Binding Modes of Thioflavin T on the Surface of Amyloid Fibrils Studied by NMR

Abstract

The mechanism for the interaction of thioflavin T (ThT) with amyloid fibrils at the molecular level is not known. Here, we used (1) H NMR spectroscopy to determine the binding mode of ThT on the surface of fibrils from lysozyme and insulin. Relayed rotating-frame Overhauser enhancements in ThT were observed, indicating that the orientation of ThT is orthogonal to the fibril surface. Importantly, the assembly state of ThT on both surfaces is different. On the surface of insulin fibrils, ThT is oligomeric, as indicated by rapid (1) H spin-lattice relaxation rate in the rotating frame (R1ρ ), presumably due to intermolecular dipole-dipole interactions between ThT molecules. In contrast, ThT on the surface of lysozyme fibrils is a monomer, as indicated by slower (1) H R1ρ . These results shed new light into the mechanism for the enhancement of ThT fluorescence and may lead to more efficient detectors of amyloid assemblies, which have escaped detection by ThT in monomer form.

Modulation of the extent of structural heterogeneity in α-synuclein fibrils by the small molecule thioflavin T

“…The binding of ThT to fibrils can occur in different ways. ThT can bind perpendicular to the long fibril axis in the cavities formed by the side chains of aromatic/hydrophobic residues across consecutive ␤-strands on the surface of the ␤-sheet in the fibrils (51,(71)(72)(73). ThT is also known to bind in a parallel orientation to the peptide strand (74).…”

Formation of distinct prion protein amyloid fibrils under identical experimental conditions

“…A commonly used method for amyloid fibril detection is a ThT assay, in which the fluorescent dye molecules specifically bind to beta-sheet grooves on the fibril's surface, causing a red-shift in their excitation/emission spectra, as well as a large increase in fluorescence intensity 39 . ThT has been shown to have distinct binding capacity on different types of fibrils, most likely due to the structure and quantity of possible binding sites [40][41][42] . This specific affinity could potentially be used as a quick primary way of differentiating between samples that contain differently structured aggregates.…”

DNA intercalators as amyloid assembly modulators: mechanistic insights

“…Further, a sharp drop in corresponding ThT yields validated S1 as preferred site for intercalator-bril interactions that plausibly prevents bril growth. Recent work on Lys and insulin amyloids 63 showed bril surface as single preferred site for ThT binding, accounting for its enhanced uorescence upon association. Drawing parallels, ThT molecules would have been incapable of displacing the intercalators from their original binding site resulting in low quantum yields.…”