| Recently, more and more attention has been paid to natural silk, which is produced by a wide variety of insects, such as silkworms and spiders. Although silk impressive mechanical properties are closely related to the primary amino acid sequence, the silk spinning conditions, such as temperature, pH, ionic strength, solvent composition and mechanical stress are possibly more important. There are several reports concerning the effect of pH and metal ions on silk fibroin (SF) conformations. However, how the metallic ions and pH impact on the SF conformation transition, that is, what mechanism is involved in the conformation conversion, still remains unclear up to now.In this work, we studied the effect of Cu(II) ions and pH on the secondary structure of Bombyx mori regenerated SF by EPR, NMR, Raman and CD. Cu(II)-EPR spectroscopy was used to determine the coordination modes of Cu(II)-SF complexes and Raman and 13C solid-state NMR spectroscopy were used to determine qualitatively and quantitatively the SF conformations. The relationship between pH, coordination modes of Cu(II)-SF complexes and SF conformations is assessed here. Meanwhile, we investigated the effect of Cu(II) ions on the conformational transition of SF in diluted solution.many researches have proved that Cu(II) plays a vital role in the folding and refolding process in the cellular isoform of prion protein (PrP) and amyloid β-peptide(Aβ), which cause the fatal neurodegenerative disease (such as Creutzfeldt-jakob disease in humans and spongiform encephalopathy in animals). Therefore, our work will be significant to medical research, on the one hand, as SF, extracted in large amounts from Bombyx mori silkworm gland, could be a proper model to study the mechanisms and medicaments for the nervous system disease. On the other hand, our results will also serve as guidance for the controllable design of high performance materials as silk fibers.There are several points obtained in this work:Firstly, Cu(II)-EPR spectroscopy indicates that Cu(II) forms stable complexes with SF and the coordination atoms in SF are changed from four-nitrogen to two-nitrogen and two-oxygen as well as to one-nitrogen and three-oxygen when the pH is lowered from 8.0 to 4.0. Based on the similarity in the relevant sequence in SF (AHGGYSGY) to the octapeptide PHGGGWGQ in PrP, we suggest that at basic and neutral pHs polypeptide AHGGYSGY in SF may form a complex with Cu(II) by coordination of imidazole Nπ of His together with two deprotonated main-chain nitrogens from two glycine residues and one nitrogen or oxygen from serine. Under weakly acidic conditions, however, Cu(Il)-amide- linkages may be broken and Cu(II) switch to bind two Nτ from two histidines in adjacent peptide chains, forming an intermolecular His(Nτ)-Cu(II)-His(Nτ) bridge.Secondly, we investigate the effect of Cu(II) and pH on the secondary structure of SF by Raman and 13C solid-state NMR spectroscopy. The conformations in the fibroin membranes were determined qualitatively by Raman and Generalized Two-Dimensional Correlation Raman Spectroscopy (2D Raman) for Amide I region (1600-1700cm~-1) of SF. Based on the qualitative results extracted from 2D Raman spectrum, the relationship between Silk I and Silk II in the studied systems were determined quantitatively by simulation of alanine Cp peak (10-30 ppm) in 13C CP-MAS solid state NMR spectrum. We draw some conclusion as follows: (1) The weak acidic condition (pH-5.2) is more favorable than neutral (pH=6.9) and weak basic (pH= 8.0) ones for the structure transition from Silk I to Silk II; (2) The introduction of a small amount of Cu(II) leads to an increase in the content of Silk II...
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