Effects of pH and Calcium Ions on the Conformational Transitions in Silk Fibroin Using 2D Raman Correlation Spectroscopy and 13C Solid-State NMR

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Abstract
Silk fibroin exists in a number of different states, such as silk I and silk II, 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 Ca2+ 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 13C 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 β-sheet and distorted β-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.