Spectral Properties of Thioflavin T and Its Complexes with Amyloid Fibrils

Comparative analysis of the absorption and fluorescence spectra and fluorescence excitation spectra of thioflavin T (ThT) in various solvents and in the composition of amyloid fibrils has shown that ThT, when excited in the region of the long-wavelength absorption band, fluoresces in the spectral region with a maximum at 478–484 nm. The appearance in aqueous and alcohol solutions of a fluorescence band with a maximum near 440 nm has been attributed to the presence in the composition of the ThT preparations of an impurity with an absorption band in the 340–350-nm range. The literature data showing that in glycerol ThT has a wide fluorescence spectrum with two maxima are due to the artifact connected with the use of a high concentration of the dye. It has been suggested that the cause of the low quantum yield of ThT aqueous and alcohol solutions is the breakage of the system of conjugated bonds due to the reorientation of the benzothiozole and benzaminic rings of ThT in the excited state with respect to one another. The main factor determining the high quantum yield of fluorescence of ThT incorporated in fibrils is the steric restriction of the rotation of the rings about one another under these conditions. The suggestions made have been verified by the quantum-chemical calculation of the ThT molecule geometry in the ground and excited states

Резонансное усиление флуоресценции квантовых точек у поверхности плазмонных пленок

Effective enhancement of the fluorescence signal of chromophores adsorbed directly onto plasmonic films can be observed under conditions of strong spectral resonance between plasmon and chromophore absorptions. This effect seems to contradict the established mechanisms of complete quenching of the fluorescence of chromophores under their adsorbtion directly onto the metal surface. However, under certain conditions, enhancement of the fluorescence signal is observed for both inorganic and organic chromophores. To understand the effect and conditions of its observation, we propose to use the quantum concept of virtual photon exchange in the near optical field - dressed photons. This concept is borrowed from the physics of elementary particles and is already well adapted to the problems of nanophotonics by M. Otsu. In this paper, we discuss exclusively the key factors responsible for enhancement of fluorescence of CdSe/ZnS nanocrystals and the effective dressed photons exchange: the size of nanoparticles, the distance between them, and the presence of spectral overlap indicating the possibility of resonant interactions between plasmons and chromophores.Communicated by Academician Sergei V. GaponenkoЭффективное усиление сигнала флуоресценции хромофоров, адсорбированных непосредственно на плазмонные пленки (ПП), можно наблюдать в условиях сильного спектрального резонанса между плазмонным поглощением и поглощением хромофора. Указанный эффект, на первый взгляд, противоречит установленным механизмам полного тушения флуоресценции хромофоров при их адсорбции непосредственно на поверхность металла. Однако при определенных условиях усиление сигнала флуоресценции наблюдается как для неорганических, так и органических хромофоров. Для понимания эффекта и условий, при которых он может наблюдаться, мы предлагаем исходить из двух основополагающих факторов, определяющих вероятности квантовых переходов, - матричного элемента перехода, ответственного за наблюдаемое излучение, и плотности конечных состояний. Однако при определении матричного элемента перехода необходимо учитывать наличие нескольких хромофорных систем (адсорбированные хромофоры и локализованные плазмоны), способных поглощать возбуждающее излучение и еще одной плазмон-поляритонной системы, имеющей светоподобную природу и способной к излучению световых квантов, а также взаимодействие между ними, которое может быть описано в рамках обмена виртуальными фотонами. Учитывая, что расстояния между наночастицами намного меньше длины волны света и дипольное приближение не работает, мы предлагаем использовать квантовую концепцию обмена виртуальными фотонами в ближнем оптическом поле - «одетыми» фотонами. Эта концепция заимствована из физики элементарных частиц и уже хорошо адаптирована к проблемам нанофотоники М. Отсу. Второй фактор, который также может давать значительный вклад в усиление, - это модификация плотности как электронных, так и фотонных состояний - эффект, который предложен С. В. Гапоненко в качестве модели, ответственной за усиление гигантского комбинационного рассеяния света хромофоров в стоксовой и антистоксовой области вблизи наночастиц с плазмонными свойствами. В данной работе обсуждаются такие ключевые факторы, ответственные за усиление флуоресценции наночастиц CdSe/ZnS и эффективный обмен виртуальными фотонами, как размер наночастиц, расстояния между ними и наличие спектрального перекрытия, указывающего на возможность резонансных взаимодействий между плазмонами и хромофорами.Представлено академиком С.В. Гапоненк

