LAUSR

Thioflavin T (ThT) is a popular fluorescent dye for detecting amyloid, a protein aggregate with a β-sheet-rich structure that causes many neurodegenerative diseases. Despite the dye's popularity, a detailed understanding of its molecular binding mechanism remains elusive. We previously reported a protein model that can bind ThT on a single-layer β-sheet and revealed that a channel formed by aromatic rings with a confined length enhanced ThT binding. One of the mutants of the model system, 5-YY/LL, showed the highest affinity with a low μM dissociation constant. Here, we investigate the residue-specific binding mechanism of ThT to 5-YY/LL. We introduced tyrosine-to-phenylalanine and tyrosine-to-histidine mutations at the channel. The mutants revealed that the fifth position of tyrosine (Y5) is important for binding of ThT. Positive charges introduced by histidine at a low pH condition at the channel repel the binding of cationic ThT. Furthermore, we found a positive-to-negative conversion in the vicinity of the binding channel increases ThT fluorescence four-fold. A detailed understanding of the ThT binding mechanism will enhance our ability to develop amyloid-specific small molecules.