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Identifying Insulin Fibril Conformational Differences by Thioflavin-T Binding Characteristics
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Biomacromolecules

Cite this: Biomacromolecules 2020, 21, 12, 4989–4997
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https://doi.org/10.1021/acs.biomac.0c01178
Published November 17, 2020

Copyright © 2020 American Chemical Society. This publication is licensed under CC-BY.

Abstract

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Amyloidogenic protein aggregation into highly structured fibrils is linked to more than 30 amyloidoses, including several neurodegenerative disorders. Despite significant progress in trying to understand the process of amyloid formation, there is still no cure or effective treatment available. A number of studies involving potential anti-amyloid compounds rely on the use of a fluorescent probe—thioflavin-T—to track the appearance, growth, or disassembly of these cytotoxic aggregates. Despite the wide application of this dye molecule, its interaction with amyloid fibrils is still poorly understood. Recent reports have shown it may possess distinct binding modes and fluorescence intensities based on the conformation of the examined fibrils. In this work, we generate insulin fibrils under four different conditions and attempt to identify distinct conformations using both classic methods, such as atomic force microscopy and Fourier-transform infrared spectroscopy, as well as their ThT binding ability and fluorescence quantum yield. We show that there is a significant variance of ThT fluorescence quantum yields, excitation/emission maxima positions, and binding modes between distinct insulin fibril conformations.

Copyright © 2020 American Chemical Society

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Introduction

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Protein misfolding and association into amyloid fibrils is linked with more than 30 amyloidoses, which lead to organ dysfunction, including neurodegenerative disorders, such as Alzheimer’s or Parkinson’s disease. (1−3) These afflictions affect millions of people worldwide, (4) and the number is projected to increase further as the average human lifespan continues to increase. (5,6) Currently, both the mechanism (7,8) and the resulting structure (9,10) of such aggregates are still not fully understood, which is one of the main reasons why there is still no effective cure or treatment available. (11)
To better understand the process of how these fibrils form, what their resulting structure is, and how potential drug molecules are able to alter the aggregation process, countless experiments are conducted in vitro using amyloidogenic proteins. (12−16) To track this process and determine whether there are amyloids present in the sample, multiple techniques are used. These include atomic force microscopy (AFM), (17,18) light scattering, (19) Fourier-transform infrared (FTIR) spectroscopy, (20) and fluorescence spectroscopy. (21) One common fluorescent probe used to investigate the kinetics of fibrilization, as well as to quantify the concentration of fibrils, is thioflavin-T (ThT). (22,23) Upon binding to β-sheet grooves located on the surface of amyloids, the conformation of ThT becomes locked, which results in both a red shift of its excitation/emission wavelengths and a significant increase in its fluorescence quantum yield. (24)
Despite the relatively simple structure and specific affinity toward amyloid fibrils, this dye molecule has been proven to be far more complex than initially perceived. One interesting aspect of ThT is its ability to bind to fibrils in more than one mode, (25) with distinct modes having specific excitation and emission wavelength maximum positions and fluorescence quantum yields. (26,27) Multiple experimental and computational reports have shown that the number of binding modes for ThT can vary from one to six, (25,28−32) depending on the protein that the fibril is composed of. It was also demonstrated that α-synuclein (33) and prion proteins (34) can form two distinct conformations that differ by their ThT fluorescence intensity. In addition to this, we have also recently observed distinct fluorescence intensities for two insulin conformations generated at different protein concentrations, (35) as well as a massive increase in ThT fluorescence when EGCG was used to alter the aggregation reaction. (36)
Insulin is a peptide hormone that regulates carbohydrate, fat, and protein metabolism. Its aggregation is associated with insulin-derived amyloidosis, localized at injection sites in patients with diabetes. (37) Considering that insulin is also a model protein used to study fibril formation, (38) as well as to screen potential anti-amyloid drugs, (39−41) such conformation-specific variation in the ThT fluorescence intensity could significantly alter the experimental results. If a compound is capable of slightly altering the structure of fibrils, which results in a different or additional ThT binding mode, this would, in turn, cause an increase or decrease in the signal’s intensity, leading to a conclusion that the molecule worked as an aggregation enhancer or inhibitor. The occurrence of such an event is not unlikely, as it is known that variations in pH (42) or concentration (35) can have a sizeable impact on the resulting structure of insulin fibrils. On the other hand, if the differences in ThT signal intensities or excitation/emission maxima positions are conformation-specific, then this factor could also be used as a quick and simple way of distinguishing between insulin fibril conformations and may be applied to other amyloidogenic proteins, such as the previously mentioned α-synuclein (33) and prion proteins. (34)

