Thioflavin T Interaction with Acetylcholinesterase: New Evidence of 1:1

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Thioflavin T Interaction with Acetylcholinesterase: New Evidence of 1:1 Binding Stoichiometry Obtained with Samples Prepared by Equilibrium Microdialysis Anna I. Sulatskaya, Georgy N Rychkov, Maksim I. Sulatsky, Natalia P. Rodina, Irina M. Kuznetsova, and Konstantin K. Turoverov ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00111 • Publication Date (Web): 13 Apr 2018 Downloaded from http://pubs.acs.org on April 15, 2018

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ACS Chemical Neuroscience

Thioflavin T Interaction with Acetylcholinesterase: New Evidence of 1:1 Binding Stoichiometry Obtained with Samples Prepared by Equilibrium Microdialysis

A.I. Sulatskaya,1 G.N. Rychkov2,3, M.I. Sulatsky,1 N.P. Rodina1,2, I.M. Kuznetsova1 and K.K. Turoverov1,2,*

1

Laboratory of Structural dynamics, stability and folding of proteins, Institute of Cytology of

the Russian Academy of Science, St. Petersburg, Tikhoretsky ave. 4, 194064, Russia, 2

Institute of Physics, Nanotechnology and Telecommunications, Peter the Great St.-Petersburg

Polytechnic University, St. Petersburg, Polytechnicheskaya 29, 195251, Russia, 3

Department of Molecular and Radiation Biophysics, Petersburg Nuclear Physics Institute,

NRC Kurchatov Institute, Orlova Roscha, Gatchina, Leningrad District, 188300, Russia. *

Corresponding author: K.K. Turoverov, e-mail: [email protected], Tel.: +7 812 297 19 57,

Fax: +7 812 297 35 41

Running head: Thioflavin T binging to acetylcholinesterase

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Abstract The aim of the present work was investigation of the fluorescent dye thioflavin T (ThT) binding to acetylcholinesterase (AChE). ThT is an effectively used test for the protease activity, as well as a probe for amyloid fibril formation. Despite the extended and active investigation of the ThT – AChE binding, there is still no common view on the stoichiometry of this interaction. In particular, there is a hypothesis explaining the spectral properties of the bound to AChE dye and high quantum yield of its fluorescence by formation of dimers or excimers of ThT. In order to confirm or deny this hypothesis we proposed a new experimental approach for examination of the ThT – AChE interaction based on spectroscopic investigation of samples prepared by equilibrium microdialysis. This approach allowed to prove the 1/1 ThT/AChE binding stoichiometry. The increase of the ThT fluorescence quantum yield and lifetime accompanying its binding to AChE can be explained by the molecular rotor nature of this dye. Together with the coincidence of the positions of free and AChE-bound ThT fluorescence spectra, the obtained results prove the groundlessness of the hypotheses about ThT aggregation while binding to AChE. The model of ThT localization in the active site of AChE was proposed by using molecular docking simulations. These results also allowed us to suggest the key role of aromatic residues in the ThT – AChE interaction, as observed for some amyloid fibrils.

Key words: acetylcholinesterase, fluorescent probe thioflavin T, amyloid fibrils, binding constants and stoichiometry, equilibrium microdialysis


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1. Introduction Acetylcholinesterase (AChE) is a serine protease that plays the key role in cholinergic synaptic transmission by hydrolyzing the neurotransmitter acetylcholine to choline and acetate with one of the highest known catalytic rate constants (1, 2). AChE is found in many types of conducting tissues: nerve and muscle, central and peripheral tissues, motor and sensory fibers, and cholinergic and noncholinergic fibers. Parafunction of AChE leads to various disorders of the nervous system (3-9), thereby investigation of AChE structure and the features of its function is an urgent problem. The three-dimensional structure of acetylcholinesterase (Figure 1) reveals a deep narrow gorge that contains two ligand binding sites: a peripheral site (P-site) near the gorge entrance and an acylation site (A-site) at the base of the gorge (10). Thioflavin T (ThT), a fluorescent probe widely used as a test for amyloid fibril formation (11-13), binds selectively to the AChE peripheral site, wherein the ThT fluorescence intensity increases significantly (14-16). The proved proportionality of the dye’s fluorescence intensity to AChE activity led to the elaboration of a rapid fluorescent method for determination of AChE activity using ThT (17). Furthermore, it was shown that while binding of another ligand to the A-site in a ternary complex and initiation of a change in the local AChE conformation at the peripheral site, the enhanced fluorescence of ThT is quenched 1.5- to 4-fold (10, 16, 18). This fact allows ThT to be used as a reporter for ligand reactions at the AChE acylation site.

Figure

1.

Three-dimensional

structure

of

Electrophorus

electricus

acetylcholinesterase (the figure was created on the basis of PDB data, file 1EEA). (A) Side view and (B) front view on the narrow and deep gorge that contains the ligand binding site are shown. Amino acid residues forming this gorge are also presented.


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Investigation of ThT interaction with AChE, as well as the correct determination of their binding parameters and characteristics of the bound dye is important not only for correct and effective application of this dye for studying of protease structure and activity. It also can contribute to understanding the mechanisms of the specific ThT-amyloid fibril binding. Surprisingly, despite the active investigation of the ThT – AChE binding, to date there is no common view on the stoichiometry of this interaction. In particular, some papers suggest that ThT binds to AChE in a monomeric form. For example, in the work of Harel (10) the crystal structure of the ThT/TcAChE complex and 1/1 stoichiometry of this complex was revealed. At the same time there is another hypothesis that suggests that the spectral properties of the bound dye and its high fluorescence quantum yield may be due to the formation of ThT dimers or excimers (19). The aim of the present work was determination and analysis of the spectral characteristics of the dye incorporated into AChE, experimental calculation of the ThT-AChE binding parameters (including their binding stoichiometry) and review the existing hypotheses about mechanism of ThT binding to AChE. All of the currently available data on the parameters of ThT – AChE binding are based on the analysis of the ThT fluorescence intensity dependence on its concentration in solutions containing AChE (18, 20-22). A significant obstacle of using the fluorescence intensity dependence on ThT concentration in solutions containing AChE for determination of the ThT- AChE binding parameters is the nonlinearity of the dependence of the fluorescence intensity on the concentration of fluorescent substance, caused by the so-called inner filter effect (see Supplement). Even experienced researchers, who do not specialize in fluorescence techniques, do not take into account the fact that a plateau of the fluorescence intensity dependence on the absorption (concentration) of a fluorescent substance could not point to the saturation of binding centers, since such a “saturable” character of the dependence is its general property. It should be noted that the estimation of this effect and linearization of recoded dependence (which, despite existing misconceptions, must be performed even when working with low concentration solutions (23)) is a difficult problem and was not conducted correctly in previous works dedicated to the investigation of ThT – AChE interaction. Another common mistake one can find in literature is regarding the determination of AChE – ThT binding parameters. Experiments based on the measurements of fluorescence intensity per se cannot, in principle, provide ACS Paragon Plus Environment

