Effects of pH and calcium ions on the conformational transitions in silk fibroin using 2D Raman correlation spectroscopy and 13C solid-state NMR

Silk fibroin exists in a number of different states, such as silk I and silk 11, with different properties largely defined by differences in secondary structure composition. Numerous attempts have been made to control the transitions from silk I to silk II in vitro to produce high-performance materials. Of all the factors influencing the structural compositions, pH and some metal ions play important roles. This paper focuses on the influence of pH and Call ions on the conformational transition from silk I to silk II in regenerated (redissolved) Bombyx mori fibroin. One- and two-dimensional correlation Raman spectroscopy was used to describe qualitatively the transitions in secondary structure in silk I, silk II, and their intermediates as pH and Ca2+ ion concentration were changed, while C-13 cross polarization magic angle spinning (CP/MAS) solid-state NMR was used to quantify these changes. We showed that conditions (low pH, pH 5.2; a defined range of Ca2+ ion concentrations; gradual water removal) that mimic natural silk spinning promote the formations of beta-sheet and distorted beta-sheet characteristic of silk II or silk II-related intermediate. In contrast, higher pH (pH 6.9-8.0) and higher Ca2+ ion concentrations maintain random coil conformations typical of silk I or silk I-related intermediate. These results help to explain why the natural silk spinning process is attended by a reduction in pH from 6.9 to 4.8 and a change in the Ca2+ ion concentration in the gland lumen as fibroin passes from the posterior division through the secretory pathway to the anterior division.