Materials and Methods

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Human recombinant insulin (Sigma-Aldrich, Cat. No. 91077C) was dissolved in either 20% acetic acid solution (AC), containing 100 mM NaCl, 100 mM sodium phosphate buffer (pH 2.0), 100 mM sodium phosphate buffer, containing 100 mM NaCl (pH 2.4) or phosphate buffer saline (pH 7.4) to a final protein concentration of 200 μM and placed into 1.5 mL test tubes (Fisher, Cat. No. 15432545) to a final volume of 1 mL (Table 1). The samples were incubated at 60 °C for 24 h in a Ditabis Thermomixer. The phosphate-buffered saline (PBS) samples also contained two glass beads each (Merck, Cat. No. 104015) and were agitated at 600 rpm throughout the incubation period.
Table 1. Insulin Sample Aggregation Reaction Conditions
sample namereaction solutionagitation
AC20% acetic acidnone
100 mM NaCl
PH20100 mM sodium phosphate buffer (pH 2.0)none
PH24100 mM sodium phosphate buffer (pH 2.4)none
100 mM NaCl
PH74phosphate buffer saline (pH 7.4)600 rpm + 2 glass beads

Atomic Force Microscopy

Each sample (30 μL) was deposited on mica disks, left to adsorb for 1 min, gently washed with 1 mL of H2O, and dried using airflow. AFM images were scanned using a Dimension Icon (Bruker) atomic force microscope (tapping mode), equipped with a silicon cantilever (40 N/m, Budget Sensors). The 1024 × 1024 pixel resolution images were acquired using a scan rate of 0.5 Hz. AFM images were analyzed using Gwyddion 2.5.5 software. The fibril height and width were determined by tracing a line perpendicular to the fibril axis, while the fibril length was determined by tracing parallel to the fibril axis. The height, width, and length distributions were calculated from 50 traces for each sample.

Fourier-Transform Infrared Spectroscopy

Each sample was centrifuged at 10 000g for 30 min, after which the supernatant was removed and fibrils were resuspended in D2O. The centrifugation and resuspension steps were repeated four times. Finally, the fibrils were resuspended in a small volume of D2O (final fibril concentration ∼10 mg/mL). The fibril samples were sonicated for 1 min using a Bandelin Sonopuls ultrasonic homogenizer with an MS 73 tip (40% total power). The FTIR spectra were recorded using a Vertex 80v (Bruker) IR spectrometer with a mercury cadmium telluride detector at room temperature under near-vacuum conditions. A total of 256 interferograms with 2 cm–1 resolution were averaged. The spectrum of D2O was subtracted from each sample’s spectrum. All spectra were normalized to the same area of amide I/I′ band (1700–1580 cm–1) using GRAMS software. To calculate the amide I/I′ band’s width at its half-height (HHBW), (43) the spectra were baseline-corrected between 1700 and 1580 cm–1 before normalization.

