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Kinetic Mechanism of Thioflavin T Binding onto the Amyloid Fibril of Hen Egg White Lysozyme Zhe Qin, Ying Sun, Baohuan Jia, Dan Wang, Yan Ma, and Gang Ma Langmuir, Just Accepted Manuscript • Publication Date (Web): 16 May 2017 Downloaded from http://pubs.acs.org on May 17, 2017

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Kinetic Mechanism of Thioflavin T Binding onto the Amyloid Fibril of Hen Egg White Lysozyme Zhe Qin, Ying Sun, Baohuan Jia, Dan Wang, Yan Ma, and Gang Ma* Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding 071002 KEYWORDS. Thioflavin T, Amyloid, Fibrillation, Lysozyme, Kinetics, Dye

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ABSTRACT.

Thioflavin T (ThT) is widely used as a fluorescent probe for amyloid fibril detection. Yet, the exact kinetic mechanism of ThT binding onto amyloid fibril remains elusive. Previously reported kinetic studies using ThT-fluorescence-detected kinetic design suggested two completely different ThT binding mechanisms. In one study, a multi-step sequential binding mechanism onto a single ThT binding site was suggested. In another study, a one-step parallel binding mechanism onto multiple ThT binding sites was suggested. The discrepancy is likely due to the incapability of ThT-fluorescence-detected kinetic design to differentiate the two abovementioned mechanisms. Considering the weakness of the ThT-fluorescence-detected approach, we here investigated ThT binding mechanism onto the amyloid fibril of hen egg white lysozyme (HEWL) using a new approach, ThT-absorbance-detected kinetic design. Our new results suggest that ThT binds to HEWL fibril through the one-step parallel binding mechanism. We hope our work can offer some new insights into the interactions between dye molecules and amyloid fibrils.


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Introduction Amyloid fibril is a unique type of protein aggregates featuring fibrillar microscopic morphology and cross-β quaternary structure.1-2 Deposition of amyloid fibrils in tissues and organs is a pathologic hallmark of a wide range of devastating human diseases, including the well-known Alzheimer’s disease, Parkinson’s disease, and type II diabetes.3 Detection of amyloid fibril is of great importance in the mechanistic studies of amyloid formation and in the clinical diagnostics of amyloid-related diseases. Though the gold standard technique to unequivocally confirm the presence of amyloid structure is to use X-ray diffraction, such approach involves a complicated sample preparation procedure to properly align amyloid fibrils, thus prohibiting its routine use.4-7 So far, the routinely employed technique to detect amyloid fibril is based on a small fluorescent dye, thioflavin T (ThT), as shown in Scheme 1.8 In aqueous solution, upon binding to amyloid fibrils, ThT fluoresces brightly at ~480 nm when excited at ~450 nm; whereas in the absence of amyloid fibrils, the fluorescence of this dye quenches under the same excitation condition. Such dramatic fluorescent enhancement is generally believed to be specific to amyloid structure, making ThT gain broad acceptance as a defining agent for amyloid structure. In history, ThT was first reported to be able to stain amyloid plague and display fluorescence by Vassar and Culling in 1959.9 Later in 1965, ThT fluorescent assay was suggested by Rogers to be used as a rapid screening technique for histologic demonstration of amyloid.10 In the 1980s and 1990s, ThT fluorescent assay were thoroughly characterized by Naiki et al and Levine and its use was further extended to in vitro detection of amyloid fibrils.8,11 Nowadays, this assay has developed into a standard quantification tool in the in vitro investigations of amyloid formation.