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information on the free dye concentration that is necessary for calculation of the binding parameters. Nonetheless, researchers replace the value of free dye concentration with the value of the input dye concentration, for the determination of the binding constants without any explanation of such a change (10, 19, 22). Therefore, as for today, we do not know any paper where the binding parameters describing the AChE-ThT interaction were determined correctly. In this work, we show how such information can be obtained using absorption spectroscopy of the sample and reference solutions prepared by equilibrium microdialysis. This approach was inherently designed for the determination of binding parameters of low molecular weight ligands to receptors (24-26). However, it has never been used for the study of the interaction of AChE with any ligands. We have shown that performed in corpore, this approach enables the determination not only of the ThT – AChE binding parameters but of the absorption spectrum, molar extinction coefficient and fluorescence quantum yield of ThT bound to AChE for the first time.

2. Results and Discussion 2.1. Equilibrium Microdialysis as a New Experimental Approach to the Preparation of Solutions for the Investigation of ThT - AChE Interaction. Absorption Spectrum of ThT Bound to AChE Equilibrium microdialysis implies allocation of two interacting agents, a ligand and receptor, in two chambers (#2 and #1, respectively) divided by a membrane permeable to the ligand and impermeable to the receptor (Figure 2 A). In our case, AChE in the buffer solution was placed in chamber #1, and ThT solution in the same buffer, with an initial concentration of С0, was placed in chamber #2. At equilibrium (Figure 2 B), the absorption spectrum of the solution in chamber #2 represents the absorption spectrum of free ThT at a concentration of Cf (Af (λ)), and the absorption spectrum of the solution in chamber #1 represents the superposition of the absorption spectra of free ThT at a concentration Cf, ThT bound to AChE at a concentration Cb (Ab(λ)), and the small apparent absorption determined by AChE light scattering (Ascat(λ)). The contribution of light scattering was eliminated as previously described(27). Thus, in chambers #1 and #2, we have sample and reference solutions for the determination of the absorption spectrum of ThT bound to AChE (Figure 2 C). The obtained results show that the absorption spectrum of ThT bound to AChE (λmax = 425 nm) is red-shifted in comparison to free ThT spectrum in buffer solution (λmax = 412 nm), however the shift is not as significant as while ThT interaction with amyloid ACS Paragon Plus Environment

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Figure 2. The use of equilibrium microdialysis for examination photophysical properties of ThT bound to AChE. (A) The sample and reference solutions were prepared using the device for equilibrium microdialysis, which consists of two chambers (500 µL each) separated by a membrane (MWCO 10,000) impermeable to particles larger than 10,000 Da. AChE in the buffer solution at concentration Cb was placed in one chamber and ThT solution in the same buffer, with an initial concentration of С0, was placed in the second chamber. These solutions were incubated in a thermostat and with constant stirring until reaching equilibrium. (B) At equilibrium we have a sample solution (containing free and AChE-bound ThT) and an optimal reference solution (containing free ThT in a concentration equal to the concentration in the sample solution) for determination of the absorption spectrum of ThT bound to AChE. (C) Photophysical properties of free and AChE-bound ThT were investigated. Absorption spectra of ThT bound to AChE (curve 3) determined with the use of absorption spectra of ThT in the sample (curve 2) and reference (curve 1) solutions after equilibrium microdialysis are shown. The absorption spectrum of free ThT at equal concentration to ThT bound to AChE (curve 4) and fluorescence spectra of ThT in the sample (curve 6) and reference (curve 5) solutions after equilibrium microdialysis are also presented.


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fibrils (for example, λmax = 449 и 450 nm for ThT bound to lysozyme and insulin amyloid fibrils, respectively) (28). The

shortwave position of the absorption

spectrum of free ThT in water solution can be explained by the orientational dipole– dipole interaction of the dye molecules with the polar solvent (see (27)). It can be assumed that the differences in the position of ThT bound to AChE and amyloid fibrils absorption spectra are caused by the dissimilarity in the polarity of the microenvironments of the dye molecules. In particular, the binding sites of ThT to AChE may be more readily available to the polar solvent whereas the binding sites of the dye to amyloid fibrils can be located in a more hydrophobic microenvironment.

2.2. ThT – AChE Binding Constant and Stoichiometry. With the use of the recorded absorption spectra of free ThT in chamber #2 (Af(λ)) for each microdialysis experiment, the concentrations of free (Cf) and bound to AChE (Cb) dye were determined (see Supplement). The dependence of bound dye concentration upon the concentration of free dye in solution is shown on the Figure 3 A. Experimental results are also represented in Scatchard coordinates (Figure 3 B). Every point on the Scatchard plot corresponds to one microdialysis experiment. The advantage of this representation of experimental results is that it allows visual estimation of the number of binding modes (different types of binding) of the ligand, which is a crucial step for the manual determination of binding parameters. In the case of ThT-AChE interaction, the linear character of this dependence indicates the existence of one binding mode. Thus, the ThT-AChE binding parameters were determined using equation (8) (Supplement) by multiple linear regressions (Graph Pad Prism 5), assuming all binding sites to be independent. Satisfactory approximation of the experimental data by the calculated curve plotting with the use of the obtained values of the binding constant (Kb) and number of binding sites (n) indicates the correctness of the chosen binding model and the determined binding parameters. The obtained results indicate 1/1 ThT/AChE binding stoichiometry. It can be noted that the calculated ThT – AChE binding constant Kb ~ 104 M-1 has the same order of magnitude as the binding constant of this dye to the one of modes of amyloid fibrils based on different proteins (28, 29) (Table 1). Binding to this mode as assumed is caused by the incorporation of the dye molecules (in monomeric form) into the grooves formed by amino acid side chains along the long ACS Paragon Plus Environment

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Figure 3. Determination of ThT – AChE binding parameters of bound to AChE dye. (A) Dependence of bound dye concentration on the concentration of free dye in solution, (B) Scatchard plot which gives visual representation of the number of binding modes. Experimental data (circles) and curves determined using calculated values of the binding constant (Kb) and number of binding sites (n) of ThT to AChE are given.

axis of the fibrils perpendicular to the β-sheets (30). Coincidence of ThT – AChE and ThT – amyloid fibrils binding parameters is unexpectedly, considering that the peripheral site of this ferment is not characterized by a β-structure typical for amyloid fibrils. Using the obtained values of absorbance (Ab) and concentration (Cb) of ThT bound to AChE, the value of the molar extinction coefficient of bound ThT (εb) was determined for the first time. The obtained results show that ThT binding to AChE is accompanied not only by the change in the position of its absorption spectra but also by the decrease in its molar extinction coefficient (εb (425)=2.3·104 cm-1M-1) compared to that of free dye in aqueous solution (εf (412)=3.2·104 cm-1M-1). These alterations might be associated with the different conformations of ThT molecules bound

to

AChE

and

free

molecules

in

solution

and

their

different

microenvironment.

