High Fluorescence Anisotropy of Thioflavin T in Aqueous Solution

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The high fluorescence anisotropy of thioflavin T in aqueous solution results from its molecular rotor nature Irina M. Kuznetsova, Anna I. Sulatskaya, Alexander A. Maskevich, Vladimir N. Uversky, and Konstantin K. Turoverov Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02747 • Publication Date (Web): 04 Dec 2015 Downloaded from http://pubs.acs.org on December 7, 2015

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Analytical Chemistry

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fluorescence

total

absorbance

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Thioflavin T fluorescence anisotropy in aqueous solutions

nonradiative transition

Low viscosity

 fl. =  int.rot. = 1ps  int.rot.  total r = 0.38



fluorescence

total

absorbance

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Thioflavin T fluorescence anisotropy in aqueous solutions

nonradiative transition

Low viscosity

 fl. =  int.rot. = 1ps  int.rot.  total r = 0.38


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The high fluorescence anisotropy of thioflavin T in aqueous solution results from its molecular rotor nature

Irina M. Kuznetsova,1 Anna I. Sulatskaya,1 Alexander A. Maskevich,2 Vladimir N. Uversky,1,3,* and Konstantin K. Turoverov1,4*

1

Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology of the Russian Academy of Sciences, St. Petersburg, 194064, Russia; 2Department of Physics, Yanka Kupala Grodno State University, Grodno, 230023, Belarus, 3Department of Molecular Medicine and USF Health Byrd Alzheimer's Research Institute, Morsani College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd, MDC07, Tampa, FL 33612, USA; 4 Department of Biophysics, Peter the Great St. Petersburg Polytechnic University, St. Petersburg, 195251, Russia

Author email addresses: [email protected], [email protected], [email protected]

*Corresponding authors: Vladimir N. Uversky and Konstantin K. Turoverov e-mails: [email protected], [email protected]


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Abstract

Thioflavin T (ThT) is widely used to study amyloid fibrils while its properties are still debated in literature. By steady-state and femtosecond time-resolved fluorescence we showed that, unlike small sized rigid molecules, the fluorescence anisotropy value of the unbound 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 neither by the internal rotation of the ThT benzothiazole and aminobenzene rings relative each other in the excited state because the axis of this rotation coincides with the direction of the transition dipole moment, nor by the rotation of the ThT molecule as a whole, because the rate of this process is three 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.

Keywords: Thioflavin T; Fluorescence anisotropy; Amyloid fibril nanostructure; Molecular rotor; Femtosecond time-resolved fluorescence


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Amyloid fibrils are regular, β-sheet-enriched, long, nanoscale aggregates of proteins with strands running perpendicular to the long axis of the fibril.1,2 Amyloidosis is a great problem of medicine because a number of human diseases, including several neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease, transmissible spongiform encephalopathies, etc., are characterized by intracellular inclusions or extracellular deposits of proteins in the form of amyloid fibrils.3-5 Proteins aggregation and formation of inclusion bodies frequently accompany over-expression of the recombinant proteins, thereby presenting a common bottleneck in modern biotechnology.6-8 There is an increasing belief that the ability to fibrillate is a generic property of a polypeptide chain, and that all proteins are potentially able to form amyloid fibrils under appropriate conditions.1,9-13 It is also believed that investigations of the structure of amyloid fibrils and molecular mechanisms of their formation are needed for better understanding of the protein folding process. Furthermore, amyloid fibrils have a unique architectures and exceptional properties (e.g., high mechanical strength) that make them an attractive subject in the materials science and nanobiotechnology.14-16 One of the proven tools for testing the appearance of amyloid fibrils in vivo and in vitro is fluorescence of the benzothiazole dye thioflavin T (ThT), which forms a highly fluorescent complex with amyloid and amyloid-like fibrils.17-20 At the same time, though ThT is a wellestablished tool for detection of amyloid fibrils, its photophysical properties and the mechanisms of its binding to amyloid fibrils are still debated in the literature. In the early works, a weak short wavelength fluorescence (ex ~ 340-350 nm, em ~ 440 nm) was recorded for the ThT in aqueous solution, while in the presence of amyloid fibrils, an enhanced fluorescence was found at longer wavelength range: ex ~ 440-450 nm, em ~ 490 nm. Based on these observations it was concluded that the short wavelength fluorescence belongs to the free ThT, and the long-