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The wide use of ThT in amyloid fibril detection has sparked great interests among physical scientists to investigate the fluorescent properties and binding mechanisms of ThT. Different theories exist to explain the unique fluorescent enhancement of ThT upon binding to amyloid fibril. Some researchers believe that the unique fluorescent enhancement of ThT upon binding to amyloid fibril is due to the molecular rotor behavior of ThT.12-15 As shown in Scheme 1, ThT has a single C-C bond connecting its benzothiazole and aniline rings. The two aromatic rings can rotate freely around this C-C bond. Upon excitation, such rotation can cause ThT relax from its radiative locally excited (LE) state to a nonradiative charge transfer (CT) state. This phenomenon, called twisted-internal-CT (TICT), is responsible for the molecular rotor behavior of ThT and results in ThT fluorescence quenching. When ThT binds onto an amyloid fibril, the rotation around the C-C bond is inhibited and the TICT process is thus suppressed. Consequently, the relaxation from the radiative excited-state to the ground state leads to ThT fluorescence. On the other hand, some researchers believe the formation of ThT dimer or excimer is responsible for the enhanced fluorescence of ThT upon binding to amyloid fibril.16-17 Considerable effort was also devoted to understand the binding mechanism of ThT. As for the binding mode, several models have been proposed, including the excimer model,16-17 the micelle model,18 and the Krebs model.19-20 The excimer model suggests that the structure of fibril-bound ThT is dimer or oligomer. The micelle model suggests that ThT binds to amyloid fibril as micelle. In the Krebs model, ThT binds in a monomeric form along the fibril axis in the grooves formed by the aromatic side chains of amino acid residues on the surface of amyloid fibril.21-23 In addition, it is now accepted that amyloid fibril can have multiple ThT binding sites;24-28 and the fibril-bound ThT may not be all fluorescence-active and some fibril-bound ThT may have very low quantum yield.29-30


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Despite the significant progresses that scientists have made towards better understanding of the interaction between ThT and amyloid fibril, one fundamental issue is still largely unexplored. That is the kinetic mechanism of ThT binding onto amyloid fibril. To our knowledge, only LeVine and Sabate et al had looked into this issue.25,

31

Yet, two completely different ThT

binding mechanisms were proposed. In the study by LeVine, he investigated the binding kinetics of ThT onto the amyloid fibrils by Aβ(1-40) peptides.31 Through multi-exponential fitting of the kinetic trace, Levine revealed the presence of four kinetic phases. To interpret his observation, Levine suggested a four-step sequential binding mechanism onto a single ThT binding site as shown in Scheme 2, including an initial bimolecular binding step and three rate-limiting consecutive steps involving amyloid tertiary or quaternary conformational changes. Intuitively, LeVine’s mechanism mimics the protein folding process involving sequential conformational changes. In the study by Sabate et al, they investigated the binding kinetics of ThT onto HET-s amyloid fibrils. They observed that the kinetic trace could be fitted into two exponentials at pH=7 and a single exponential at pH=2. They used a one-step parallel binding mechanism onto multiple ThT binding sites to interpret the observed two-exponential fitting at pH=7. Intuitively, the mechanism proposed by Sabate et al’s mimics the ligand binding process onto multiple independent sites on a receptor. The generalized form of this parallel binding mechanism is described in Scheme 3. In addition, Sabate et al speculated that the observed single-exponential fitting at pH=2 was due to the fact that there is only one predominated ThT binding site at low pH. In both of the two studies, researchers used ThT-fluorescence-detected kinetic design. Namely, ThT was kept in excess of amyloid fibrils to satisfy the pseudo-first-order condition and the binding kinetics of ThT was monitored by measuring the fluorescence increase of fibrilbound ThT. We believe that the discrepancy between the two previous studies is likely due to the


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fact that the ThT-fluorescence-detected kinetic design is unable to differentiate the two proposed mechanisms as both the multi-step sequential binding mechanism and the one-step parallel binding mechanism can interpret the observed multi-exponential fitting of the kinetic trace. Considering the weakness of the ThT-fluorescence-detected kinetic design, we here performed a kinetic investigation of ThT binding onto amyloid fibril of hen egg white lysozyme (HEWL) using a new kinetic design, ThT-absorbance-detected kinetic design. Namely, we monitor the absorbance decrease of free unbound ThT rather than the fluorescence increase of fibril-bound ThT during amyloid binding. Our new results suggest that the kinetic interaction between ThT and the amyloid fibril of HEWL follow the one-step parallel binding mechanism.