2.3. Conformation of ThT bound to AChE To assess the possible values of angles between aromatic rings of ThT bound in the P-site of AChE, we used flexible docking in ICM Pro 3.8 (31) (see Supplement for details). This analysis revealed 14 different poses of ThT in the Psite of the protein. In general, two orientations of ThT relative to the active site were obtained: 1) the benzothiazole ring is in the active site gorge and the phenyl ring is ACS Paragon Plus Environment

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Table 1. Binding parameters of ThT to AChE and to amyloid fibrils on the basis of different proteins and photophysical properties of the bound dye

Object ThT bound to acetylcholinesterase

λmax, nm

εi, maxx10-4, Kbi x10-5, M-1 M-1cm-1

ni

qi

1

2.3

0.2

1.0

0.04

1

7.9

78

0.02

0.72

2

2.3

0.4

0.14

0.27

1

5.3

72

0.11

0.44

Fibrils (28)

2

6.2

0.60

0.25

0.0001

ThT bound amyloid fibrils formed from Aβ42 (28)

1

8.7

70

0.004

0.18

2

1.4

0.2

0.26

0.03

1

1.8

71

0.08

0.24

2

1.4

0.21

0.92

0.08

ThT bound to insulin amyloid fibrils(28)

ThT bound to lysozyme amyloid

425

mode

450

449

440

ThT bound Sup35p amyloid fibrils (29)

440

Free ThT in aqueous solution (32)

412

-

3.2

-

-

0.0001

Free ThT in 99 % glycerol (33)

424

-

3.6

-

-

0.066

near the gorge entrance, and 2) the inverse orientation, where the phenyl ring is in the active site gorge and the benzothiazole ring near the gorge entrance. Comparing binding scores (31), which estimate the binding free energy of ThT, the first orientation is preferential (the minimal binding score for the pose in the first orientation is about 18.7 kcal/mol and in the second is about 12.3 kcal/mol). The pose of ThT in the P-site with the minimal binding score is shown in Figure 4. Noticeable flexibility of amino acid residues was observed for D72, L74, Y330, H440, and substantial flexibility was observed for W84 and W279. These residues adopt the most suitable conformations to fit different poses of ThT. The torsion angle between phenyl and ACS Paragon Plus Environment

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Figure 4. Possible location of ThT at the P-site of AChE. The pose with the minimal binding score is illustrated. The dimethylamino-phenyl ring of ThT lies outside the active site gorge. (A) General view of AChE spatial structure is presented; ThT molecule is in ball representation. (B) Close view of ThT in the P-site is shown. ThT and amino acid residues in 5 Å vicinity of ThT are in ball-and-stick representations. Residues Asp72 and W279 in the P-site are emphasized.

benzothiazole rings varied from 26.0° to 45.7°. The mean value of the angle, ~39.7° (with root-mean-square deviation 6.57°), is close to the value of the angle calculated earlier for the free dye in aqueous solution, ~37° (27, 34). The conformation of the ThT molecules bound to AChE have been studied previously (10). Crystallographic structure of TcAChE (AChE from Torpedo californica) in complex with ThT (PDB entry 2J3Q) demonstrates ThT position in P-site having the described above orientation of the second type. However, the main calling an attention structural peculiarity of ThT in this complex is the exact planarity of the molecule. This contrasts with relatively high values of ThT atomic B-factors (~55 Å2), characterizing atomic displacements from mean position, in comparison with B-factors of atoms of amino acids forming the binding site of TcAChE (~25 Å2). Although the authors modeled the two-ring system as coplanar, there are some papers where the possibility of existence of angles about 20° is noted (34). Furthermore the study of ThT interaction with two alternative states of β-2microglobulin (one monomeric, the other an amyloid-like oligomer), clearly shows the possibility of formation of the non-planar ThT conformation in the complex with β-2-microglobulin (PDB entries 3MYZ and 3MZT) (34). Thus, the calculated value ACS Paragon Plus Environment

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of the angle between ThT fragments in the bound to AChE from Electrophorus electricus (~39.7°) and nonplanar conformation of the dye molecule seems quite plausible.

2.4. Model of ThT – AChE Binding Despite the fact that we experimentally proved the existence of one binding mode of ThT to AChE and showed a 1:1 stoichiometry of this interaction (which agrees with the ideas of other authors (10)), we would like to consider other existing hypnotizes about the model of ThT – AChE binding. In particular, there is a hypothesis that the spectral properties of the bound to AChE dye and high quantum yield of its fluorescence may be due to the formation of ThT dimers or excimers (19). This suggestion grew up from an early work (11) where a weak short wavelength fluorescence band (λex ~ 340-350 nm, λem ~ 440 nm) was erroneously attributed to free ThT in solution and an intense long wavelength fluorescence band was assigned to ThT aggregates bound to amyloid fibrils (λex ~ 412 nm, λem ~ 490 nm). This work and subsequent papers (35, 36) ignored the lack of the required equivalence between the short wavelength band of the ThT fluorescence excitation spectrum and the absorption spectrum of the free dye. On the contrary, the maximum of the fluorescence excitation spectrum of this spectral band corresponds to a minimum in the absorption spectrum(27). Consequently, not the long wavelength band of the ThT fluorescence spectrum in aqueous solution, but the short one requires a special explanation. Later, an explanation for the short wavelength spectral band of ThT in alcohol and in aqueous solutions based on the results of quantum chemical calculations and using the molecular rotor model to describe the nature of ThT was given (27). Quantum chemical calculations showed that the photophysical properties of ThT molecules are most determined by the ϕ angle between the benzothiazole and aminobenzene rings of the dye. It has been shown that the presence of a methyl group attached to the nitrogen atom of benzothiazole ring, preclude the existence of strict planar conformation of the ThT molecule in the ground state, expose the barrier when ϕ = 0 (180)° and increases energy ThT molecules in the ground state. Thus reducing the amount energy barrier at ϕ = 90 (270)° between the conformations of ThT, corresponding to a minimum of energy. At ϕ close to 90° and 270° the system of conjugated double bonds of ThT decomposes into π-conjugated systems of aminobenzene and benzothiazole rings, which then may act as independent chromophores. The barrier is low (700 cm-1) (27) and, therefore, in the ground state a certain fraction of molecules will have a conformation with ACS Paragon Plus Environment

