Sequential Order of the Secondary Structure Transitions of Proteins under External Perturbations: Regenerated Silk Fibroin under Thermal Treatment.

Whether the process of protein folding/unfolding is fully cooperative or it contains sequential elements has long been a fundamental issue in protein science. This issue seemingly became straightforward since the appearance of generalized two-dimensional (2D) correlation spectroscopy in 1990s, because 2D correlation analysis has been considered as a convenient and powerful analytical tool to determine the sequential order of events under external physical or chemical perturbations. In this work, the sequential order of the secondary structure transitions of regenerated silk fibroin under thermal treatment from 130 to 220 °C was first studied using generalized 2D correlation spectroscopy, but an apparently doubtful sequential order was obtained; β-sheet was the first one to change at low temperature, then the random coil, followed by the nonamide C═O and, finally, the α-helix. A subsequent detailed in situ infrared spectral analysis showed that the main secondary structures of silk fibroin, including α-helix, β-turn, random coil and β-sheet (high-wavenumber component), all changed with a fully cooperative manner at a relatively low temperature of 150 °C. But the low-wavenumber component of β-sheet started to change at a higher temperature of 180 °C. Besides, it has also been found that, before 200 °C, the loss of α-helix and random coil was transformed into β-turn, β-sheet, and nonamide C═O. After 200 °C, some β-turn structure was also disruptured and transformed into β-sheet and nonamide C═O.