Experimental Materials Hen egg white lysozyme (HEWL) (L6876) was purchased from Sigma-Aldrich (Saint Louis, USA). Thioflavin T (ThT) of ultrapure grade was purchased from AnaSpec (Fremont, USA). Congo red (CR) with >85% purity was purchased from Sigma-Aldrich (Saint Louis, USA). Guanidine hydrochloride (GdnHCl) with >99% purity was purchased from Sigma-Aldrich (Saint Louis, USA). Potassium phosphate dibasic with >99% and potassium phosphate monobasic with >99% were purchased from Aladdin (Shanghai, China). Deionized water with a resistivity of 18.2 MΩ·cm was obtained from a Millipore system (Billerica, USA). Sulfuric acid (H2SO4) (98%) and hydrogen peroxide (H2O2) (30%) were obtained from local vendors. HEWL amyloid fibril preparation and sample pretreatment HEWL amyloid fibrils were prepared according to previously published method by Vernaglia et al.32 Briefly, a 2mg/mL HEWL incubation solution was prepared in 20 mM potassium phosphate buffer (pH=6.3) with 3M GdnHCl. The incubation solution was first filtered with a


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0.22 µm filter and then incubated at 50 °C in a thermal mixer with 900 rpm for 4 hrs. Upon completion, the HEWL incubation solution turned from transparent to turbid. The glass incubation vials were thoroughly cleaned using Piranha solution (3 parts of concentrated H2SO4 and 1 part of 30% H2O2) before use. The as-prepared HEWL fibril was characterized by ThT fluorescence assay, CR assay, and atomic force microscopy (AFM), and Fourier transform infrared (FTIR) spectroscopy. It should be pointed out that throughout the work the concentration of HEWL amyloid fibril was denoted using the concentration of HEWL monomer that forms fibrils. To prepare the fibril sample for kinetic study, the sample was further processed according to the following protocol. The fibril sample first went through centrifugation at 10000 rpm for 10 mins. The supernatant was discarded and the fibril sample was suspended again in pure water. This centrifugation-washing procedure was repeated for three times to ensure the complete removal of GdnHCl and phosphate salts. The desalted fibril sample was further suspended in pure water and was then subjected to sonication for 5 mins in an ice bath using an ultrasonic probe sonicator (Ningbo, China). After such sample pretreatment, the fibril suspension become a stable colloidal system and is ready for kinetic studies. ThT Fluorescence Assay The assay was used to monitor the completion of HEWL fibrillation as well as to confirm the amyloid nature of the HEWL fibrils. The fluorescence measurement was performed with a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan). An excitation wavelength of 450 nm with a slit width of 5 nm and the emission wavelength range of 460-600 nm with a slit width of 10 nm were chosen throughout the experiment. The PMT voltage of the detector was set to be 500V. For the measurement, 10 µL of HEWL fibril solution was added into 1 mL of 10 µM ThT


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solution in a 1.0 cm quartz cuvette. The spectrum of ThT solution with native HEWL was also measured under identical conditions for comparison. CR Assay The assay was performed with an Implen UV−Vis nanophotometer (München, Germany). The concentration of CR solution was 5 µM. The buffer was 10 mM phosphate saline buffer (pH = 7.4) containing 0.5% ethanol. For the measurement, 30 µL of 2mg/ml HEWL fibrils solution was added into 1 mL of CR solution in a 1.0 cm quartz cuvette. The spectrum of CR solution with native HEWL was also measured under identical conditions for comparison. Atomic Force Microscopy (AFM) Upon completion of HEWL fibrillation, the incubation solution was taken out of the incubation vial and diluted with deionized water from a Millipore system (Billerica, MA). A 100 µL aliquot of the diluted solution was then deposited onto a freshly cleaved mica surface. After a 10 min waiting time, the mica surface was rinsed with deionized water and dried in a desiccator. The AFM scanning on the mica surface was performed in air with NT-MDT Solver P47 scanning probe microscope (Zelenograd, Russia) in tapping mode. A 100 µm×100 µm scanner was used throughout the AFM experiment. The AFM probe (model # HC_NC) was purchased from NT-MDT. The probe cantilever has a resonance frequency of ∼140 kHz and a force constant of ∼3.5 N/m. The AFM image analysis was performed by the NT-MDT software, NOVA. FTIR Spectroscopy All measurements were carried out on a Bruker Vertex 70 FTIR spectrometer (Ettlingen, Germany) equipped with a DLaTGS detector. The HEWL amyloid fibril prepared in solution was first gone through a centrifugation-washing procedure to remove GdnHCl. And then, the


