Dynamic Protein−Water Relationships during β-Sheet Formation

We investigated the polymer−water interaction in a model fibrous protein. Bombyx mori silk fibroin film, a typical model of biodegradable material, was cast from aqueous solution and analyzed in this study. Differential scanning calorimetry (DSC), its temperature-modulated variant (TMDSC), and the time-resolved technique of Fourier transform infrared spectroscopy (FTIR) were used for the first time to monitor the detailed structural changes of silk fibroin during heating and during isothermal crystallization above the glass transition temperature, Tg ∼ 451 K (178 °C). Results show that intermolecular bound water molecules, acting as a plasticizer, will strongly affect the secondary structure of silk fibroin. DSC study shows that silk fibroin initially displays a water-induced glass transition around 80 °C during heating, resulting from a temporary bound water-silk structure. Quantitative thermal analysis of the heat capacity changes of this system during heating revealed that no β-sheet crystals were formed below Tg. FTIR scans also confirmed that no β-sheets were formed during heating and removal of bound water below Tg. During water removal, the amide II region shifts to lower frequency, and the 1515 cm−1 band increases slightly, indicating change of the microenvironment in the silk fibroin chains. During isothermal crystallization above Tg, the amide I region of silk fibroin spectra showed a phase transition from the secondary structures of noncrystalline random coils and α-helices to the β-pleated-sheet crystals, while the amide II region and the 1515 cm−1 band remain stable during β-sheet formation. Analogy is made between the crystallization of synthetic polymers according to the four-state scheme of Strobl and the crystallization process of silk fibroin, which we suggest proceeds through an intermediate precursor stage associated with water removal. This study provides a deeper understanding of the formation of β-pleated sheets during the crystallization process in silk fibroin, with implications for the crystallization of naturally occurring silk fibers from animals such as the silkworm and spider.