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wavelength fluorescence is determined by the complexes of ThT with the amyloid fibril.17,20 Thus, a twofold problem was put forward: how ThT binds to amyloid fibrils and what is the origin of new enhanced fluorescence. In several subsequent studies it was suggested that ThT binds to amyloid fibrils in some aggregated form, and that these ThT aggregates are responsible for the emergence of new enhanced fluorescence band. In fact, it was proposed that ThT binds to fibrils as dimers, excimers, or even micelles.18,21-23 Surprisingly, none of these studies addressed the fact that the excitation spectrum of the ThT short wavelength fluorescence does not coincide with the absorption spectrum of the dye as it should. Excitation spectrum differs from the ThT absorption spectrum in such a way that an excitation spectrum maximum corresponds to a minimum in the absorption spectrum. At the same time, ThT in aqueous solution, when being excited in the range of its absorption band, emits in the same spectral range as the ThT bound to amyloid fibrils, though this fluorescence has very low quantum yield.24 Consequently, the long-wavelength fluorescence is determined by ThT molecules, and it is short wavelength fluorescence of ThT in aqueous solution that requires special explanation but not the long-wavelength fluorescence of this dye. According to some modern views on this problem, all spectroscopic properties of ThT (including the occurrence of short wavelength band of fluorescence excitation and fluorescence) can be explained within the frames of its molecular rotor nature.25 This model was firstly proposed by Voropay et al,26 and later was supported by the quantum-chemical calculations of the ThT molecule energy dependence on the angle between the benzothiazole and aminobenzene rings in the ground and excited state.27,28 These calculations revealed that the presence of the methyl groups at the nitrogen atom of bezothiazole ring prevents the formation of strictly planar


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conformation of the dye molecule in its ground state. The energy minimum is observed when the

 angle between the planes of benzothiazole and aminobenzene rings equals to 37°. The difference between the molecule energy in this conformation and the conformation with  = 90° (270°) is not too large (about 700 cm-1).28 This means that in ground state a part of ThT molecules has  angle close to 90° (270°) and, consequently, is characterized by damaged electron conjugated system. It was suggested that just these molecules are responsible for the short wavelength component of the ThT fluorescence.28 This weak short wavelength fluorescence can be observed only in the case when a normal ThT fluorescence intensity is low, e.g. in aqueous media. Quantum-chemical calculations have also shown that in the excited state, ThT has energy minimum at  = 90°, and that there is no energy barrier (or such a barrier is negligibly small) between the conformations with  = 37° and  = 90°. Due to the intersection between ground and excited states at  = 90° there is a high probability of the radiationless transition to the ground state at  = 90°.25 Therefore, the rotational relaxation of the ThT benzothiazole and aminobenzene rings relative to each other in the excited state is the main cause of the radiationless deactivation of the dye. When ThT binds to amyloid fibrils, the restrictions on the rotation of its fragments relative to each other lead to a sharp increase in the quantum yield of the ThT fluorescence. In parallel with the quantum-chemical calculations, experimental studies of the dependence of the dye fluorescence intensity on the solvent temperature, viscosity and pressure24,29-31 and the ThT time-resolved fluorescence studies25,29-35 have been carried out. These investigations provided strong support to the validity of the ThT molecular rotor model. The popularity of ThT as a probe for amyloid fibrils detection is based on its high specificity of interaction with amyloid fibrils: the dye does not bind to proteins in their native