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the unbound general system of delocalized π-electrons of aminobenzene and benzothiazole rings. It is known that the increase of the size of π-conjugated bonds system shifts the absorption and fluorescence spectra to longer wavelengths. Therefore, any fragments of ThT, e.g. benzothiazole ring, must have a shorter wavelength absorption spectrum (and fluorescence), as compared to that of the ThT molecule. Thus, the existence of short wavelength bands of fluorescence and excitation of fluorescence in alcohol and aqueous solutions of ThT can be attributed to the existence in the ground state of molecules, which behave as independent fragments of ThT along with full-size ThT molecules. Despite all aforesaid the hypothesis about aggregation of ThT molecules while binding to AChE is based on the assumption that free monomer dye molecules have a shortwavelength fluorescence spectra (λem ~ 440 nm), and fluorescence spectra of ThT bound to AChE due to the aggregates of the dye molecules are red shifted to a maximum λem ~ 490 nm (19). For an experimental prove of the invalidity of this assumption the fluorescence spectra of the samples and references solutions prepared by equilibrium microdialysis were recorded (Fig. 2 C). The obtained results show the coincidence of the position of the fluorescence spectra of free ThT monomers in water solution and AChE-bound dye and their difference from the spectra of ThT excimers in water solution (λem ~ 570 nm) (37) (Figure 5 A, B). Fluorescence spectrum of ThT bound to AChE also coincides to that of the dye bound to amyloid fibrils (Figure 5 C). These results confirm the groundlessness of the hypotheses of dye aggregation while it binds to AChE and amyloid fibrils. The reasons of the ThT fluorescence intensity increase when the dye binds to AChE will be explained in the next section.

2.5. Fluorescence Quantum Yield of ThT Bound to AChE The registration of fluorescence of solutions prepared by equilibrium microdialysis can also be used for the determination of the fluorescence quantum yield of ThT bound to AChE. For this aim the inner filter effect problem (23) was primarily solved. In this work, we used a Cary Eclipse spectrofluorimeter, which essentially simplifies the correction of the detected fluorescence intensity for this effect, allowing an analytical determination of the correction coefficients (see Supplement). With the use of corrected on the primary inner filter effect fluorescence intensity values, the fluorescence quantum yield of ThT bound to AChE was determined (q = 0.04). This value is considerably greater than the corresponding value for the free dye in aqueous solution (q = 0.0001), comparable to that for the dye in 99% glycerol and bound to one of the modes (with lower affinity) of Sup35p


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Figure 5. Normalized absorption (dashed lines) and fluorescence (solid lines) spectra of ThT bound to AChE (A), dye monomers (blue lines) and excimers (red lines) in water solution (B) and probe incorporated into insulin amyloid fibrils (C).

and Abeta-peptide (1-42) amyloid fibrils, however, smaller than that for ThT bound to the another mode (with higher affinity) of amyloid fibrils (Table 1). The low fluorescence quantum yield of free ThT in aqueous solution is caused by its molecular rotor nature. The benzothiazole and aminobenzene rings of the dye can rotate relative to one another in the excited state (27, 32), and the transition of the excited dye molecules in a state with an angle between fragments close to 90° leads to its nonradiative transition to the ground state. We believe that the fluorescence quantum yield of the dye when it binds to AChE significantly increases due to the restriction of the ThT fragments rotation relative to one another in an excited state. The difference in the values of the fluorescence quantum yield of ThT associated with AChE and one of the modes (with higher affinity) of amyloid fibrils formed from different proteins can be explained by different rigidity of the microenvironment of the bound dye molecules. ACS Paragon Plus Environment

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2.6. Fluorescence Lifetime and Anisotropy of ThT Bound to AChE Rotation of ThT fragments relative to each other in the excited state leads to the conformation with the disturbed π-electron conjugated system (ϕ ≈ 90°) and, consequently, to the radiation-less deactivation of the excited state of ThT molecules. In aqueous solutions, the rate of this process is much higher than the rotation of ThT molecule as a whole, and the fluorescence lifetime is determined by the rate of internal rotation of benzothiazole and aminobenzene rings relative to each other. In our recent works, we experimentally determined the fluorescence lifetime of the excited state of free ThT in aqueous solutions with the use of the values of radiative lifetime and fluorescence quantum yield of ThT (1 ps) (32) and the dye fluorescence decay curves (0.98 ps) (38). In this work, we showed that while ThT binding to AChE the dye florescence lifetime increases about 3 orders of magnitude (Fig. 6 A). This can be caused by the restriction of the rotational motions of ThT fragments relative to each other in the excited state and by the decrease of the rate of the radiation-less deactivation of the dye molecules. For the same reason, there is an increase in the dye fluorescence lifetime (approximately to the same values) when it binds to amyloid fibrils. Fluorescence anisotropy of ThT in aqueous medium is very high, close to the limiting value (38). Direction of the transition dipole moment of ThT coincides with the axis of the internal rotation of the benzothiazole and aminobenzene rings relative to each other. Therefore, the relative rotation of fragments cannot change the direction of the transition dipole moment of ThT. As relative rotation of the dye fragments (that leads to radiationless deactivation of the dye molecules) is two orders of magnitude faster than the rotation of the ThT molecule as a whole, the molecule does not have enough time to change its spatial orientation during the lifetime of the excited state. In this work we showed that while ThT binding to AChE the characteristic time of both process (rotations) increases and the florescence anisotropy of ThT remain extremely high (Fig. 6 B) as while binding to amyloid fibrils or being diluted in 99 % glycerol.

