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 micromolar dissociation constant. Here, we investigate the residue-specific mechanism of binding of ThT to 5-YY/LL. We introduced tyrosine to phenylalanine and tyrosine to histidine mutations into the channel. The mutants revealed that the fifth position of tyrosine (Y5) is important for binding of ThT. Positive charges introduced by histidine under 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 4-fold. A detailed understanding of the ThT binding mechanism will enhance our ability to develop amyloid-specific small molecules.
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