Thioflavin T as a Molecular Rotor: Fluorescent Properties of Thioflavin T in Solvents with Different Viscosity

Abstract

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.

Anle138b and related compounds are aggregation specific fluorescence markers and reveal high affinity binding to α-synuclein aggregates

“…For these molecules the increase in fluorescence is assumed to originate from the rigid and/or hydrophobic environment at the binding site of the aggregates [30][31][32]. Although these compounds have been studied extensively, the fluorescence mechanism and the origin of the fluorescence increase upon binding are still controversial [31,[33][34][35]. For the DPPcompounds one may assume that a rigid environment at the binding sites of the fibrillar aggregates [30] could reduce the intrinsic flexibility of the compounds and with that could slow down internal conversion processes.…”

Quantitative Viscosity Mapping Using Fluorescence Lifetime Measurements

“…When ThT is excited by light, the fragments twist relative to each other. The excited ThT has two routes to relaxation: (1) fluorescence emission and (2) a non-radiative twisted intermolecular charge transfer between the fluorescent LE (locally excited) state and the non-fluorescent TICT (twisted intermolecular charge transfer) state [26]. The latter involves the change in rotation angle between the two fragments [27].…”

Caracterização de lipoproteína de baixa densidade (LDL) por meios espectroscópicos

“…Estudos recentes demonstraram que a ThT comporta-se com um Rotor Molecular [88], [89], [90]. Rotores moleculares são moléculas fluorescentes caracterizados pela capacidade de formar estados torcidos através da rotação de um dos seus fragmentos com relação ao restante da molécula [91].…”

“…Rotores moleculares são moléculas fluorescentes caracterizados pela capacidade de formar estados torcidos através da rotação de um dos seus fragmentos com relação ao restante da molécula [91]. Toda molécula tem uma tendência a encontrar a conformação de menor energia e normalmente é uma conformação planar, entretanto na Tioflavina T, o estado fundamental a molécula apresenta um ângulo de torção entre os fragmentos (1) e (2) de φ = 37º [90].…”

“…Kelényi e colaboradores [86] Entretanto publicações recentes demonstraram que a molécula de ThT é um rotor molecular e na presença das fibrilas de amiloidose a liberdade de rotação intramolecular diminui, não ocorrendo mais a transição do estado excitado LE para o estado excitado TICT, diminuindo assim a perda não radioativa e consequentemente aumentando a emissão fluorescente do corante [88,90,91,100].…”