Sample Preparation for ThT Binding Assays

Each sample was centrifuged at 10 000g for 30 min, after which the supernatant was removed and the fibrils were resuspended in MilliQ H2O. The centrifugation and resuspension procedure was repeated five times. Each sample was then sonicated for 1 min using a Bandelin Sonopuls ultrasonic homogenizer with an MS 73 tip (40% total power). The samples were then combined to a final protein concentration of 200 μM. The combined solution was then further sonicated for 10 min using the previously described method with 30 s sonication/rest intervals. The resuspension and sonication procedure resulted in highly dispersed and fragmented aggregates (Figure S1A–D), with a greatly reduced average fibril length (Figure S2A,B). The four fibril samples had similar width, and the height distribution remained comparable to the untreated fibrils (Figure S1E,F).
ThT (Sigma-Aldrich, Cat. No. T3516) was dissolved in MilliQ water to a final concentration of 10 mM. The exact concentration was determined by taking an aliquot of the dye solution, diluting it 200 times with MilliQ water and scanning its absorbance at 412 nm (ε412 = 23 250 M–1 cm–1). The ThT solution was then diluted to prepare 200, 20, and 2 μM stock solutions. The sonicated fibrils were mixed with ThT stock solutions and H2O to a range of ThT concentrations (final protein concentration was 100 μM in all cases).

ThT Excitation–Emission Matrix (EEM) Analysis

Each fibril–ThT solution (100 μL) was placed in a 3 mm pathlength cuvette, and its excitation–emission matrix was scanned using a Varian Cary Eclipse fluorescence spectrophotometer (excitation range was 435–465 nm with 1 nm steps and 5 nm slit width, emission range was 460–500 nm with 1 nm steps and 2.5 nm slit width; all other device parameters were kept the same for all sample scans). Three EEMs were scanned for every sample, the background spectrum was subtracted, and the resulting matrices were averaged. Due to slightly different fibril cross-interactions in the presence of ThT, the EEM areas where light scattering is prevalent cannot be accurately subtracted. Therefore, for further data analysis, the EEM region present 7 nm or less away from the excitation wavelength was not taken into account.
To correct for the inner filter effect caused by ThT, the absorbance spectra of each sample were scanned from 300 to 600 nm using 1 nm steps (each spectrum was the average of three repeats). The spectrum of fibrils without ThT was subtracted from each fibril–ThT spectrum. Because of ThT-induced differences in fibril association, the fibril spectrum could not be subtracted by a factor of 1 in certain cases (light absorbance differences induced by fibril clumping). The fibril spectrum was therefore subtracted by multiplying it by a certain factor (usually between 0.9 and 1.1) until there was no slope observed in the 550–600 nm range in the resulting spectrum.
The inner filter effect was corrected for every EEM point by using the following equation
(1)
where AEx is the sample’s absorbance at the excitation wavelength, AEm is the sample’s absorbance at the emission wavelength, Im is the signal intensity observed during measurement, and Ic is the corrected signal intensity.
The EEM intensity “center of mass” was calculated by selecting the top 10% intensity values and using the following equation
(2)
where λ is the wavelength of either the excitation or emission center of mass, λn is the excitation or emission wavelength, ∑In is the sum of all signal intensities at λn, and ∑Ia is the sum of all signal intensities.
This method helps avoiding the discrepancy caused by variations in light scattering at emission wavelengths close to the excitation wavelength, as well as EEM maxima position deviations due to background noise.