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desalted fibril sample was lyophilized. The FTIR spectrum of the lyophilized HEWL fibril was measured in attenuated total reflection (ATR) mode with 4 cm-1 resolution and 32 scans. A Pike Technologies MIRacle single-reflection ATR accessory (Madison, USA) with a diamond element was used. The final spectrum was further smoothed with Bruker’s OPUS software (version 7.2) to remove water vapor interference and noise using Savitzky-Golay algorithm with a 25-point window size. For comparison, the FTIR spectrum of the powder of native HEWL directly from the vendor was also measured. In addition, the two FTIR spectra were normalized. Kinetic Experiment The kinetic experiment was performed by a Bio-Logic SFM-300 stopped-flow instrument (Paris, France). A FC-15 type stopped-flow cuvette was used. In ThT-absorbance-detected stopped-flow experiment, one sample syringe contained ThT stock solution with a concentration of 30 µM and the two other sample syringes contained the stock solutions of HEWL fibrils. In this experiment, HEWL fibril is in excess of ThT to satisfy pseudo-first-order kinetic condition. When performing the experiments, the three solutions were forced from the syringes into the stopped-flow cuvette. After mixing, the concentration of ThT was 2 µM. The concentrations of ThT in both the stock solution and the stopped-flow cuvette are both below the critical micelle concentration of ThT in water of 31 µM.25 This means that in our kinetic study, ThT existed as a monomer in its aqueous solution. Several different final concentrations of HEWL fibrils were used, which were 133 µM, 267 µM, 400 µM, 533 µM and 665 µM, respectively. The reaction dead time was about 2.5 ms. The absorbance decay of ThT upon binding to HEWL fibrils was monitored at 370 nm. The experiments were performed at 20 °C using a circulating water bath for temperature control. The kinetic trace shown in the manuscript is a representative one, whereas the obtained Kobs is the average of at least eight measurements. Before conducting


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kinetic experiments, the suitable detection wavelength and the optimum pseudo-first-order kinetic condition were determined using conventional UV-Vis absorption spectroscopy with an Implen UV-vis nanophotometer (München, Germany). Results and Discussion We choose the amyloid fibrils formed by HEWL to investigate the kinetic binding mechanism of ThT. HEWL is widely used as a model system in amyloid research. The HEWL amyloid fibrils were prepared using the method proposed by Vernaglia et al.32 By putting HEWL in a partially denatured state using GdnHCl and incubating HEWL at a moderately high temperature under constant shaking, HEWL can form amyloid fibrils in a few hours. The HEWL fibrils were characterized using AFM, ThT fluorescent assay, and CR assay to confirm its amyloid nature. The results are shown in Fig. 1. The AFM result shown in Fig. 1A confirms the fibrillar morphology of HEWL fibril. The fibril height is ∼20 nm. The morphology of the GdnHClinduced fibrils prepared here looks quite different from that of the amyloid fibrils prepared from HEWL reported by us and others previously.33-37 This is due to the fact that the amyloid fibrils of HEWL in these previous studies were prepared under heat and acidic conditions.35, 37 Under these conditions, HEWL undergoes hydrolysis to generate a series of amyloidogenic peptides and it is the amyloidogenic peptide rather than the full-length HEWL that forms the amyloid fibrils. In this study, the GdnHCl-induced fibril is formed merely by full-length HEWL. Furthermore, the binding properties of ThT on the GdnHCl-induced HEWL fibril has been well characterized by Sulatskaya et al using UV-Vis spectroscopy combined with equilibrium microdialysis.29, 38 Their results indicate that GdnHCl-induced fibril has two ThT binding sites with Kb1 = 7.5×106 M-1, Kb2 = 5.6×104 M-1, and n1 = 0.11, n2 = 0.24. ThT bound to the first site has a high quantum yield of 0.44; while ThT bound to the second site has a very low quantum


