Silk Fiber Assembly Studied by Synchrotron Radiation SAXS/WAXS and Raman Spectroscopy

Abstract

We have characterized the steps involved in silk assembly from the protein solution into beta-type fibers by a combination of small-angle and wide-angle X-ray scattering and Raman spectroscopy. The aggregation process was studied in a concentric flow microfluidic cell, which allows mimicking the spinning duct. The fibroin molecule in solution shows an elongated shape with a maximum diameter of 38 nm. During the pH-driven initial assembly step, large-scale aggregates of fibroin molecules with a maximum diameter of about 260 nm are formed. Raman spectroscopy on the dried, fibrous material shows a principally alpha-helical silk I secondary structure, which is transformed gradually into beta-type silk II by increasing immersion times in water. The formation of crystalline beta-sheet domains within the fiber is confirmed by wide-angle X-ray scattering. The assembly process resembles the peptide condensation-ordering model proposed for amyloid cross-beta formation.

Self-Assembly Mechanism of pH-Responsive Glycolipids: Micelles, Fibers, Vesicles, and Bilayers

“…Similarities are found in the formation of twisted/ helical ribbons from gemini surfactant systems, which form platelets in the presence of a racemate mixture of D-, L-tartrate counterions but form ribbons in the enantiomeric excess of one or the other. 13 More generally, recent studies on the fibrillation mechanism of silk fibers (using SAXS/WAXS/Raman), 69 peptide nanotubes (using SAXS, cryo electron tomograghy, and cryo TEM), 16 and amyloid fibers (Monte Carlo simulation) 70 all seem to exclude the nucleation process from micelles but rather converge toward an aggregation/conversion (for silk and amyloid) or oriented attachment (peptide nanotubes) set of mechanisms. We conclude then that micelles, present at basic pH, serve as a reservoir of matter but not as nucleation points.…”

Structure of silk by raman spectromicroscopy: From the spinning glands to the fibers

“…68 The Fermi resonance doublet of the tyrosyl ring at 830/850 cm 21 has often been used to assess the strength of hydrogen bonding of the hydroxyl group of Tyr residues, its ionization state and the hydrophobic/hydrophilic character of its environment. 17,65,69,70 To get insights into the spinning process, the effect of pH 71 on the fibroin folding was also investigated by combining Raman and NMR spectroscopy. As the pH decreases from 6.8 to 4.8, a structural transition has been observed from the silk I conformation to the silk II conformation (the b-sheet form).…”

“…70 In an attempt to reproduce the flow in the gland canal, the aggregation process was studied in a concentric flow microfluidic cell that mimics the geometry of the silkworm spinning duct and the acidification occurring in natural assembly, using small-angle and wide-angle X-ray scattering in conjunction with Raman spectroscopy. 69 Changes in the intensity ratio of the bands at 1083 and 1003 cm 21 were interpreted as a conversion of a a-helical/silk I-rich structure into a silk II-rich structure.…”

Structural Transition of Bombyx mori Liquid Silk Studied with Vibrational Circular Dichroism Spectroscopy

“…4 Thus, much attention has been focused on the mechanism of the structural transition from liquid silk to silk fibers not only to understand the structural origin of the expression of their excellent mechanical properties and biocompatibility but also to develop materials that are more advanced than silk fibers. [5][6][7][8][9][10][11] For the spectroscopic study of the mechanism, silk fibroin concentrated aqueous solutions that are formed by the lysis of silk fibers in LiBr or CaCl2/C2H5OH solutions at high temperatures, called regenerated silk fibroin solutions, have been used. [6][7][8][9]11 By electronic circular dichroism (ECD) and infrared (IR) absorption measurements and X-ray scattering and NMR ones under shear, it was observed that regenerated silk fibroin solutions adopted a random-coil structure and they were converted into crystalline fibers with a Silk II structure, which mainly comprised a β-sheet structure.…”

“…[5][6][7][8][9][10][11] For the spectroscopic study of the mechanism, silk fibroin concentrated aqueous solutions that are formed by the lysis of silk fibers in LiBr or CaCl2/C2H5OH solutions at high temperatures, called regenerated silk fibroin solutions, have been used. [6][7][8][9]11 By electronic circular dichroism (ECD) and infrared (IR) absorption measurements and X-ray scattering and NMR ones under shear, it was observed that regenerated silk fibroin solutions adopted a random-coil structure and they were converted into crystalline fibers with a Silk II structure, which mainly comprised a β-sheet structure. [6][7][8]11 However, in the studies on the transition to silk fibers, only the regenerated silk fibroin solutions have been used in the initial state of the transition.…”