(II) and SF. Moreover, chemical shifts of N 1s and O 1s of SF were also detected, implying that Cu(II) may coordinate with both N and O atoms in the SF. In addition, Raman spectra of the same series of Cu(II)-RSF films recorded the conformation transition of SF that may occur by the coordination of Cu(II) and SF macromolecular chains.

Effect of Metallic Ions on Silk Formation in the Mulberry Silkworm, Bombyx mori

A protein conformation transition from random coil and/or helical conformation to beta-sheet is known to be central to the process used by silk-spinning spiders and insects to convert concentrated protein solutions to tough insoluble threads. Several factors including pH, metallic ions, shear force, and/or elongational flow can initiate this transition in both spiders and silkworms. Here, we report the use of proton induced X-ray emission (PIXE), inductively coupled plasma mass spectroscopy (ICP-MS) and atomic adsorption spectroscopy (AAS) to investigate the concentrations of six metal elements (Na, K, Mg, Ca, Cu, and Zn) at different stages in the silk secretory pathway in the Bombyx mori silkworm. We also report the use of Raman spectra to monitor the effects of these six metallic ions on the conformation transition of natural silk fibroin dope and concentrated regenerated silk fibroin solution at concentrations similar to the natural dope. The results showed that the metal element contents increased from the posterior part to the anterior part of silk gland with the exception of Ca which decreased significantly in the anterior part. We show that these changes in composition can be correlated with (i) the ability of Mg2+, Cu2+, and Zn2+ to induce the conformation transition of silk fibroin to beta-sheet, (ii) the effect of Ca2+ in forming a stable protein network (gel), and (iii) the ability of Na+ and K+ to break down the protein network.

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.

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.

Structural changes and their effect on mechanical properties of silk fibroin/chitosan blends

Silk fibroin/chitosan blend films were prepared by the solvent casting method. Miscibility between silk fibroin and chitosan was examined by dynamic mechanical thermal analysis. Structural changes of silk fibroin by the addition of chitosan were investigated by IR spectroscopy. The conformational transition of silk fibroin from random coil form to β-sheet structure induced by blending with chitosan resulted in the increase of crystallinity and density of the blend films. The blend film containing 30 wt % chitosan exhibited a maximum increase in crystallinity and density. It was found that the tensile strength and initial tensile modulus of blend films were greatly enhanced with increasing the chitosan content and showed a maximum value at the composition of 30 wt % chitosan. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 74: 2571–2575, 1999

Conformation transition of silk fibroin induced by blending chitosan

The conformation of silk fibroin in silk fibroin/chitosan (SF/CS) blend membrane was analyzed by infrared spectrum, X-ray diffractometry, and Raman spec- trum. The results demonstrated that the SF could show b-sheet conformation when the SF content in blend membranes was 10% (w/w) and 60-80% ( w/w), while the pure SF membrane showed random coil conformation. A mechanism of the conformation transition was suggested in that the SF chain could use the rigid CS chain as a mold plate to stretch itself to form a b-sheet structure according to the strong hydrogen bond between CS and SF. Therefore, a new concept, named ''Polymer-Induced Conformation Transition,'' was proposed. q 1997 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 35: 2293-2296, 1997

Preparation and characterization of silk fibroin powder and its application to enzyme immobilization

AbstractThe powders of Bombyx mori silk fibroin were prepared from aqueous solutions of silk fibroin by several kinds of the insolubilization methods, some of which used methanol. The insolubilization appeared to be effected by the conformational transition of silk fibroin from the random coil to antiparallel β‐sheet form, which was monitored with IR spectroscopy. In order to characterize the structure of the power in the swollen state in water, a spin‐labeled silk fibroin powder was prepared and the ESR spectra were observed, as well as 13C‐NMR. The heterogeneous structure was analyzed quantitatively in terms of the fraction of fast, slow, and very slow components in the ESR spectra. Finally, an enzyme, invertase, was immobilized with the silk fibroin powder. The thermal stability of the enzyme was much improved by the immobilization.