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yield similar to that of free unbound ThT. In Fig. 1B, ThT fluorescent assay result shows the fluorescent enhancement around 480 nm under the excitation of 450 nm when ThT binds HEWL fibril, which is a typical feature of amyloid fibrils. The CR assay result in Fig. 1C reveals an absorption increase of CR at about 540 nm when CR binds HEWL fibril, which is also an indicator of the presence of amyloid structure.39 The HEWL fibrils were additionally characterized by FTIR spectroscopy and the result shown in Fig. 1D. The FTIR spectrum of HEWL fibrils features a prominent absorption at 1628 cm-1, indicating the formation of β-sheet structure; while the FTIR spectrum of native HEWL features a broad peak at 1644 cm-1 due to its dominant α-helix and loop structures. We propose to use ThT-absorbance-detected kinetic design to study the kinetics mechanism of ThT binding onto HEWL amyloid fibril. Namely, we keep amyloid fibril to be in excess of ThT to satisfy the pseudo-first-order condition. The kinetic trace of ThT binding is recorded by monitoring the absorbance decay of free unbound ThT (rather than fibril-bound ThT) during binding. This new design allows us to avoid the mechanistic ambiguity in previous studies in differentiating sequential binding mechanism and parallel binding mechanism. Furthermore, this ThT-absorbance-detected technique allows the monitoring of both ThT-fluorescence-active species and ThT-fluorescence-inactive species as it monitors the concentration decay of free unbound ThT; while ThT-fluorescence-detected technique can only monitor the fibril-bound ThT that is fluorescence-active. Before conducting the ThT-absorbance-detected kinetic experiments, the suitable detection wavelength and pseudo-first-order kinetic condition were determined using conventional UV-Vis absorption spectroscopy. Fig. 2A shows the spectra of free ThT and ThT bound to HEWL fibrils. The spectrum of free THT was obtained by measuring ThT aqueous solution with a


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concentration of 10 µM; and the spectrum of fibril-bound ThT was obtained by measuring the aqueous solution containing 4 µM of ThT and 46.7 µM of HEWL fibrils. Since HEWL fibril scatters light, the presented absorption spectrum of fibril-bound ThT in Fig. 1A has been corrected to remove the scattering contribution according to the method proposed by Sulatskaya et al.38 As can be seen, the interaction of ThT with HEWL fibril leads to a spectral red shift in the absorption spectrum. The absorption maximum of free unbound ThT locates at 412 nm; while the absorption maximum of fibril-bound ThT appears at ~450 nm, similar to the absorption maximum reported by others.30, 38, 40 The spectra in Fig. 2A further indicate that the spectral region of free ThT spectrum below 370 nm is negligibly affected by the absorption of fibrilbound ThT. Thus, we chose 370 nm as the detection wavelength to monitor the kinetic decay of free unbound ThT during binding. The reason that we did not choose a wavelength of fibrilbound ThT (e.g. at 480 nm) as the detection wavelength is due to the fact that ThT molecules bound at different fibril sites can display different absorption spectra with different absorption coefficients. This is well established in the work by Sulatskaya et al.38 It is thus difficult for us to directly relate the measured total absorbance of fibril-bound ThT to its concentration during kinetic analysis. We also performed a titration experiment to determine the excess amount of fibrils that is needed to satisfy the pseudo-first-order kinetic condition. In Fig. 2B, we let the concentration of HEWL fibril fixed at 46.6 µM and then added increasing amount of concentrated ThT solution and recorded the UV-Vis spectra of ThT. We used the absorbance at 480 nm to quantify the amount of fibril-bound ThT as the spectral region above 480 nm is minimally affect by the absorbance of free ThT. The titration results in Fig. 2B showed that 8 µM ThT would saturate the binding sites on HWEL fibril. This clearly indicates that the binding of ThT to HEWL is not stoichiometric (1:1). The results let us to determine the concentration


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ratio between ThT and HEWL fibril for saturated binding. When performing kinetic studies, we used a fibril concentration that is at least ten times higher than needed for saturation binding. This ensures that the concentration of binding sites on HEWL fibrils satisfies pseudo-first-order condition. For example, if we use 2 µM ThT in the kinetic study, we would choose the concentration of HEWL fibril to be above 116.5 µM. Fig. 3 shows the ThT binding investigation results using ThT-absorbance-detected kinetic design. We keep the concentration of ThT in the mixing cuvette in a stopped-flow apparatus at a fixed value of 2 µM and studied the pseudo-first-order kinetics of ThT binding onto HEWL fibrils. It should be pointed out that we here used HEWL monomer concentration to denote the concentration of the binding sites on HEWL fibrils as the two concentrations are proportional to each other. Fig. 3A, 3B, 3C, 3D, and 3E show the kinetic traces of ThT absorbance decay when the concentrations of HEWL are at 133 µM, 267 µM, 400 µM, 533 µM and 665 µM, respectively. As we can see, upon binding to HEWL fibril, the absorbance of the free unbound ThT quickly decreases to a baseline value. Here one may wonder why the absorbance of free unbound ThT after the completion of fibril binding did not reach zero. This is simply due to the fact that HEWL fibrils scatter light and the non-zero baseline value is due to fibril scattering. As the scattering of HEWL fibril is relevant to the size of the HEWL fibril and it is very unlikely for ThT to change the particle size of HEWL fibril during binding, the scattering of HEWL is believed to be constant during ThT binding and have no effect on the following-up kinetic fitting and analysis. Another feature of the kinetic decay trace is that the absorbance change after binding is small (in the order of 0.001). This is due to the fact that we use 370 nm as the detection wavelength to avoid the interference from the absorbance of fibril-bound ThT and this wavelength is away from the absorption maximum of free ThT at 412 nm. So the absorbance


