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

Area of Science:

• Biochemistry
• Chemical Biology
• Structural Biology

Background:

• Thioflavin-T (ThT) is a crucial dye for detecting amyloid structures, widely used for over 50 years.
• The precise molecular mechanism underlying ThT's interaction with amyloid fibrils, particularly polyglutamine (polyQ) peptides, remains incompletely understood.

Purpose of the Study:

• To elucidate the molecular mechanism of Thioflavin-T (ThT) binding to short polyglutamine (polyQ) peptides.
• To identify the specific polyQ chain length required for ThT staining and characterize the binding sites.

Main Methods:

• Chemically synthesized polyglutamine peptides (Q(n), n=5-10) were used in ThT staining assays.
• Solid-state NMR techniques, specifically (13)C{(2)H}REDOR and (13)C{(2)H}DQF-REDOR, were employed to probe ThT-binding sites in Q(8) peptide aggregates.
• Isotopically labeled ThT and polyQ peptides were utilized to determine intermolecular contacts.

Main Results:

• The minimum polyglutamine peptide length for positive ThT staining was determined to be Q(6).
• Two distinct ThT-binding sites were identified in short polyQ peptides (n=6-9): a high-affinity site (Kd1 = 0.1-0.17 μM) and a low-affinity site (Kd2 = 5.7-7.4 μM).
• NMR experiments revealed that all five carbons of the glutamine residue are involved in ThT binding sites, with the N-terminal glutamine residue directly interacting with ThT at high-affinity sites.

Conclusions:

• The study provides critical insights into the molecular basis of ThT-amyloid interactions, specifically for polyglutamine peptides.
• The findings clarify the role of polyQ chain length and N-terminal interactions in ThT binding, advancing our understanding of amyloid detection methods.