Short polyglutamine peptide forms a high-affinity binding site for thioflavin-T at the N-terminus

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 mu M) and a low-affinity site (k(d2) = 5.7-7.4 mu M), were observed in short polyQs (n = 6-9). C-13{H-2}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-C-13(2)] labeled Q(8)s. C-13{H-2} 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. C-13{H-2}DQF-REDOR experiments of [3-Me-d(3)]ThT/residue-specific [1,2-C-13(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.