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change due to ThT decay is intrinsically small. Yet, owing to the high sensitivity of the photomultiplier tube (PMT) of the stopped-flow apparatus, we were able to reproducibly detect these decay traces with confidence. Furthermore, in Fig. S1 and S2 in the supporting information we have included the results from two controls to demonstrate that the observed absorbance changes in Fig. 3 (in the order of 0.001), though small, are well above the noise level in our kinetic design. In Fig. 3, we have shown that all of the five traces can be successfully fitted with a single-exponential function. The obtained fitting correlation coefficients, R2, are displayed inside the figures. Furthermore, in Fig. 3F, we showed that the obtained and the concentration of protein (i.e., the concentration of binding sites) assume a linear relationship passing through origin. In addition, one issue that we would like to further point out is about choosing 370 nm as the detection wavelength. As we discussed above, choosing 370 nm as the detection wavelength is very important in our kinetic investigation because the absorbance of the reaction mixture at this wavelength is negligibly affected by the absorption of fibril-bound ThT and thus only due to the absorption of free unbound ThT. If we choose a different detection wavelength, e.g. 445 nm as shown in Fig. S3 in the supporting information, the absorbance change of the kinetic trace will contain the absorption contributions from both the free unbound ThT and the fibril-bound ThT. Such kinetic trace cannot be fitted with a single-exponential function and is not suitable for our kinetic analysis either.

In the following, we argue that the above kinetic observations can be interpreted using the onestep parallel binding mechanism described in Scheme 3. In Scheme 3, we use L to denote ThT, use F to denote a particular type of the binding site on HEWL fibril, use LF to denote ThT bound onto type binding site, and use to denote the associate reaction rate of ThT onto type


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binding site. The binding reaction between ThT and amyloid fibril in general should be a reversible process. Yet, as we mentioned above, according to the previous study by Sulatskaya et al about the binding constant of ThT onto the HEWL amyloid fibril used here, the value of ThT binding constant is in the order of 104 to 106.38 Considering the relatively high binding constant and the excess amount of binding sites over ThT in our pseudo-first-order kinetic design, we can neglect the backward dissociation reaction in our kinetic reasoning and only consider the forward association reaction. If we define the initial (at time zero) concentration of free unbound ThT to be a, the concentrations of fibril-bound ThT on type i binding site and the total fibril-bound ThT at time during binding to be and , the initial concentration of type i binding site to be , the rate law for the proposed one-step parallel binding mechanism can be expressed in the following. =

= ∑

(1)

= ( − − − ⋯ − − ⋯ )( − ) (2)

Under pseudo-first-order condition, fi is in excess of and can be considered as a constant. The total decay rate of ThT, r, in eq. (1) can then be written in the following way. = ∑

= (∑ )( − ∑ ) (3)

If we define = ∑ , considering = ∑ , we have eq. (4). =

= ( − ) (4)

Through integration, we can get eq. (5). − = (− ) (5)


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For ThT-absorbance-detected kinetic design, we have eq. (6), where

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!,

,

"

correspond to

the absorbance of free unbound ThT at time 0, and ∞. Due to the scattering of HEWL fibril, "

would not be zero after the completion of binding, but a constant number.

−

"

=(

!

