Binding of thioflavin T by albumins: An underestimated role of protein oligomeric heterogeneity.

Amyloid fibrils formation is the well-known hallmark of various neurodegenerative diseases. Thioflavin T (ThT)-based fluorescence assays are widely used to detect and characterize fibrils, however, if performed in bioliquids, the analysis can be biased due to the presence of other, especially abundant, proteins. Particularly, it is known that albumin may bind ThT, although the binding mechanism remains debatable.

Thioflavin T fluoresces as excimer in highly concentrated aqueous solutions and as monomer being incorporated in amyloid fibrils.

Fluorescence of thioflavin T (ThT) is a proven tool for amyloid fibrils study. The correct model of ThT binding to fibrils is crucial to clarify amyloid fibrils structure and mechanism of their formation. Although there are convincing evidences that ThT has molecular rotor nature, implying it's binding to fibrils in monomer form, speculations concerning ThT binding to fibrils in aggregated forms appear in literature so far.

High Fluorescence Anisotropy of Thioflavin T in Aqueous Solution Resulting from Its Molecular Rotor Nature.

Thioflavin T (ThT) is widely used to study amyloid fibrils while its properties are still debated in the literature. By steady-state and femtosecond time-resolved fluorescence we showed that, unlike small sized rigid molecules, the fluorescence anisotropy value of the free ThT in aqueous solutions is very high, close to the limiting value. This is determined by the molecular rotor nature of ThT, where the direction of the ThT transition dipole moment S₀ → S₁* is not changed either by the internal rotation of the ThT benzothiazole and aminobenzene rings relative to each other in the excited state, because the axis of this rotation coincides with the direction of the transition dipole moment, or by the rotation of the ThT molecule as a whole, because the rate of this process is 3 orders of magnitude smaller than the rate of the internal rotation which leads to the fluorescence quenching.

Fluorescence quantum yield of thioflavin T in rigid isotropic solution and incorporated into the amyloid fibrils.

In this work, the fluorescence of thioflavin T (ThT) was studied in a wide range of viscosity and temperature. It was shown that ThT fluorescence quantum yield varies from 0.0001 in water at room temperature to 0.

Charge transfer process determines ultrafast excited state deactivation of thioflavin T in low-viscosity solvents.

Here we provide first direct experimental results about photoinduced TICT-state formation for Thioflavin T (ThT). In this work, femtosecond transient absorption spectra dynamics for ThT, dissolved in low-viscosity solvents (water, ethanol, 2-propanol, butanol) was investigated. It was found that decay lifetime of fluorescent LE-state for ThT in low-viscous solvents does not exceed 12 ps, and its value correlates well with rising time of the absorption band at 470 nm.

Thioflavin T as a molecular rotor: fluorescent properties of thioflavin T in solvents with different viscosity.

The effect of solvent viscosity on thioflavin T (ThT) fluorescent properties is analyzed to understand the molecular mechanisms of the characteristic increase in ThT fluorescence intensity accompanying its incorporation into the amyloid-like fibrils. To this end, the dependencies of the ThT quantum yield and fluorescence lifetime on temperature and glycerol content in the water-glycerol mixtures are studied. It has been found that fluorescent properties of ThT are typical for the specific class of fluorophores known as molecular rotors.

Computational study of thioflavin T torsional relaxation in the excited state.

Quantum-chemical calculations of the Thioflavin T (ThT) molecule in the ground S0 and first excited singlet S1 states were carried out. It has been established that ThT in the ground state has a noticeable nonplanar conformation: the torsion angle phi between the benzthiazole and the dimethylaminobenzene rings has been found to be approximately 37 degrees. The energy barriers of the intramolecular rotation appearing at phi = 0 and 90 degrees are quite low: semiempirical AM1 and PM3 methods predict values approximately 700 cm-1 and ab initio methods approximately 1000-2000 cm(-1).

Spectral properties of thioflavin T in solvents with different dielectric properties and in a fibril-incorporated form.

The increase in the solvent polarity induces a significant shift of the long-wavelength absorption band of the thioflavin T (ThT) to the shorter wavelengths. This is due to the fact that the positive charge of the ThT molecule (Z = +1e) is unequally and very differently distributed between the benzthiazole and aminobenzene rings in the ground and excited states. Therefore, ThT ground state is stabilized by the orientational interactions of the polar solvent dipoles with the positively charged ThT fragments, whereas the configuration of the solvation shell of the ThT molecule in the excited Franck-Condon state is likely far from being equilibrium.