Bound ThT Concentration Determination

Two methods used to determine the concentration of fibril-bound ThT include sample centrifugation (26) or sample dialysis. (29) Since each sample is sonicated, certain fibril types become difficult to separate from solution by centrifugation. Microdialysis techniques can lack accuracy at low ThT concentrations due to dye–membrane association and the relatively long time needed that can result in fibril clumping. To determine the concentration of bound ThT, a different method was devised based on two ThT–fibril interaction factors. First, based on the reported dye binding constants, (29) when the sample contains 100 μM insulin aggregates and 1 μM or less ThT, the majority of it should be bound to fibrils. Second, the difference between the absorbance spectra of free and bound ThT is much greater than the difference between spectra of ThT bound in distinct binding modes.
In this case, the absorbance spectra of ThT in the range from 0.1 to 1.0 μM should be the result of bound ThT molecules. By taking the absorbance values at 412 nm (free ThT spectrum maximum position) and 450 nm (bound ThT absorbance spectrum shifts toward higher wavelengths), we can calculate the extinction coefficients of bound ThT at these two wavelengths (ε412 and ε450) for all four fibril samples. Since the ε412 and ε450 values are known for free ThT (23 250 and 5880 M–1 cm–1 respectively), the concentration of bound ThT can be calculated using the following system of equations
(3)
(4)
where cF is the concentration of free ThT, cB is the concentration of bound ThT, ε412 and ε450 are the extinction coefficients of bound ThT, and A412 and A450 are sample absorbance values at 412 and 450 nm, determined by subtracting the fibril absorbance spectrum from the fibril–ThT spectrum, as described previously.
The concentration of bound ThT determined using this method may lose accuracy if distinct binding modes have vastly different ε412 and ε450 values; however, the calculated total ThT concentration has a linear dependence on total ThT present in the sample (Figure S3), which indicates that there are no drastic changes to bound ThT extinction coefficients.

Results

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Four distinct conformation insulin amyloid fibrils were generated by aggregating the protein under four different environmental conditions: 20% acetic acid solution (further referred to as AC), (35) pH 2.0 and pH 2.4 sodium phosphate buffers (PH20 and PH24, respectively), (42) as well as pH 7.4 phosphate buffer saline (PH74). (44) To evaluate the effectiveness of identifying insulin amyloid fibrils with different conformations, control experiments had to be carried out using well-established methods, such as Fourier-transform infrared spectroscopy and atomic force microscopy.

Atomic Force Microscopy

The four fibril samples were first evaluated based on their AFM images, as well as by comparing the height and width of aggregates. The AC (Figure 1A), PH20 (Figure 1B), and PH24 (Figure 1C) samples contain long, linear fibrils, with some self-association observed in the case of AC and PH24 conditions. The PH74 sample (Figure 1D), on the other hand, contains highly fragmented and short aggregates. When considering the height distribution (Figure 1E), PH20 and PH74 aggregates have the smallest height (average value is 4 nm). The AC fibril height is slightly bigger—5 nm—and the pH 2.4 sample contains the highest fibrils—7 nm. Based on the width of aggregates (Figure 1F), all four samples have different values, with PH74 having the lowest (13 nm), PH20 (14 nm), pH 2.4 (16 nm), and AC (18 nm). By combining the height and width measurements and the visual inspection of AFM images, the PH20 and PH74 samples are easy to differentiate from the rest, while AC and PH24 are quite similar to one another.

Figure 1

Figure 1. Atomic force microscopy images and fibril height and width distributions of insulin samples prepared under different conditions. Insulin fibrils were prepared under AC (A), PH20 (B), PH24 (C), and PH74 (D) conditions. Fibril height (E) and width (F) distribution (n = 50), where box plots indicate the interquartile range and error bars are 1 standard deviation.

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Fibril length was not used as a means of distinguishing between insulin fibril conformations due to fragmentation having a substantial effect on this factor, as can be seen in the case of PH74, where the sample was agitated during aggregation (Figure S2A,B). There were also no clear periodicity patterns observed for any of the fibrils, where such a parameter could be determined (Figure S4). Prior to further experiments, the samples were sonicated, which resulted in all four aggregates having a comparable length distribution (Figure S2B). The width distribution experienced a slight increase (Figure S1F), likely due to the lateral association of small fibril fragments. (45) Sonication had a very minimal influence on all four aggregate type height distributions (Figure S1E).