−

" )exp (− )

(6)

Furthermore, since = ∑ and each is proportional to total fibril concentration ( (or the total concentration of binding sites), we will have eq. (7) if we define = ) (. = (∑ ) )( (7) As ∑ ) in eq. (7) is a constant, eq. (7) shows that is directly proportional to (. The kinetic reasoning shown above is a general description for the simple one-step parallel binding mechanism of ThT onto multiple sites of amyloid fibril. In the case of HEWL fibril used in this study, the total binding site number should be two. As we can see, eq. (6) and eq. (7) match the experimental observations and fittings in Fig. 3 very well, thus providing strong support to the proposed parallel binding mechanism through a simple one-step bimolecular reaction at each site as shown in Scheme 3. Furthermore, the single-exponential fitting in Fig. 3 indicates that the reaction order of ThT in the binding reaction is one. Namely, it is the ThT monomer that is bound onto the binding site. This excludes other binding modes, such as dimer or oligomer binding, and micelle binding. The proposed one-step parallel binding mechanism in this study using ThT-absorbancedetected kinetic design appears to be able to interpret previous observations in a unified manner. For example, in the case of HET-s fibril in the study by Sabate et al,25 it is likely that at pH=2 only one ThT binding site is favored and the kinetic binding trace is thus fitted into a single exponential; while at pH=7, there are two favored ThT binding sites and the kinetic binding trace is thus fitted into two exponentials. In the case of Aβ1-40 fibril in LeVine’s study, there are four


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ThT binding sites favored during binding and each binding follows the one-step bimolecular binding mechanism. Therefore, the kinetic binding trace can be fitted into four exponentials. Certainly, it should be emphasized that more amyloid fibril systems (e.g., Aβ1-40 fibril and HET-s fibril) need to be explored using the newly proposed ThT-absorbance-detected kinetic design before we can make the one-step parallel binding mechanism as a general mechanism for ThT binding onto amyloid fibril. Conclusions In summary, using ThT-absorbance-detected kinetic design, we studied the kinetic mechanism of ThT binding onto amyloid fibril using HEWL fibril as a model system. Our kinetic observations support the one-step parallel binding mechanism for the kinetic interaction between ThT and HEWL amyloid fibril. In this mechanism, ThT monomer binds onto HEWL amyloid fibril through a simple bimolecular step. We hope our work can shed new light on the interaction between dye molecule and amyloid fibril.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (G.M.). ACKNOWLEDGMENT We gratefully acknowledge the financial support from the National Natural Science Foundation of China (No. 21075027), the Natural Science Foundation of Hebei Province (No. B2011201082 and B2016201034), Juren plan, and Program for Changjiang Scholars and Innovative Research Team in University (No. IRT_15R16).


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Supporting Information Available. Figure S1, Figure S2, Figure S3 for the control experiments in the kinetic investigation. This information is available free of charge via the Internet at http://pubs.acs.org/.

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Scheme 1. Molecular structure of Thioflavin T.


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Scheme 2. Four-step sequential binding mechanism of ThT onto amyloid fibril. L: ThT; F: amyloid fibril; LF: ThT-fibril complex; k1: association rate constant for bimolecular binding; k2, k3, k4: rate constants for amyloid conformational change.


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Scheme 3. One-step parallel binding mechanism onto multiple ThT binding sites. L: ThT; Fi: a particular type of binding site on HEWL fibril; LFi: ThT bound onto type i binding site; : association rate constant for the binding step.


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Figure 1. (A) AFM characterization of HEWL fibril; (B) ThT fluorescence assay; (C) CR assay; (D) FTIR characterization of HEWL fibril and native HEWL. a.u.: arbitrary unit.


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Figure 2. (A) Absorption spectra of free ThT (10 µM) (red) and fibril-bound ThT (4 µM) (black); (B) Titration curve for ThT binding onto HEWL fibril (46.6 µM) measured using the absorbance at 480 nm. Solid line: linear fit.


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Figure 3. Kinetic results for ThT (2 μM) binding onto HEWL amyloid fibril using ThTabsorbance-detected kinetic design. (A) Kinetic trace with 133 μM HEWL; (B) Kinetic trace with 267 μM HEWL; (C) Kinetic trace with 400 μM HEWL; (D) Kinetic trace with 533 μM HEWL; (E) Kinetic trace with 665 μM HEWL; (F) Dependence of kobs on HEWL fibril concentration. Dot: experimental; red line: fitted; R2: correlation coefficient.


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SYNOPSIS


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Kinetic Mechanism of Thioflavin T Binding onto the ... - ACS Publications

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