Redesign of a thioflavin-T-binding protein with a flat ß-sheet to evaluate a thioflavin-T-derived photocatalyst with enhanced affinity.

Amyloids, proteinous aggregates with ß-sheet-rich fibrils, are involved in several neurodegenerative diseases such as Alzheimer's disease; thus, their detection is critically important. The most common fluorescent dye for amyloid detection is thioflavin-T (ThT), which shows on/off fluorescence upon amyloid binding. We previously reported that an engineered globular protein with a flat ß-sheet, peptide self-assembly mimic (PSAM), can be used as an amyloid binding model. In this study, we further explored the residue-specific properties of ThT-binding to the flat ß-sheet by introducing systematic mutations. We found that site-specific mutations at the ThT-binding channel enhanced affinity. We also evaluated the binding of a ThT-based photocatalyst, which showed the photooxygenation activity on the amyloid fibril upon light radiation. Upon binding of the photocatalyst to the PSAM variant, singlet oxygen-generating activity was observed. The results of this study expand our understanding of the detailed binding mechanism of amyloid-specific molecules.

Residue-Specific Binding Mechanisms of Thioflavin T to a Surface of Flat ß-Sheets within a Peptide Self-Assembly Mimic.

Thioflavin T (ThT) is a popular fluorescent dye for detecting amyloid, a protein aggregate with a ß-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 ß-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.