3. Conclusion In the current work, an approach for investigation of acetylcholinesterase (AChE) interaction with the specific fluorescent probe thioflavin T (ThT) based on the using of solutions prepared by equilibrium microdialysis was proposed. The comparative study of the photophysical properties of free and AChE-bound fluorescent probe ThT and the evaluation of the binding parameters of ThT – AChE complex determined by this approach allowed us to prove the 1:1 ThT – AChE stoichiometry and the groundlessness of the ACS Paragon Plus Environment

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Figure 6. Time dependence of fluorescence of ThT bound to AChE. (a) Decay curve of the bound to AChE dye fluorescence. The excitation laser impulse profile (1), experimental decay curve of the bound dye fluorescence (2), best-fit calculated fluorescence decay curve (3), and deviation between the experimental and calculated decay (4) are shown. The fluorescence decay curve show best fit to a biexponential decay model. (b) Anisotropy of the bound to AChE dye. The excitation laser impulse profile (1), the decay curves of the vertical (2) and horizontal (3) components of the fluorescence, and the change in fluorescence anisotropy (4) over time are shown.

hypotheses regarding the aggregation of bound dye. We have shown that performed in corpore this approach enables the determination not only of ThT – AChE binding parameters but also, for the first time, of the absorption spectrum, molar extinction coefficient and fluorescence quantum yield of ThT bound to AChE. A significant increase of ThT fluorescence lifetime and quantum yield when it binds to AChE (that was determined with the use of the fluorescence intensity of the specially prepared solutions corrected on the inner filter effect) was explained by the dye’s molecular rotor nature. The results of the docking simulation allowed us to propose a model of ThT location in the active site of AChE and suggest a key role of aromatic residues in ThT – AChE interaction, as observed for some amyloid fibrils (39-41). ACS Paragon Plus Environment

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4. Materials and Methods 4.1. Materials Thioflavin T "UltraPure Grade" from AnaSpec (USA); N-acetyl-tryptophan amide (NATA) and Electrophorus electricus acetylcholinesterase (AChE, purified from the electric organ of the eel E. electricus) from Sigma-Aldrich (USA) and ATTO-425 from ATTO-TEC (Germany) were used. All samples were used without additional purification. Distilled water (for ThT) or PBS (for NATA and ATTO-425) were used as the solvent. Commercial chemical of AChE (1256 U/mg protein, 827 U/mg solid) was dissolved in 40 mM sodium phosphate buffer at pH 7.0 at a concentration of 1 mg/mL, yielding a clear solution. AChE concentration was controlled by absorption spectroscopy using the extinction coefficient ε1% = 1.8. The purity of the commercial preparation was monitored using electrophoresis in native and denaturing conditions.

4.2. Spectral Measurements The absorption spectra were recorded using a U-3900H spectrophotometer (Hitachi, Japan). For the experiments with a wide range of concentrations, Helma cells (Germany) with different optical path lengths (0.1, 0.2, 0.5, 1, and 5 cm) were used. The concentration of ThT in solutions was determined using a molar extinction coefficient of ε412 = 3.16·104 M-1cm-1 (according to the results of our measurements). Fluorescent measurements were performed using a Cary Eclipse spectrofluorimeter (Agilent

Technologies,

intensity F ( λex ) =

∫ F (λ λ

ex

Australia).

The

total

fluorescence

, λ em )d λ em (where F(λex,λem) is the fluorescence intensity excited

em

at the wavelength λex and recorded at the wavelength λem) and the fluorescence spectra were determined using 425-nm wavelength excitation light. The spectral slits 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 (see below and detailed description in (23)). PBS solutions of the fluorescent dye АТТО-425 were used as a reference to normalize the recorded and corrected values of the ThT fluorescence intensity. These values are presented as the product of the absorbance and the fluorescence quantum yield of the dye. The fluorescence quantum yield of АТТО425 was taken as 0.9 (ATTO-TEC Catalogue 2009/2010 p.14). All experiments were performed at room temperature (23°C).


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4.3. Equilibrium Microdialysis 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 10000).

4.4. Molecular Modeling Rough spatial model of thioflavin T (ThT) was obtained using Molsoft ICM version 3.8 (42) and Avogadro (43) on the basis of record 16062 (thioflavin T) in the ChemSpider database (chemspider.com). The structure of ThT was further optimized in the Gamess US program (44). The charge state of the molecule was treated as +1. As the Protein Data Bank contains only low-resolution spatial structures for AChE from Electrophorus electricus (EeTChE), which was used in our experimental measurements, for our molecular modeling, we chose the highly homologous AChE from Torpedo californica (TcAChE), with a better quality crystal structure at 2.8 Å resolution (PDB code 2J3Q (10)). In this entry, ThT is located at the entrance to the active site of TcAChE. Chemicals and ions cocrystallized with the protein were removed. A standard regularization procedure was performed to eliminate minor van der Waals clashes of atoms. Twelve amino acid substitutions were made to mimic the amino acid sequence of EeTChE at the active site and in its vicinity. Each substitution was followed by conformational optimization of the mutated amino acid and its neighbors within 5 Å. The optimized TcAChE model was used for flexible docking with ThT, conducted in Molsoft ICM version 3.8(31). Mulliken point charges calculated during ab initio geometry optimization were assigned to each atom of ThT prior to docking. Ten independent runs of molecular docking, starting from different initial spatial locations of ThT in the vicinity of the protein active center, were performed. The obtained poses of ThT were ascribed to the A- and P-site depending on their location relative to the active site entrance. The distribution of the torsion angle between the phenyl and benzothiazole rings was calculated for the ThT poses in the P-site.

4.5. Time-resolved Fluorescence Measurements The fluorescence decay curves in the subnanosecond and nanosecond ranges were recorded by a spectrometer FluoTime 300 (Pico Quant, Germany) with the Laser Diode Head LDH-C-440 (λex = 440 nm). The measured emission decays were fit to a multiexponential function using the standard convolute-and-compare nonlinear leastACS Paragon Plus Environment

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squares procedure (45). 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. The fitting routine was based on the nonlinear leastsquares method. Minimization was performed according to Marquardt (46). Fluorescence anisotropy was determined as: r = ( FVV − GFHV )

(F

V V

+ 2GFHV ) , where

FVV and FHV are vertical and horizontal components of fluorescence intensity excited by vertical polarized light, and G = FV

H

FHH is coefficient which determines the different

sensitivity of the registering system for vertical and horizontal components of fluorescence intensity.

ASSOCIATED CONTENT Supporting information There supplementary material contains 1) the explanation of the nonlinearity of the sample fluorescence intensity dependence on its concentration and the method of the fluorescence intensity correction for the primary inner filter effect, 2) the method of the determination of ThT-AChE binding parameters and photophysical characteristics of the bound dye and 3) the description of molecular modeling performing.

AUTHOR INFORMATION Corresponding Author * Mailing address: Laboratory of Structural dynamics, stability and folding of proteins, Institute of Cytology of the Russian Academy of Science, St. Petersburg, Tikhoretsky ave. 4, 194064, Russia. E-mail: [email protected]. Phone: +7 812 297 19 57. ORCID Anna I. Sulatskaya 0000-0002-1207-3384 Georgy N. Rychkov 0000-0003-2767-9175 Maxim I. Sulatsky 0000-0003-4665-3181 Natalia P. Rodina 0000-0001-5860-7014 Irina M. Kuznetsova 0000-0002-3336-4834 Konstantin K. Turoverov 0000-0002-6977-1896


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Author Contributions IMK, KKT conceived and AIS, GNR designed the experiments. AIS, MIS, NPR performed the spectroscopic experiments and GNR performed the molecular modeling. AIS, GNR, MIS, NPR, IMK, KKT analyzed the data and wrote the paper.