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state (with at least one exception, acetylcholinesterase36), denatured proteins in partially folded states (e.g., the molten globule state), or amorphous aggregates. It is also important that fluorescence intensity of free dye in aqueous solution is very low (the fluorescence quantum yield is approximately 0.0001),24 and the incorporation of ThT into amyloid fibrils causes a substantial increase in its fluorescence intensity (in some cases, several thousand fold). In recent years, new ThT fluorescence-based approaches have been elaborated for the analysis of the amyloid fibril structure.18,37-39 Many of them were aimed at the determination of the binding stoichiometry and binding parameters of specific ThT – amyloid fibril complexes. The earlier works were based solely on ThT fluorescence (reviewed in ref.18), while in the later studies, a new approach that combines absorption spectroscopy and fluorescence was proposed. The key point of this approach was sample preparation by equilibrium microdialysis. This method provided a means for characterizing the interaction of ThT with amyloid fibrils and for determining the binding stoichiometry and binding constants, absorption spectrum, molar extinction coefficient, and fluorescence quantum yield of the ThT bound to the sites of fibrils with different binding modes.37-39 In some studies, it was proposed that the anisotropy of ThT fluorescence can be used to study amyloid fibril formation.40-44 In one of these works,40 it was shown that ThT fluorescence anisotropy can serve as a useful tool for examining the nanostructural organization of the amyloid fibrils by polarized near-field scanning optical microscopy.40 The results of this work provides a good prove of the ThT monomeric incorporation into amyloid fibrils along fibril axis.45 In other studies,41-44 it was shown that, as it could be expected, the fluorescence anisotropy of ThT bound to amyloid fibrils was high, and it was suggested that the ThT fluorescence anisotropy has to increase on its binding to fibrils.41-44 Curiously, the fluorescence


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anisotropy of free ThT has not been specially examined, apparently because the fluorescence anisotropy of similarly sized rigid molecules usually equals to zero. We believe that since ThT is a molecule rotor, it must have high fluorescence anisotropy in the unbound state in aqueous solutions. The aim of this work was to verify this hypothesis and to check the suitability of the ThT fluorescence anisotropy as a test for the amyloid fibril formation.

Experimental section Materials: "Ultrapure Grade" ThT from AnaSpec (USA), glycerol from Merck (Germany) and lysozyme (Sigma) were used without further purification. Lysozyme amyloid fibrils were prepared as previously described.46 The glycerol concentration was determined using an Abbe refractometer (LOMO, Russia) at 23C from the dependence of the refractive index on the glycerol content. The ThT concentration in the solutions was 1.4x10-5 M. Steady-state fluorescence measurements: Fluorescence measurements were performed with a Cary Eclipse spectrofluorometer (Agilent Technologies, Australia) and a homemade spectrofluorometer.47 The total fluorescence intensity F  ex  

 F 

ex

, em d em , where

em

F(λex,λem) is the fluorescence intensity that is excited at the wavelength λex and recorded at the wavelength em, and the fluorescence spectra, were determined using 412 nm wavelength excitation light. The fluorescence excitation spectra were recorded at emission wavelengths of 490 nm. The spectral slit width was 10 nm for most experiments. Changing the slits width did not influence the experimental results. The recorded values for total fluorescence intensity were corrected for the inner filter effect.48 Fluorescence anisotropy was determined as: r   IVV - GI HV   IVV  2GI HV  , where IVV and I HV are vertical and horizontal components of fluorescence intensity excited by vertical polarized


