Multimodal characterization of temperature-induced structural remodeling and aggregation of regenerated silk fibroin.

Regenerated silk fibroin (RSF) is a protein biomaterial whose molecular conformation, aggregation behavior, and functional properties are strongly influenced by processing conditions. The current study investigated the structural response of RSF to thermal treatment following incubation at 50-100 °C for 3 h. Intrinsic fluorescence increased progressively with treatment temperature without an appreciable displacement of the emission maximum, while fluorescence lifetime measurements at selected conditions indicated altered excited-state behavior of the intrinsic fluorophore population. Increased Thioflavin T and Congo Red responses were accompanied by enhanced ANS fluorescence, indicating increased accessibility of ordered dye-binding structures and hydrophobic sites. Secondary-structure analyses revealed thermal redistribution of conformational components. FTIR showed non-monotonic changes in β-sheet, α-helix, β-turn, and random-coil contributions, whereas circular dichroism demonstrated retention of the β-sheet-rich structural framework with a modest increase in estimated antiparallel β-sheet content. Quantitative X-ray powder diffraction analysis showed an increase in relative crystallinity from 45.41% in untreated RSF to 52.33% and 70.85% after treatment at 50 and 100 °C, respectively, with a corresponding decrease in the amorphous fraction. Dynamic light scattering revealed an increase in hydrodynamic size and polydispersity, while microscopic analyses showed the formation of larger, more interconnected, and morphologically heterogeneous assemblies at elevated temperatures. Surface roughness also increased most prominently in the 100 °C-treated sample. These findings demonstrate that thermal treatment promotes coupled changes in the local molecular environment, hydrophobic exposure, secondary-structure distribution, molecular ordering, and higher-order assembly of RSF, providing an integrated description of its thermally induced structural evolution.