Acknowledgements This work was supported by the "Molecular and Cell Biology" Program of the Russian Academy of Sciences, Russian Foundation of Basic Research (grants numbers 1604-01614 and 18-54-00022_Bel), the RF President Fellowship (number SP-841.2018.4).

Conflict of interest The authors have declared no conflicts of interest.


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References 1.

Pohanka, M. (2009) Cholinesterases, a target of pharmacology and

toxicology, Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 155, 219-229. 2.

Martyn, J. A., Fagerlund, M. J., and Eriksson, L. I. (2009) Basic principles

of neuromuscular transmission, Anaesthesia 64 Suppl 1, 1-9. 3.

Inestrosa, N. C., Alvarez, A., Perez, C. A., Moreno, R. D., Vicente, M.,

Linker, C., Casanueva, O. I., Soto, C., and Garrido, J. (1996) Acetylcholinesterase accelerates assembly of amyloid-beta-peptides into Alzheimer's fibrils: possible role of the peripheral site of the enzyme, Neuron 16, 881-891. 4.

Atack, J. R., Perry, E. K., Bonham, J. R., Perry, R. H., Tomlinson, B. E.,

Blessed, G., and Fairbairn, A. (1983) Molecular forms of acetylcholinesterase in senile dementia of Alzheimer type: selective loss of the intermediate (10S) form, Neurosci Lett 40, 199-204. 5.

Gilboa-Geffen, A., Hartmann, G., and Soreq, H. (2012) Stressing

hematopoiesis and immunity: an acetylcholinesterase window into nervous and immune system interactions, Front Mol Neurosci 5, 30. 6.

Mahadeva, B., Phillips, L. H., 2nd, and Juel, V. C. (2008) Autoimmune

disorders of neuromuscular transmission, Semin Neurol 28, 212-227. 7.

Younkin, S. G., Goodridge, B., Katz, J., Lockett, G., Nafziger, D., Usiak,

M. F., and Younkin, L. H. (1986) Molecular forms of acetylcholinesterases in Alzheimer's disease, Fed Proc 45, 2982-2988. 8.

Arendt, T., Bruckner, M. K., Lange, M., and Bigl, V. (1992) Changes in

acetylcholinesterase and butyrylcholinesterase in Alzheimer's disease resemble embryonic development--a study of molecular forms, Neurochem Int 21, 381-396. 9.

Fishman, E. B., Siek, G. C., MacCallum, R. D., Bird, E. D., Volicer, L., and

Marquis, J. K. (1986) Distribution of the molecular forms of acetylcholinesterase in human brain: alterations in dementia of the Alzheimer type, Ann Neurol 19, 246-252. 10.

Harel, M., Sonoda, L. K., Silman, I., Sussman, J. L., and Rosenberry, T. L.

(2008) Crystal structure of thioflavin T bound to the peripheral site of Torpedo californica acetylcholinesterase reveals how thioflavin T acts as a sensitive fluorescent reporter of ligand binding to the acylation site, J Am Chem Soc 130, 7856-7861. 11.

LeVine, H., 3rd. (1993) Thioflavine T interaction with synthetic Alzheimer's

disease beta-amyloid peptides: detection of amyloid aggregation in solution, Protein Sci. 2, 404-410.


20

Page 21 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


12.

LeVine, H., 3rd. (1999) Quantification of beta-sheet amyloid fibril

structures with thioflavin T, Methods Enzymol. 309, 274-284. 13.

Naiki, H., Higuchi, K., Hosokawa, M., and Takeda, T. (1989) Fluorometric

determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1, Anal. Biochem. 177, 244-249. 14.

Auletta, J. T., Johnson, J. L., and Rosenberry, T. L. (2010) Molecular basis

of inhibition of substrate hydrolysis by a ligand bound to the peripheral site of acetylcholinesterase, Chem Biol Interact 187, 135-141. 15.

Dounin, V., Constantinof, A., Schulze, H., Bachmann, T. T., and Kerman,

K. (2011) Electrochemical detection of interaction between Thioflavin T and acetylcholinesterase, Analyst 136, 1234-1238. 16.

Sultatos, L. G., and Kaushik, R. (2008) Altered binding of thioflavin t to the

peripheral anionic site of acetylcholinesterase after phosphorylation of the active site by chlorpyrifos oxon or dichlorvos, Toxicol Appl Pharmacol 230, 390-396. 17.

Antokhin, A. M., Gainullina, E. T., Ryzhikov, S. B., Taranchenko, V. F.,

and Yavaeva, D. K. (2009) Rapid method for measurement of acetylcholinesterase activity, Bull Exp Biol Med 147, 109-110. 18.

Rosenberry, T. L., Sonoda, L. K., Dekat, S. E., Cusack, B., and Johnson, J.

L. (2008) Monitoring the reaction of carbachol with acetylcholinesterase by thioflavin T fluorescence and acetylthiocholine hydrolysis, Chem Biol Interact 175, 235-241. 19.

Groenning, M., Olsen, L., van de Weert, M., Flink, J. M., Frokjaer, S., and

Jorgensen, F. S. (2007) Study on the binding of Thioflavin T to beta-sheet-rich and nonbeta-sheet cavities, J. Struct. Biol. 158, 358-369. 20.

Rosenberry, T. L., Sonoda, L. K., Dekat, S. E., Cusack, B., and Johnson, J.

L. (2008) Analysis of the reaction of carbachol with acetylcholinesterase using thioflavin T as a coupled fluorescence reporter, Biochemistry 47, 13056-13063. 21.

De Ferrari, G. V., Mallender, W. D., Inestrosa, N. C., and Rosenberry, T. L.

(2001) Thioflavin T is a fluorescent probe of the acetylcholinesterase peripheral site that reveals conformational interactions between the peripheral and acylation sites, The Journal of biological chemistry 276, 23282-23287. 22.

Johnson, J. L., Cusack, B., Davies, M. P., Fauq, A., and Rosenberry, T. L.

(2003) Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate, Biochemistry 42, 5438-5452.


21


23.

Page 22 of 34

Fonin, A. V., Sulatskaya, A. I., Kuznetsova, I. M., and Turoverov, K. K.

(2014) Fluorescence of dyes in solutions with high absorbance. Inner filter effect correction, PloS one 9, e103878. 24.

Oravcova, J., Bohs, B., and Lindner, W. (1996) Drug-protein binding sites.

New trends in analytical and experimental methodology, J Chromatogr B Biomed Appl 677, 1-28. 25.