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light, and G  IVH I HH is coefficient which determines the different sensitivity of the registering system for vertical and horizontal components of fluorescence intensity. Time-resolved fluorescence measurements: Ultrafast fluorescence decays were measured using a femtosecond fluorescence up-conversion device (FOG100, CDP, Russia). The fluorescence up-conversion technique was employed to measure the time-resolved emission of ThT in aqueous solutions at concentrations of 3·10-5 (absorbance ≈ 1.0). For excitation, we used a cavity-dumped titanium:sapphire femtosecond laser (Mira, Coherent), which provided short (120 fs) pulses at a repetition rate of 85 MHz. The samples were excited with second harmonic light (420 nm), and the 490 nm fluorescence from the samples was up-converted by mixing it with the fundamental light pulse (gate pulse ~ 840 nm). The up-converted signal was measured using a photon counter after passing through a proper bandpass filter and a double monochromator. All of these measurements were performed in a rotating cell (1 mm path length) to ensure better heat dissipation and to avoid dye photodegradation. The fluorescence decay curves and the time dependence of the fluorescence anisotropy in 99% glycerol were recorded by a spectrometer Pico Quant Fluotime 300 (Germany) with Laser Diode Head LDH-C-440 (ex = 440nm) or by a homemade device with pulsed discharge lamp as the source of excitation light. The measured emission decays were fit to a multiexponential function using the standard convolute-and-compare nonlinear least-squares procedure.49 In this method, the convolution of the model exponential function with the instrument response function (IRF) was compared to the experimental data until a satisfactory fit was obtained. The IRF was measured using cross correlation of the excitation and fundamental gate pulse. A special program was used to analyze the decay curves.47 The fitting routine was based on the nonlinear least-squares method. Minimization was performed according to Marquardt.50


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Absorption measurements: The absorption spectra were recorded using a U-3900H spectrophotometer (Hitachi, Japan). For obtaining the absorption spectra of ThT bound to amyloid fibrils the sample and reference solutions were prepared by equilibrium microdialysis. The processing of the experimental data described earlier.24,37,38 Equilibrium microdialysis was performed using a Harvard Apparatus/Amika (USA) device that consists of two chambers (500 μL each) that are separated by a membrane (MWCO 10 000) that is impermeable to particles larger than 10 000 Da.

Results and discussion In the early studies on the interaction of ThT with amyloid fibrils,17,20 due to the unknown reasons, fluorescence excitation and fluorescence spectra of ThT in aqueous solution were recorded and excited at em = 440 nm and ex = 340 nm, respectively. Our experiments show that such choice of ex and em indeed leads to the appearance of weak "short-wavelength” fluorescence with the maximal fluorescence excitation and emission at 360 nm and 440 nm, respectively (Figure 1A). Although these ThT fluorescence parameters in aqueous solution are commonly used in research, almost nobody paid attention to the fact that "short-wavelength" fluorescence excitation spectrum does not coincide with the ThT absorption spectrum (em = 412 nm). Such discrepancy between the absorption and fluorescence excitation spectra is only possible if along with the dye molecules solution contains some additional molecules with different spectral characteristics. Such additional molecules can be different origin. For example, the appearance of the fluorescence excitation spectrum which does not coincide with the absorption spectrum of 9-(2-carboxy-2-cyanovinyl) indolidine (CCVJ), which similar to ThT is a molecular rotor, was determined by the dye photo-induced isomerization.51 In the case of ThT,


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the existence of "short-wavelength fluorescence" is determined by the fragments of ThT dye molecules with the distorted -electron conjugated system (the angle  between benzothiazole and aminobenzene rings equals to 90°); i.e., rigid small molecules (Figure 1A, right panel). The appearance of such molecules is possible because the ground state energy difference between the ThT molecules with  = 37° (corresponding to energy minimum) and  = 90° is not high.28 When more natural excitation and registration wavelengths are selected, namely with the excitation wavelength corresponding to the maximum of the absorption spectrum (ex = 412 nm) and with the registration wavelength corresponding to the maximum of the ThT fluorescence spectrum (em = 490 nm), one can detect the "long-wavelength" ThT fluorescence in aqueous solution (Figure 1B). In this case, after correction for the primary inner filter effect,48 the fluorescence excitation spectrum coincides with the absorption spectrum (Figure 1B). For ThT bound to insulin amyloid fibrils, fluorescence excitation and emission spectra have maxima at 450 and 490 nm, respectively (Figure 1C). The idea that free ThT in aqueous solution is characterized by the "short-wavelength" fluorescence is a misconception that led to a number of erroneous conclusions, such as a hypothesis that ThT is bound to fibrils in aggregated state and that ThT fluorescence anisotropy can be used for detection of amyloid fibril formation.41,42,44 In one of the studies, it was suggested that individual amyloid fibrils can be detected by capillary electrophoresis using the ThT fluorescence anisotropy.43 In that work, it was shown that the fluorescence anisotropy was very high through the entire length of the electropherogram, and not only in the region where fibrils were localized. These high levels of the background fluorescence anisotropy are in a good agreement with our data. However, since the authors were sure that the fluorescence anisotropy of free ThT should be close to zero, they ascribed the noisy


