Distance-Dependent Tryptophan-Induced Quenching of Thioflavin T Defines the Amyloid Core Architecture.

Thioflavin T (ThT) is widely employed as a fluorogenic marker for amyloid formation. ThT fluorescence is utilized to detect amyloid fibrils as well as to follow aggregation kinetics. Here, we make a unique case to demonstrate that site-specific tryptophan-induced fluorescence quenching of ThT bound to the α-synuclein amyloid can define the central amyloid core. We show that distance-dependent quenching of amyloid-bound ThT by site-specifically incorporated tryptophan maps the proximal and distal locations of the polypeptide chain within amyloid fibrils. Our studies indicate that tryptophan-induced fluorescence quenching is dominated by the static quenching mechanism. Our findings underscore the utility of site-specific amino acid-based quenching of ThT fluorescence to characterize the core architecture of amyloid derived from a wide range of proteins.