title: Short polyglutamine peptide forms a high-affinity binding site for thioflavin-T at the N-terminus.
abstract: 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.
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan. matsuokas11@chem.sci.osaka-u.ac.jp
authors.forename: Shigeru
authors.initials: S
authors.lastname: Matsuoka
authors.forename: Motoki
authors.initials: M
authors.lastname: Murai
authors.forename: Toshio
authors.initials: T
authors.lastname: Yamazaki
authors.forename: Masayuki
authors.initials: M
authors.lastname: Inoue
mesh_terms: D020816:Amino Acid Motifs; D052160:Benzothiazoles; D001665:Binding Sites; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D055503:Protein Multimerization; D013844:Thiazoles / Q000378:metabolism*
chemical_list: D052160:Benzothiazoles; D010455:Peptides; D013844:Thiazoles; C009462:thioflavin T; C097188:polyglutamine