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signal of high fluorescence anisotropy to the background of unknown nature and artificially eliminated it. To clarify the situation with binding-induced changes in the ThT fluorescence anisotropy, we examined fluorescence anisotropy of free dye in different solutions and of the amyloid fibril bound ThT. Figure 2 shows the results of the steady-state measurements of the fluorescence anisotropy for ThT in aqueous solution, in 99% glycerol, and for ThT bound to lysozyme amyloid fibrils. Panels A and B present data obtained with the excitation at 435 and 365 nm, respectively. The wavelength of 435 nm corresponds to the long wavelength band of the ThT absorption spectrum, whereas the wavelength of 365 nm practically coincides with the maximum of the excitation fluorescence spectrum of the short wavelength fluorescence (see above and Figure 1). In both cases, the fluorescence spectra (curves 1, 2 and 3) and the dependence of the fluorescence anisotropy on the emission wavelength (curves 4, 5 and 6) were recorded for ThT in aqueous solution (curves 1 and 4), ThT in 99% glycerol (curves 2 and 5), and for the ThT bound to the lysozyme amyloid fibrils (curves 3 and 6). As it was expected, fluorescence spectra (with ex = 435 nm) of ThT in aqueous solution, 99% glycerol, and in the presence of amyloid fibrils were close to each other. At the same time, when fluorescence was excited at 365 nm, the fluorescence spectra of ThT in aqueous solution was significantly blue shifted in comparison with the spectra determined for the ThT in viscous solutions and for ThT bound to the amyloid fibrils. This short wavelength band is determined by the dye molecules with the disturbed system of -electron conjugated bonds (see Figure 1). It is seen that just this molecules have low fluorescence anisotropy. The fluorescence of the dye molecules with disturbed system of electron conjugated bonds is rather weak and can be detected only when the fluorescence of ThT is very low, namely when it is measured in aqueous solutions. The fluorescence of the dye


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molecules with the disturbed system of -electron conjugated bonds is negligibly small and cannot be recorded in viscous solutions or in the presence of amyloid fibrils. As we expected, when fluorescence was excited at 435 nm (in the spectral range of the ThT absorption band), the fluorescence anisotropy of free ThT in aqueous solution was high, similar to that in 99% glycerol, and was close to the anisotropy limiting value of 0.4 (Figure 2A). The obtained data are in good agreement with the view that ThT is a molecular rotor. The high value of the fluorescence anisotropy is observed in solutions with relatively low viscosity not only for ThT but also for other molecular rotors.52,53 We found that the fluorescence anisotropy of the ThT bound to the lysozyme amyloid fibrils, though being very high, is slightly lower than that of the ThT anisotropy in aqueous solution (Figure 2A). At first glance it seems strange. However, the ThT bound to amyloid fibrils loses its peculiarity of molecule rotor, as the rotation of benzothiazole and aminobenzene rings are restricted in the bound state. First of all, this is manifested in the significant increase in the fluorescence lifetime and the fluorescence intensity on ThT binding to fibrils. Furthermore, in the excited state, the ThT molecule would tend to achieve the energy minimum at  = 90°, whereas the rigid environment could hinder this process. This can lead to the molecule deformation, which causes changes in the direction of the transition dipole moment that result in the decrease in the fluorescence anisotropy. Figure 2B shows the changes in the fluorescence anisotropy together with the fluorescence spectrum when the emission is excited at 365 nm. As it might be expected, the fluorescence anisotropy is very low in the wavelength range corresponding to the fluorescence of the ThT fragments, i.e. small rigid molecules. In the schematic presentation of the ThT molecule in a twisted state, only the benzothiazole ring is colored, indicating that only this ring is responsible for fluorescence. The small peak at 416 nm is due to the Raman scattering of water. The


