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.

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. It indicates that LE-state of ThT initially formed upon photoexcitation is quite rapidly converted to a transient state characterized by absorption at 470 nm. We associate this emerging intermediate state with nonfluorescent TICT-state of the dye. Rate of LE --> TICT process significantly depends on viscosity and is comparable to the rate of solvent relaxation resulting in time-dependent Stokes shift of ThT stimulated emission band. TICT-state deactivation was found to be also viscosity dependent and its lifetime changed from 3.8 +/- 0.1 ps (in H(2)O) to 360 +/- 60 ps (in butanol). It was proposed that a nonradiative deactivation process proceeds through a conical intersection between TICT(S(1)') and S(0) energy levels. The results obtained confirm the earlier proposed model that twisted internal charge transfer process takes place in the excited state of the dye and that ThT behaves as a molecular rotor (Stsiapura, V. I.; Maskevich, A. A.; Kuzmitsky, V. A.; Uversky, V. N.; Kuznetsova, I. M.; Turoverov, K. K. J. Phys. Chem. B 2008, 112, 15893-15902).

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 phi = 37 degrees between the benzthiazole and aminobenzene rings and in the excited-state twisted conformation of ThT with phi = 90 degrees 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.

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. Here the role of low-order albumin oligomers in ThT binding is investigated using time-resolved fluorometry and size-exclusion chromatography. Under conditions used, the fraction of dimers in human (HSA) and bovine (BSA) serum albumin solutions is as low as ∼7%, however, it is responsible for ∼50% of ThT binding. For both albumins, the binding affinity was estimated to be ∼200 and ∼40µM for monomeric and dimeric species, respectively. Molecular docking suggested that ThT preferentially binds in the hydrophobic pocket of subdomain IB of albumin monomer in a similar position but with a variable torsion angle, resulting in a lower fluorescence enhancement (∼40-fold) compared to amyloid fibrils (∼1000-fold). Dimerization of albumin presumably creates an extra binding site at the subunit interface. These results demonstrate the underestimated role of low-order albumin oligomers that can be highly relevant when analyzing drugs binding using fluorescence spectroscopy.

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. The elaborated approach for fluorescence intensity correction on the inner filter effects applied to ThT aqueous solutions with a wide range of concentration allowed characterizing ThT excimers fluorescence and showing its difference from that of ThT bound to fibrils. Obtained results experimentally prove the monomer model of ThT binding to amyloid fibrils and demonstrate wide capacity of the used approach in the spectroscopy of other fluorescent dyes for examination of concentration self-quenching and deformation of fluorescence spectra, dye molecules interaction, dimers and excimers formation.

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.

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). The INDO/S calculations of vertical transitions to the S1(abs) excited state have revealed that energy ES1(abs) is minimal for the twisted conformation with phi = 90 degrees and that the intramolecular charge-transfer takes place upon the ThT fragments' rotation from phi = 0 to 90 degrees. Ab initio CIS/RHF calculations were performed to find optimal geometries in the excited S1 state for a series of conformers having fixed phi values. The CIS calculations have predicted a minimum of the S1 state energy at phi approximately 21 degrees; however, the energy values are 1.5 times overestimated in comparison to experimental data. Excited state energy dependence on the torsion angle phi, obtained by the INDO/S method, reveals that ES1(fluor) is minimal at phi = approximately 80-100 degrees, and a plateau is clearly observed for torsion angles ranging from 20 to 50 degrees. On the basis of the calculation results, the following scheme of photophysical processes in the excited S1 state of the ThT is suggested. According to the model, a twisted internal charge-transfer (TICT) process takes place for the ThT molecule in the excited singlet state, resulting in a transition from the fluorescent locally excited (LE) state to the nonfluorescent TICT state, accompanied by torsion angle phi growth from 37 to 90 degrees. The TICT process effectively competes with radiative transition from the LE state and is responsible for significant quenching of the ThT fluorescence in low-viscosity solvents. For viscous solvents or when the ThT molecule is located in a rather rigid microenvironment, for example, when it is bound to amyloid fibrils, internal rotation in the dye molecule is blocked due to steric hindrance, which results in suppression of the LE --> TICT quenching process and in a high quantum yield of 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 phi angle close to 90 or 270 degrees, serve as independent chromophores. On the basis of the results of the quantum-chemical calculations, it is proposed that at phi = 90 degrees (270 degrees), 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 pi-conjugated bonds of the benzthiazole and aminobenzene rings.