Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.
Org Biomol Chem. 2012 Aug 14;10(30):5787-90. doi: 10.1039/c2ob07157f. Epub 2012 Mar 12.
Thioflavin-T is one of the most important amyloid specific dyes and has been used for more than 50 years; however, the molecular mechanism of staining is still not understood. Chemically synthesized short polyglutamine peptides (Q(n), n = 5-10) were subjected to the thioflavin-T (ThT) staining assay. It was found that the minimum Q(n) peptide that stained positive to ThT was Q(6). Two types of ThT-binding sites, a high-affinity site (k(d1) = 0.1-0.17 μM) and a low-affinity site (k(d2) = 5.7-7.4 μM), were observed in short polyQs (n = 6-9). (13)C{(2)H}REDOR NMR experiments were carried out to extract the local structure of ThT binding sites in Q(8) peptide aggregates by observing the intermolecular dipolar coupling between [3-Me-d(3)]ThT and natural abundance Q(8) or residue-specific [1,2-(13)C(2)] labeled Q(8)s. (13)C{(2)H}REDOR difference spectra of the [3-Me-d(3)]ThT/natural abundance Q(8) (1/9) complex indicated that all of the five carbons of the glutamine residue participated in the formation of ThT-binding sites. (13)C{(2)H}DQF-REDOR experiments of [3-Me-d(3)]ThT/residue-specific [1,2-(13)C(2)] labeled Q(8) (1/50) complexes demonstrated that the N-terminal glutamine residue had direct contact with the ThT molecule at the high-affinity ThT-binding sites.
硫黄素 T 是最重要的淀粉样蛋白特异性染料之一，已经使用了 50 多年；然而，其染色的分子机制仍不清楚。我们对化学合成的短聚谷氨酰胺肽（Q(n)，n=5-10）进行了硫黄素-T（ThT）染色试验。结果发现，能被 ThT 染色阳性的最短 Q(n)肽是 Q(6)。在短聚 Q（n=6-9）中观察到两种类型的 ThT 结合位点，高亲和力结合位点（k(d1)=0.1-0.17 μM）和低亲和力结合位点（k(d2)=5.7-7.4 μM）。（13）C{(2)H}REDOR NMR 实验通过观察[3-Me-d(3)]ThT 与天然丰度 Q(8)或残基特异性[1,2-(13)C(2)]标记 Q(8)之间的分子间偶极偶合，对 Q(8)肽聚集体中 ThT 结合位点的局部结构进行了提取。[3-Me-d(3)]ThT/天然丰度 Q(8)(1/9)复合物的（13）C{(2)H}REDOR 差谱表明，谷氨酰胺残基的所有五个碳原子都参与了 ThT 结合位点的形成。[3-Me-d(3)]ThT/残基特异性[1,2-(13)C(2)]标记 Q(8)(1/50)复合物的（13）C{(2)H}DQF-REDOR 实验表明，N 端谷氨酰胺残基在高亲和力 ThT 结合位点与 ThT 分子直接接触。