Rahman, M. H., Maruyama, T., Okada, T., Yamasaki, K., and Otagiri, M.

(1993) Study of interaction of carprofen and its enantiomers with human serum albumin--I. Mechanism of binding studied by dialysis and spectroscopic methods, Biochemical pharmacology 46, 1721-1731. 26.

Shcharbin, D., Szwedzka, M., and Bryszewska, M. (2007) Does

fluorescence of ANS reflect its binding to PAMAM dendrimer?, Bioorg Chem 35, 170174. 27.

Maskevich, A. A., Stsiapura, V. I., Kuzmitsky, V. A., Kuznetsova, I. M.,

Povarova, O. I., Uversky, V. N., and Turoverov, K. K. (2007) Spectral properties of thioflavin T in solvents with different dielectric properties and in a fibril-incorporated form, J. Proteome Res. 6, 1392-1401. 28.

Kuznetsova, I. M., Sulatskaya, A. I., Uversky, V. N., and Turoverov, K. K.

(2012) A new trend in the experimental methodology for the analysis of the thioflavin T binding to amyloid fibrils, Molecular neurobiology 45, 488-498. 29.

Sulatskaya, A. I., Kuznetsova, I. M., Belousov, M. V., Bondarev, S. A.,

Zhouravleva, G. A., and Turoverov, K. K. (2016) Stoichiometry and Affinity of Thioflavin T Binding to Sup35p Amyloid Fibrils, PloS one 11, e0156314. 30.

Krebs, M. R., Bromley, E. H., and Donald, A. M. (2005) The binding of

thioflavin-T to amyloid fibrils: localisation and implications, J. Struct. Biol. 149, 30-37. 31.

Neves, M. A., Totrov, M., and Abagyan, R. (2012) Docking and scoring

with ICM: the benchmarking results and strategies for improvement, J Comput Aided Mol Des 26, 675-686. 32.

Sulatskaya, A. I., Maskevich, A. A., Kuznetsova, I. M., Uversky, V. N., and

Turoverov, K. K. (2010) Fluorescence quantum yield of thioflavin T in rigid isotropic solution and incorporated into the amyloid fibrils, PloS one 5, e15385. 33.

Rodina, N. P., Sulatsky, M. I., Sulatskaya, A. I., Kuznetsova, I. M.,

Uversky, V. N., and Turoverov, K. K. (2017) Photophysical properties of fluorescent probe thioflavin T in crowded milieu, Journal of Spectroscopy 2017, ID 2365756: 23657512365710. ACS Paragon Plus Environment

22

Page 23 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


34.

Wolfe, L. S., Calabrese, M. F., Nath, A., Blaho, D. V., Miranker, A. D., and

Xiong, Y. (2010) Protein-induced photophysical changes to the amyloid indicator dye thioflavin T, Proc Natl Acad Sci U S A 107, 16863-16868. 35. fluorescence

LeVine, H., 3rd. (2005) Mechanism of A beta(1-40) fibril-induced of

(trans,trans)-1-bromo-2,5-bis(4-hydroxystyryl)benzene

(K114),

Biochemistry 44, 15937-15943. 36.

Khurana, R., Coleman, C., Ionescu-Zanetti, C., Carter, S. A., Krishna, V.,

Grover, R. K., Roy, R., and Singh, S. (2005) Mechanism of thioflavin T binding to amyloid fibrils, J. Struct. Biol. 151, 229-238. 37.

Sulatskaya, A. I., Lavysh, A. V., Maskevich, A. A., Kuznetsova, I. M., and

Turoverov, K. K. (2017) Thioflavin T fluoresces as excimer in highly concentrated aqueous solutions and as monomer being incorporated in amyloid fibrils, Scientific reports 7, 2146. 38.

Kuznetsova, I. M., Sulatskaya, A. I., Maskevich, A. A., Uversky, V. N., and

Turoverov, K. K. (2016) High Fluorescence Anisotropy of Thioflavin T in Aqueous Solution Resulting from Its Molecular Rotor Nature, Analytical chemistry 88, 718-724. 39.

Marek, P., Abedini, A., Song, B., Kanungo, M., Johnson, M. E., Gupta, R.,

Zaman, W., Wong, S. S., and Raleigh, D. P. (2007) Aromatic interactions are not required for amyloid fibril formation by islet amyloid polypeptide but do influence the rate of fibril formation and fibril morphology, Biochemistry 46, 3255-3261. 40.

Padrick, S. B., and Miranker, A. D. (2001) Islet amyloid polypeptide:

identification of long-range contacts and local order on the fibrillogenesis pathway, J Mol Biol 308, 783-794. 41.

Kajava, A. V., Aebi, U., and Steven, A. C. (2005) The parallel superpleated

beta-structure as a model for amyloid fibrils of human amylin, J Mol Biol 348, 247-252. 42.

Abagyan, R., Totrov, M., Kuznetsov, D. (1994) ICM-A New Method for

Protein Modeling and Design: Applications to Docking and Structure Prediction from the Distorted Native Conformation, Journal of computational chemistry 15, 488-506. 43.

Hanwell, M. D., Curtis, D. E., Lonie, D. C., Vandermeersch, T., Zurek, E.,

and Hutchison, G. R. (2012) Avogadro: an advanced semantic chemical editor, visualization, and analysis platform, J Cheminform 4, 17. 44.

Schmidt, M. W., Baldridge, K.K., Boatz, J.A., Elbert, S.T., Gordon, M.S., et

al. (1993) General atomic and molecular electronic structure system, Journal of computational chemistry 14, 1347-1363.


23


45.

Page 24 of 34

O’Connor, D. V. P., D. (1984) Time-correlated Single Photon Counting,

New-York, Academic Press, 37-54. 46.

Marquardt, D. W. (1963) An algorithm for least-squares estimation of non

linear parameters, J. Soc. Ind. Appl. Math. 11, 431-441.


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Figure Legends: Figure 1. Three-dimensional structure of Electrophorus electricus acetylcholinesterase (the figure was created on the basis of PDB data, file 1EEA). (A) Side view and (B) front view on the narrow and deep gorge that contains the ligand binding site are shown. Amino acid residues forming this gorge are also presented.

