Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jyonan, Yonezawa, Yamagata 992-8510, Japan.
Department of Chemistry, Graduate School of Science, Kyushu University, Fukuoka 812-8582, Japan.
Biochemistry. 2020 Aug 4;59(30):2782-2787. doi: 10.1021/acs.biochem.0c00280. Epub 2020 Jun 10.
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 (Y) 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.
硫黄素 T（ThT）是一种常用于检测淀粉样蛋白的荧光染料，淀粉样蛋白是一种富含β-折叠结构的蛋白质聚集体，会引起许多神经退行性疾病。尽管该染料很受欢迎，但人们对其分子结合机制仍知之甚少。我们之前曾报道过一种可以在单层β-折叠上结合 ThT 的蛋白质模型，并揭示了由具有一定限制长度的芳环形成的通道增强了 ThT 的结合。模型系统的一个突变体 5-YY/LL 与低微摩尔解离常数表现出最高的亲和力。在这里，我们研究了 ThT 与 5-YY/LL 结合的残基特异性机制。我们将通道中的苯丙氨酸突变为酪氨酸，将组氨酸突变为酪氨酸。这些突变体表明，酪氨酸（Y）的第五位对于 ThT 的结合很重要。在通道处的低 pH 条件下引入的组氨酸的正电荷排斥带正电荷的 ThT 的结合。此外，我们发现结合通道附近的正电荷到负电荷的转换将 ThT 荧光增加了 4 倍。对 ThT 结合机制的深入了解将提高我们开发淀粉样蛋白特异性小分子的能力。
