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 0.28 in rigid isotropic solution (T/η→0). The deviation of the fluorescence quantum yield from unity in rigid isotropic solution suggests that fluorescence quantum yield depends not only on the ultra-fast oscillation of ThT fragments relative to each other in an excited state as was suggested earlier, but also depends on the molecular configuration in the ground state. This means that the fluorescence quantum yield of the dye incorporated into amyloid fibrils must depend on its conformation, which, in turn, depends on the ThT environment. Therefore, the fluorescence quantum yield of ThT incorporated into amyloid fibrils can differ from that in the rigid isotropic solution. In particular, the fluorescence quantum yield of ThT incorporated into insulin fibrils was determined to be 0.43. Consequently, the ThT fluorescence quantum yield could be used to characterize the peculiarities of the fibrillar structure, which opens some new possibilities in the ThT use for structural characterization of the amyloid fibrils

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. Consequently, ThT fluorescence anisotropy cannot be directly used to study amyloid fibrils formation, as it was proposed by some authors

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. It has been established that the low ThT fluorescence intensity in the solvents with low viscosity is caused by the nonradiative deactivation of the excited state associated with the torsional motion of the ThT benzthiazole and aminobenzene rings relative to each other, which results in the transition of ThT molecule to nonfluorescent twisted internal charge transfer (TICT) state. The rate of this process is determined by the solvent viscosity, whereas the emission does occur from the nonequilibrium locally excited (LE) state. High polarization degree of the ThT fluorescence (P = 0.45) observed for glycerol solutions of different viscosity confirms the nonequilibrium character of the emission from the LE state and testifies that rotational correlation time of the whole molecule is considerably greater than the time required to accomplish transition to the nonfluorescent TICT state. Torsional movements of the ThT fragments take place in the same temporal interval as solvent relaxation, which leads to nonexponential fluorescence decay of the dye in viscous solvents. This photophysical model successfully explains the fluorescent properties of ThT in solvents with different viscosities. The model is confirmed by the results of the quantum-chemical calculations, which showed that energy minimum for the ground state of ThT corresponds to conformation with torsional angle φ = 37° between the benzthiazole and aminobenzene rings and in the excited-state twisted conformation of ThT with φ = 90° has minimal energy. These data support the idea that the reason for the characteristic increase in the ThT fluorescence intensity accompanying its incorporation into the amyloid fibrils is determined by the rigidity of the dye environment, which prevents the rotation of the benzthiazole ring relative to the aminobenzene ring in the excited state

Aggregation of thioflavin T and its new derivative in the presence of anionic polyelectrolyte

Spectral properties of aqueous solutions of thioflavin T (ThT) and its new derivative, trans-2-[4-(dimethylamino) styryl]-3-ethyl-1,3-benzothiazolium perchlorate (DMASEBT) were studied in the presence of anionic polyelectrolyte sodium polystyrene sulfonate (PSS). It was shown that PSS promote the dye dimerization process. Quantum–chemical analysis of DMASEBT in monomer and dimer forms allowed to suggest that the dimers have a sandwich-like structure, i.e. H-aggregates can be formed. DMASEBT dimer formation in the presence of PSS leads to a 40 nm hypsochromic shift of the dye absorption spectrum and to quenching of its fluorescence. The PSS interaction with the monomeric dye leads to a 26 nm bathochromic shift of the absorption spectrum and to one order of magnitude increase in its fluorescence

Aggregation of thioflavin T and its new derivative in the presence of anionic polyelectrolyte

Spectral properties of aqueous solutions of thioflavin T (ThT) and its new derivative, trans-2-[4-(dimethylamino) styryl]-3-ethyl-1,3-benzothiazolium perchlorate (DMASEBT) were studied in the presence of anionic polyelectrolyte sodium polystyrene sulfonate (PSS). It was shown that PSS promote the dye dimerization process. Quantum–chemical analysis of DMASEBT in monomer and dimer forms allowed to suggest that the dimers have a sandwich-like structure, i.e. H-aggregates can be formed. DMASEBT dimer formation in the presence of PSS leads to a 40 nm hypsochromic shift of the dye absorption spectrum and to quenching of its fluorescence. The PSS interaction with the monomeric dye leads to a 26 nm bathochromic shift of the absorption spectrum and to one order of magnitude increase in its fluorescence

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. ThT absorption spectrum has the shortest (412 nm) and the longest (450 nm) wavelengths in water and in water being incorporated to the amyloid fibrils, respectively. Intriguingly, the position of the ThT fluorescence spectrum depends on the polarity of solvent to a significantly lesser degree than its absorption spectrum:  being excited at 440 nm, ThT has emission with maxima at 493 and 478 nm in water and fibrils, respectively. This can be due to the fact that, in the excited state, the rotational oscillations of the ThT fragments relative to each other prevent establishing equilibrium with the solvent and fluorescence occurs from the partially equilibrium excited stated to the partially equilibrium ground state. For the fibril-incorporated ThT, the maximum of the fluorescence excitation spectrum coincides with the maximum of the long wavelength absorption band (450 nm), whereas for ThT in aqueous and alcohol solutions, additional short-wavelength bands of fluorescence and fluorescence excitation spectra were described (Naiki et al. Anal. Biochem.1989, 177, 244−249; Le Vine Methods Enzymol.1999, 309, 274−284). These bands could result either from some fluorescent admixtures (including free benzthiazole and aminobenzene) or from the specific ThT conformers in which benzthiazole and aminobenzene rings, being oriented at φ angle close to 90 or 270°, serve as independent chromophores. On the basis of the results of the quantum-chemical calculations, it is proposed that at φ = 90° (270°), the relatively low barrier (only 700 cm-1) of the internal rotation of the benzthiazole and aminobenzene rings relative to each other gives rise to a subpopulation of ThT molecules possessing a violated system of the π-conjugated bonds of the benzthiazole and aminobenzene rings

Absorption spectra of thioflavin T in water-glycerol mixtures.

<p>Curves 1–5 correspond to 13, 35, 56, 83 and 99% wt glycerol content, respectively.</p

Comparison of activation energy of the solvent viscous flow (Δ<i>E_η</i>) with the activation energy of the non-radiative deactivation of excited state (Δ<i>E_q</i>) (determined by the temperature dependence of fluorescence quantum yield at high glycerol content in solution).

<p>Comparison of activation energy of the solvent viscous flow (Δ<i>E_η</i>) with the activation energy of the non-radiative deactivation of excited state (Δ<i>E_q</i>) (determined by the temperature dependence of fluorescence quantum yield at high glycerol content in solution).</p

Emission Quantum Yield, Excited-State Lifetime and Radiative Lifetime of Thioflavin T in Solutions with Different Glycerol Content at Different Temperatures.

a<p>the value was determined by extrapolation of the dependence given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015385#pone-0015385-g002" target="_blank">Figure 2</a>;</p>b<p>the value was obtained for thioflavin T in 99% glycerol at 77 K <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015385#pone.0015385-Stsiapura2" target="_blank">[31]</a>.</p>c<p>the value was evaluated as <i>τ</i>_r q, average <i>τ</i>_r was taken as 7.8 ns.</p

Model of thioflavin T molecule.

<p>Model of thioflavin T molecule.</p