Fibril Secondary Structure

FTIR was used to detect differences in the fibril secondary structure, relying on the amide I/I′ region (Figure 2). The AC and PH20 samples share similarities in the fact that they both contain a small peak at 1729 cm–1, which is associated with deuterated carboxyl groups. The appearance of this peak can be associated to the disappearance of salt bridge interactions and subsequent deuteration of the resulting carboxyl group. (46) They also both have the main minima in the second derivative at 1628 cm–1, which can be associated with stronger hydrogen bonds in the β-sheet structure, while the bands at 1640 cm–1 for AC and 1641 cm–1 for PH20 are related to weaker hydrogen bonds. The difference between AC and PH20 fibrils lays in more expressed bands at 1659 and 1673 cm–1 (associated with turns and/or loops) in the second derivative spectrum of PH20. There is also a considerable difference between half-height band widths (43) of AC and PH20 amide I/I′ bands (Figure 2). The PH24 sample displays the only main minimum at 1630 cm–1, which suggests a single hydrogen bonding profile in the β-sheet structure. This spectrum also has no peak associated with deuterated carboxyl groups. The most distinct FTIR spectrum belongs to the PH74 sample, which has the main minimum at 1636 cm–1, with smaller bands at 1628–1630 cm–1, indicating a dominant presence of weaker and a smaller fraction of stronger hydrogen bonds in the β-sheet structure. Based on these observations, it is not difficult to separate the PH24 and PH74 samples from the rest; however, the AC and PH20 FTIR spectra have quite a few similarities and can only be accurately distinguished based on different HHBW values. All four types of fibrils contain parallel β-sheets, and there is no clear indication of the presence of antiparallel ones. (47)

Figure 2

Figure 2. Insulin sample’s FTIR spectra (A), second derivatives (B), and spectrum positions associated with β-sheets, turns, loops, deuterated carboxyl groups, and each spectrum’s band’s width at its half-height (table inserted).

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Bound ThT Intensities

Before conducting experiments with ThT binding, all fibril samples were repeatedly resuspended into MilliQ water and sonicated to negate any effect that the solution’s ionic strength, additives, or fibril superstructural organization may have on dye binding or fluorescence.
After measuring each sample’s fluorescence EEMs and absorbance spectra under a range of ThT concentrations, the maximum fluorescence intensities, as well as bound ThT concentrations, were determined. Since fibrils may possess different dye binding modes or have parts of their surface covered due to cluster formation, the absolute fluorescence intensity could not be used to differentiate between samples. Instead, to accurately compare each sample, the ratios between ThT fluorescence intensities and bound dye concentrations (I/cB) were calculated by dividing the fluorescence intensity with the concentration of bound dye molecules at each examined ThT concentration.
In the case of AC (Figure 3A), the I/cB ratio is around 110 μM–1 when the total concentration of ThT is low. Once the dye’s concentration reaches 3–4 μM, the ratio begins to decrease, eventually reaching 50 μM–1. Such reductions in the fluorescence quantum yield are associated with self-quenching of dye molecules due to binding in close proximity on the fibril’s surface. (48−50) For PH20 (Figure 3B), both the initial (30–40 μM–1) and final (20–30 μM–1) ratio values are quite low in comparison and they do not experience such a drastic drop upon an increase of ThT concentrations. The PH24 sample (Figure 3C) initial and final ratios are within the margin of error (35–50 μM–1), and there is almost no reduction in values throughout the entire ThT concentration range. The PH74 (Figure 3D) ratio dependence on total ThT concentration has a sigmoidal shape, with initial values being 60–80 μM–1, which then drop to 10 μM–1. Based on I/cB ratios, all four samples have distinct dependencies on the total ThT concentration. AC and PH74 have similar shapes but different initial and final values, as do PH20 and PH24 samples.

Figure 3

Figure 3. Insulin sample’s ThT fluorescence intensity and bound ThT concentration ratios (I/cB) at different total dye concentrations. Insulin fibrils were prepared under AC (A), PH20 (B), PH24 (C) and PH74 (D) conditions.