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presence of this peak at 416 nm in the fluorescence anisotropy spectrum highlights the low fluorescence intensity. The contribution of the ThT molecules to the bulk fluorescence increases with the increase in the registration wavelength (see above and Figure 1). This leads to the observed increase in the fluorescence anisotropy (Figure 2 B). The pulsed excitation experiment provided additional, stronger evidence for the limiting high fluorescence anisotropy value of the ThT in aqueous solutions. The experimentally determined fluorescence lifetime of the excited state of 0.98 ps (Figure 3A), which is in a good agreement with the earlier determined value of 1 ps.34 As this relative rotation of the dye fragments is two orders of magnitude faster than the rotation of the ThT molecule as a whole, the molecule does not have time to change it spatial orientation during the lifetime of the excited state. That is why the fluorescence anisotropy in aqueous medium is very high, close to the limiting value. Figure 3B shows the decay of the vertical and horizontal components of the fluorescence intensity and the time dependence of the fluorescence anisotropy of ThT in aqueous solution. The fluorescence was excited by vertically polarized laser impulses at 420 nm. The duration of excitation impulse was much shorter than the fluorescence decay. The fluorescence anisotropy was calculated on the basis of the vertical and horizontal components of the fluorescence intensity. The fluorescence anisotropy remained obviously unchanged until the fluorescence completely decays. Although in viscous isotropic solutions (99% glycerol), the characteristic time of both process (rotations) increases (Figure 4A), the fluorescence anisotropy of ThT remain the same (Figure 4B). In conclusion we propose scheme illustrating photophysical properties of ThT (Figure 5). The axis of internal rotation of the ThT fragments relative to each other coincides with the


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direction of the transition dipole moment S0  S1* .27,54 Therefore, this relative rotation of fragments cannot change the direction of the transition dipole moment of ThT. However, such rotation of the ThT fragments relative to each other in the excited state leads to the conformation with the disturbed -electron conjugated system (  90°), and, consequently, to

the

radiationless deactivation of ThT molecules.25 The obtained experimental data provided further support to the hypothesis that ThT is a molecular rotor. Furthermore, our data indicated that ThT was a unique molecular rotor because the fluorescence anisotropy of molecular rotors with different mutual orientation of the axis of internal rotation and direction of the transition dipole moment can depend on the viscosity of the dye environment (see, e.g., refs.55,56).

Acknowledgments This work was supported in part by the Program "Molecular and Cell Biology" of the Russian Academy of Sciences, Russian Foundation for Basic Research, grants 14-04-90024_Bel (K.K.T.) and 13-04-02068 (A.I.S.)

and Russian Foundation President Fellowship SP-1982.2015.4

(A.I.S.).

References (1) Dobson, C. M. Trends Biochem. Sci. 1999, 24, 329-332. (2) Dobson, C. M. Methods 2004, 34, 4-14. (3) Carrell, R. W.; Gooptu, B. Curr. Opin. Struct. Biol. 1998, 8, 799-809.