Figure 2. The use of equilibrium microdialysis for examination photophysical properties of ThT bound to AChE. (A) The sample and reference solutions were prepared using the device for equilibrium microdialysis, which consists of two chambers (500 µL each) separated by a membrane (MWCO 10,000) impermeable to particles larger than 10,000 Da. AChE in the buffer solution at concentration Cb was placed in one chamber and ThT solution in the same buffer, with an initial concentration of С0, was placed in the second chamber. These solutions were incubated in a thermostat and with constant stirring until reaching equilibrium. (B) At equilibrium we have a sample solution (containing free and AChE-bound ThT) and an optimal reference solution (containing free ThT in a concentration equal to the concentration in the sample solution) for determination of the absorption spectrum of ThT bound to AChE. (C) Photophysical properties of free and AChE-bound ThT were investigated. Absorption spectra of ThT bound to AChE (curve 3) determined with the use of absorption spectra of ThT in the sample (curve 2) and reference (curve 1) solutions after equilibrium microdialysis are shown. The absorption spectrum of free ThT at equal concentration to ThT bound to AChE (curve 4) and fluorescence spectra of ThT in the sample (curve 6) and reference (curve 5) solutions after equilibrium microdialysis are also presented.

Figure 3. Determination of ThT – AChE binding parameters of bound to AChE dye. (A) Dependence of bound dye concentration on the concentration of free dye in solution, (B) Scatchard plot which gives visual representation of the number of binding modes. Experimental data (circles) and curves determined through the use of calculated values of the binding constant (Kb) and number of binding sites (n) of ThT to AChE are given. Figure 4. Possible location of ThT at the P-site of AChE. The pose with the minimal binding score is illustrated. The dimethylamino-phenyl ring of ThT lies outside the active site gorge. (A) General view of AChE spatial structure is presented; ThT molecule is in ball representation. (B) Close view of ThT in the P-site is shown. ThT and amino acid ACS Paragon Plus Environment

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residues in 5 Å vicinity of ThT are in ball-and-stick representations. Residues Asp72 and W279 in the P-site are emphasized.

Figure 5. Normalized absorption (dashed lines) and fluorescence (solid lines) spectra of ThT bound to AChE (A), dye monomers (blue lines) and excimers (red lines) in water solution (B) and probe incorporated into insulin amyloid fibrils (C).

Figure 6. Time dependence of fluorescence of ThT bound to AChE. (a) Decay curve of the bound to AChE dye fluorescence. The excitation laser impulse profile (1), experimental decay curve of the bound dye fluorescence (2), best fit calculated fluorescence decay curve (3), and deviation between the experimental and calculated decay (4) are shown. The fluorescence decay curve show best fit to a biexponential decay model. (b) Anisotropy of the bound to AChE dye. The excitation laser impulse profile (1), the decay curves of the vertical (2) and horizontal (3) components of the fluorescence, and the change in fluorescence anisotropy (4) over time are shown.


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For table of contents use only Thioflavin T Interaction with Acetylcholinesterase: New Evidence of 1:1 Binding Stoichiometry Obtained with Samples Prepared by Equilibrium Microdialysis

A.I. Sulatskaya, G.N. Rychkov, M.I. Sulatsky, N.P. Rodina, I.M. Kuznetsova and K.K. Turoverov


27


Figure 1. Three-dimensional structure of Electrophorus electricus acetylcholinesterase (the figure was created on the basis of PDB data, file 1EEA). (A) Side view and (B) front view on the narrow and deep gorge that contains the ligand binding site are shown. Amino acid residues forming this gorge are also presented. 60x25mm (300 x 300 DPI)


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Figure 2. The use of equilibrium microdialysis for examination photophysical properties of ThT bound to AChE. (A) The sample and reference solutions were prepared using the device for equilibrium microdialysis, which consists of two chambers (500 µL each) separated by a membrane (MWCO 10,000) impermeable to particles larger than 10,000 Da. AChE in the buffer solution at concentration Cb was placed in one chamber and ThT solution in the same buffer, with an initial concentration of С0, was placed in the second chamber. These solutions were incubated in a thermostat and with constant stirring until reaching equilibrium. (B) At equilibrium we have a sample solution (containing free and AChE-bound ThT) and an optimal reference solution (containing free ThT in a concentration equal to the concentration in the sample solution) for determination of the absorption spectrum of ThT bound to AChE. (C) Photophysical properties of free and AChE-bound ThT were investigated. Absorption spectra of ThT bound to AChE (curve 3) determined with the use of absorption spectra of ThT in the sample (curve 2) and reference (curve 1) solutions after equilibrium microdialysis are shown. The absorption spectrum of free ThT at equal concentration to ThT bound to AChE (curve 4) and fluorescence spectra of ThT in the sample (curve 6) and reference (curve 5) solutions after equilibrium microdialysis are also presented. 117x128mm (300 x 300 DPI)




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Figure 3. Determination of ThT – AChE binding parameters of bound to AChE dye. (A) Dependence of bound dye concentration on the concentration of free dye in solution, (B) Scatchard plot which gives visual representation of the number of binding modes. Experimental data (circles) and curves determined through the use of calculated values of the binding constant (Kb) and number of binding sites (n) of ThT to AChE are given. 143x59mm (300 x 300 DPI)



Figure 4. Possible location of ThT at the P-site of AChE. The pose with the minimal binding score is illustrated. The dimethylamino-phenyl ring of ThT lies outside the active site gorge. (A) General view of AChE spatial structure is presented; ThT molecule is in ball representation. (B) Close view of ThT in the Psite is shown. ThT and amino acid residues in 5 Å vicinity of ThT are in ball-and-stick representations. Residues Asp72 and W279 in the P-site are emphasized. 82x39mm (300 x 300 DPI)


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Figure 5. Normalized absorption (dashed lines) and fluorescence (solid lines) spectra of ThT bound to AChE (A), dye monomers (blue lines) and excimers (red lines) in water solution (B) and probe incorporated into insulin amyloid fibrils (C). 104x179mm (300 x 300 DPI)



Figure 6. Time dependence of fluorescence of ThT bound to AChE. (a) Decay curve of the bound to AChE dye fluorescence. The excitation laser impulse profile (1), experimental decay curve of the bound dye fluorescence (2), best fit calculated fluorescence decay curve (3), and deviation between the experimental and calculated decay (4) are shown. The fluorescence decay curve show best fit to a biexponential decay model. (b) Anisotropy of the bound to AChE dye. The excitation laser impulse profile (1), the decay curves of the vertical (2) and horizontal (3) components of the fluorescence, and the change in fluorescence anisotropy (4) over time are shown. 105x117mm (300 x 300 DPI)


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For table of contents use only

Thioflavin T Interaction with Acetylcholinesterase: New Evidence of 1:1 Binding Stoichiometry Obtained with Samples Prepared by Equilibrium Microdialysis A.I. Sulatskaya, G.N. Rychkov, M.I. Sulatsky, N.P. Rodina, I.M. Kuznetsova and K.K. Turoverov

80x39mm (300 x 300 DPI)


Thioflavin T Interaction with Acetylcholinesterase: New Evidence of 1:1

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