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

Analyzing Thioflavin T Binding to Amyloid Fibrils by an Equilibrium Microdialysis-Based Technique

A new approach for the determination of the amyloid fibril – thioflavin T (ThT) binding parameters (the number of binding modes, stoichiometry, and binding constants of each mode) is proposed. This approach is based on the absorption spectroscopy determination of the concentration of free and bound to fibril dye in solutions, which are prepared by equilibrium microdialysis. Furthermore, the proposed approach allowed us, for the first time, to determine the absorption spectrum, molar extinction coefficient, and fluorescence quantum yield of the ThT bound to fibril by each binding modes. This approach is universal and can be used for determining the binding parameters of any dye interaction with a receptor, such as ANS binding to proteins in the molten globule state or to protein amorphous aggregates

Effects of oral gamma-aminobutyric acid (GABA) administration on stress and sleep in humans: a systematic review

Gamma-aminobutyric acid (GABA) is a non-proteinogenic amino acid and is the main inhibitory neurotransmitter in the mammalian brain. GABA's stress-reducing, and sleep enhancing effects have been established. However, although several human clinical trials have been conducted, results regarding the role of natural and/or biosynthetic oral GABA intake on stress and sleep are mixed. We performed a systematic review to examine whether natural and/or biosynthetic oral GABA intake has an effect on stress and sleep. We systematically searched on PubMed database for studies published up to February 2020 following PRISMA guidelines. Only placebo-controlled human trials that assessed stress, sleep, and related psychophysiological outcomes as a response to natural GABA (i.e., GABA that is present naturally in foods) or biosynthetic GABA (i.e., GABA that is produced via fermentation) intake were included. Fourteen studies met the criteria and were included in the systematic review. Although more studies are needed before any inferences can be made about the efficacy of oral GABA consumption on stress and sleep, results show that there is limited evidence for stress and very limited evidence for sleep benefits of oral GABA intake

Extrinsic Fluorescent Dyes as Tools for Protein Characterization

Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization

Formation of trans-2-[4-(dimethylamino)styryl]-3-ethyl-1,3-nzothiazolium perchlorate dimers in the presence of sodium polystyrene sulfonate formation of trans-2-[4-(dimethylamino)styryl]-3-ethyl-1,3-benzothiazolium perchlorate dimers in the presence of sodium polystyrene sulfonate

The spectral properties of a novel thiofl avin T derivative, trans-2-[4-(dimethylamino)styryl]-3-ethyl-1,3- benzothiazolium perchlorate (DMASEBT), were studied in aqueous solutions in the presence of sodium polystyrene sulfonate (SPS). It was shown that SPS either could interact with dye monomers or initiate the formation of non-fl uorescent dye dimers depending on the concentration ratio of dye and polyelectrolyte. DMASEBT dimer formation in the presence of SPS produced a hypsochromic shift by 40 nm in the absorption spectrum and quenched fl uorescence. A bathochromic shift of the absorption spectrum and an increase of the fl uorescence intensity by an order of magnitude were observed if DMASEBT monomers interacted with SPS. Quantum-chemical analysis found that sandwich dimers (H-aggregates) were most stable. A comparison of DMASEBT spectra in the presence of SPS and amyloid fi brils showed that DMASEBT molecules were incorporated into amyloid fi brils as monomers. The spectral changes associated with this incorporation could not be explained by the formation of dye aggregates

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