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Figure legends Figure 1. The absorption, fluorescence excitation and fluorescence spectra of ThT in aqueous solution and incorporated into amyloid fibrils. Panel A. "Short wavelength" fluorescence of ThT in aqueous solutions, which in reality is determined by fluorescence of ThT fragments presented on the right of spectra. Absorption spectrum (solid line), fluorescence excitation spectrum (dashed line) recorded at em = 440 nm and fluorescence spectrum (dotted line) excited at ex = 360 nm. Panel B. True ThT fluorescence in aqueous solution. Absorption, fluorescence excitation (em = 490 nm), and fluorescence (ex = 412 nm) spectra are given. Designations are the same as on the Panel A. The ThT molecule structure corresponding to the energy minimum (the angle between benzothiazole and aminobenzene rings equals to 37°) is presented on the right of the spectra. Panel C. The absorption, fluorescence excitation (em = 490 nm),

and

fluorescence (ex = 450 nm) spectra of ThT bound to insulin amyloid fibrils. Designations are the same as on the Panel A. Important, here is true absorption spectrum of ThT bound to amyloid fibrils obtained using solutions prepared by microdialysis after removal of free ThT absorption,37,38,57,58 but not absorption spectrum of ThT in the presence of amyloid fibrils which is very close to absorption spectrum of free ThT molecules in aqueous solutions. Scheme illustrating excitation energy deactivation and heterogeneity of emitting center of ThT molecules in polar solution is given on the right of the spectra. Arrows denote absorption (1), fluorescence from Locally Excited (LE) state (2), torsional relaxation process from LE to Twisted Internal Charge Transfer (TICT) state (3), and nonradiative transition from TICT state to the ground state (4). E and N are energy of equilibrium and nonequilibrium with solvent states. Vertical arrows are transition with absorption and emission of light. S0 and S1* are energy levels corresponding ground and excited states of ThT molecule. W0 and W1 are stabilizing energy of ThT molecule in


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ground and excited equilibrium states due to orientation interaction of positively charged fragments of ThT and dipoles of polar solvent. Gradient painting between equilibrium and nonequilibrium states characterizes heterogeneity of emitting centers. Mostly populated are partially-equilibrium states. Fluorescence corresponds to transitions from partially-equilibrium excited states to partially-equilibrium ground states. Thickness of the arrow characterizes the probability of the transition.

Figure 2. Fluorescence anisotropy of ThT measured at steady-state excitation by the vertically polarized light. The fluorescence spectra (1, 2, 3) and the dependence of fluorescence anisotropy on the emission wavelength (4, 5, 6) for ThT in aqueous solution (1,4), in 99% glycerol (2, 5), and for ThT bound to lysozyme amyloid fibrils (3, 6) are presented. The excitation wavelengths were 435 nm (Panel A) and 365 nm (panel B).

Insets show structures of the molecules

responsible for the absorption of the excitation light. The description of structures presented on panels A and B are given in legend to Figure 1.

Figure 3. Time dependence of fluorescence of ThT in aqueous solution. Panel A. Decay curves of ThT fluorescence in aqueous solution. The excitation laser impulse profile (1), experimental decay curves of the fluorescence of ThT solutions recorded at 490 nm (2), best-fit calculated fluorescence decay curves (3), and the deviation between the experimental and calculated decay curves (4) are shown. The fluorescence decay curves show the best fit to a biexponential decay model. Panel B. Anisotropy of ThT in aqueous solutions. The excitation laser impulse profile (1), the decay curves of the vertical (2) and horizontal (3) components of the fluorescence


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excited by vertically polarized laser impulses at 420 nm and the change in fluorescence anisotropy (4) over time are shown.

Figure 4. Time dependence of fluorescence of ThT in 99% glycerol. Designations as in the legend to Figure 4.

Figure 5. Scheme illustrating photophysical properties of ThT. ThT molecule is represented in the ground state, excited Franck-Condon state and conformation with angle between benzothiazole and aminobenzene rings equals to 90° (dark state). Direction of the transition dipole moment of ThT coincides with the axis of internal rotation benzothiazole and aminobenzene rings relative to each other. In solutions of low viscosity, the fluorescence lifetime is determined by the rate of internal rotation of benzothiazole and aminobenzene rings relative to each other  fl 

int .rot . In isotropic solutions (aqueous solution or 99% glycerol) the

rate of internal rotation of the ThT fragments is much lower than the rotation of the ThT molecule as a whole ρint.rot.

High Fluorescence Anisotropy of Thioflavin T in Aqueous Solution

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