Synergic molecular engineering based chemical structural analysis of amyloid formation with Thioflavin T by probing mechanism

The emergence of amyloid and protein misfolding is closely associated with neurodegenerative diseases such as Parkinson's and Alzheimer's. In this study, we present a novel application of fluorescence resonance energy transfer (FRET) using Thioflavin T (THT) as an amyloid probe to monitor the amyloid conversion of hen egg white lysozyme (HEWL), a protein implicated in hereditary systemic amyloidosis. Under acidic conditions, at low HEWL concentrations (2.5 mg/mL) and elevated temperatures (65 °C), we observed a progressive increase in THT fluorescence emission, indicating amyloid β-sheet formation. Transmission electron microscopy and dynamic light scattering confirmed fibril formation with an approximate size of 100 nm. The zero-order kinetics of the unfolding process suggests that unfolding is the rate-limiting step in amyloid formation. Additionally, the photoluminescence lifespan of THT increased and reached saturation at 10 ns after eight hours of incubation, marking the completion of amyloid aggregation. Our study uniquely establishes the effectiveness of THT-based FRET in real-time monitoring of amyloid formation, offering new insights into protein misfolding mechanisms. These findings provide a valuable platform for in vitro amyloid research and hold potential for future in vivo applications, contributing to the broader understanding of neurodegenerative disease pathology.