Thioflavin T Interaction with Acetylcholinesterase: New Evidence of 1:1 Binding Stoichiometry Obtained with Samples Prepared by Equilibrium Microdialysis

Abstract

The aim of the present work was investigation of the fluorescent dye thioflavin T (ThT) binding to acetylcholinesterase (AChE). ThT is an effective test for protease activity, as well as a probe for amyloid fibril formation. Despite the extended and active investigation of ThT-AChE binding, there is still no common view on the stoichiometry of this interaction. In particular, there is a hypothesis explaining the spectral properties of bound to AChE dye and high quantum yield of its fluorescence by formation of dimers or excimers of ThT. In order to confirm or deny this hypothesis, we proposed a new experimental approach for examination of ThT-AChE interaction based on spectroscopic investigation of samples prepared by equilibrium microdialysis. This approach allowed us to prove 1/1 ThT/AChE binding stoichiometry. The increase of ThT fluorescence quantum yield and lifetime accompanying its binding to AChE can be explained by the molecular rotor nature of this dye. Together with the coincidence of the positions of free and AChE-bound ThT fluorescence spectra, the obtained results prove the groundlessness of the hypotheses about ThT aggregation while binding to AChE. The model of ThT localization in the active site of AChE was proposed by using molecular docking simulations. These results also allowed us to suggest the key role of aromatic residues in ThT-AChE interaction, as observed for some amyloid fibrils.

Mind Your Dye: The Amyloid Sensor Thioflavin T Interacts with Sulfated Glycosaminoglycans Used To Induce Cross-β-Sheet Motifs

“…ThT was originally considered as a β-sheet specific dye that does not interact with proteins in their native folded state. In recent times, however, this view has been considerably challenged due to the accumulation of experimental data on the binding of ThT to a non-β-sheet cleft of acetylcholinesterase, serum albumins and into the β-barrel of chicken α 1 -acid glycoprotein . Moreover, nucleic acid interactions of ThT have also been demonstrated as its intercalation between DNA base pairs and/or association with DNA grooves. − Chemically, ThT is the chloride salt of a quaternary nitrogen cation of an N -methylated benzothiazole fragment linked to an N , N -dimethylaniline ring via a single C–C bond, which allows the two rings to rotate relative to each other (Scheme ).…”

“…4 ThT was originally considered as a β-sheet specific dye that does not interact with proteins in their native folded state. In recent times, however, this view has been considerably challenged due to the accumulation of experimental data on the binding of ThT to a non-β-sheet cleft of acetylcholinesterase 5 , serum albumins 6 and into the β-barrel of chicken α 1 -acid glycoprotein. 7 Moreover, nucleic acid interactions of ThT have also been demonstrated as its intercalation between DNA base pairs and/or association with DNA grooves.…”

Effects of Modifying Thioflavin T at the N3-Position on Its G4 Binding and Fluorescence Emission

“…The excited state of free ThT (ϕ = 90 • ) does not emit fluorescence (quantum yield: Φ < 0.001) owing to the radiationless deactivation caused by non-radiative twisted internal charge transfer [14,15]. However, when ThT binds to a target, ϕ is restricted to 20 • -40 • in the excited state and fluorescence emission derived from the locally excited state can be observed (Φ = 0.3-0.8) [16]. Therefore, high contrast images with low background fluorescence can be obtained using ThT without removing excess probe molecules.…”

High-Contrast Facile Imaging with Target-Directing Fluorescent Molecular Rotors, the N3-Modified Thioflavin T Derivatives

“…The excited state of free ThT (φ = 90°) does not emit fluorescence (quantum yield Φ < 0.001) because of the radiation-less deactivation caused by nonradiative twisted internal charge transfer. Meanwhile, when ThT binds to targets and thereby φ is restricted to be approximately 20–40° at the excited state, fluorescence emission derived from the locally excited state can be observed (Φ = 0.3–0.8). − Therefore, high-contrast images having low background fluorescence can generally be obtained using ThT without removing excess probe molecules …”