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To verify the distinct ways of dye–fibril association, the bound ThT absorbance (29) and fluorescence spectra at 1 μM ThT (where most of the dye molecules are in their bound state) were compared (Figure S5A,B). The PH20 sample had the lowest absorbance values, as well as the lowest maximum absorbance wavelength (420 nm). PH24 had a wider peak, but the absorbance intensity was similar to PH20. Both AC and PH74 had the highest absorbance peaks, which were also shifted toward 445 nm. This partial similarity between PH20 and PH24, as well as between AC and PH74, is in line with the observed I/cB ratio dependencies on total ThT concentration. Under these conditions, the fluorescence intensities correlate with the absorbance intensities (Figure S5B).

EEM Maxima Positions

Different modes of ThT binding may possess specific excitation and emission wavelength maximum positions, which was also used to differentiate between the four samples. In the case of AC (Figure 4A), at low ThT concentrations, the maximum positions are located at 443/480 nm excitation/emission wavelengths. As the dye’s concentration increases, the excitation wavelength shifts toward 448 nm and the emission wavelength shifts to 478 nm. At higher ThT concentrations, the emission wavelength remains stable, but the excitation wavelength shifts toward 481 nm. This type of maximum position movement is a likely indicator of three ThT binding modes. For PH20 (Figure 4B), there is minimal variation in the excitation wavelength, which remains at 445–447 nm; however, there is a shift in the emission wavelength—from 479 to 485 nm. The PH24 sample’s (Figure 4C) EEM maximum position moves from 450/479 to a similar position to the PH20 sample (446/485). This means that PH20 and PH24 fibrils likely have one different and one similar ThT binding mode. The PBS sample’s (Figure 4D) EEM maximum position experiences the most significant movement, forming an arc-shape from 456/478 to 445/486. It has partial overlap with PH20 and PH24 sample positions at higher ThT concentrations, suggesting the existence of a similar binding mode for all three cases. The position at low ThT values, however, is different from all of the other samples. Such a significant movement and the arc-shape is also a likely indicator of more than two modes of ThT binding.

Figure 4

Figure 4. Insulin sample’s ThT fluorescence EEM intensity center of mass positions at different total ThT concentrations. Insulin fibrils were prepared under AC (A), PH20 (B), PH24 (C), and PH74 (D) conditions.

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Discussion

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Using the two classical methods (AFM and FTIR) to compare insulin fibrils formed at different conditions, it can be identified that the four samples possess distinct morphologies and secondary structures. In some instances, there were similarities between AFM images or fibril heights and widths, and in others, there were similarities between FTIR spectra. Combining both methods, however, allows us to very clearly differentiate between the four insulin conformations.
When we examine the sample fluorescence intensity and bound ThT concentration ratios (I/cB), we see that there are clear distinctions between the four samples. Besides the fact that this allows us to differentiate between the distinct conformations, there is also an interesting I/cB ratio dependence on total ThT concentrations. In two of the cases, namely, AC and PH74, the ratio decreases with increasing ThT concentrations. Such an event is to be expected, as ThT is known to induce a self-quenching effect. (48) However, PH20 and PH24 samples do not experience such a significant decrease, even at the highest dye concentrations. It is possible that ThT binds in such a manner that it does not allow for a self-quenching effect to occur. The absorbance spectra of 1 μM bound ThT displays significant distinctions between all four samples, further supporting the idea that these four fibril conformations have specific ThT binding characteristics.
Comparing each sample’s excitation–emission matrix and their intensity center of mass also leads to four different EEM maximum position distributions. At low ThT concentrations, all four samples have clearly distinct positions. In three of the cases, namely, PH20, PH24, and PH74, increasing the total ThT concentration leads to a maximum position convergence at 446/485 nm. This means that, while each fibril conformation possibly has unique ThT binding modes, some conformations may share a similar binding mode.
Since these ThT binding/fluorescence characteristics stem from either morphological or secondary structure variations, we have to examine which of them could be responsible for such dye–fibril association. In the case of morphology, the only real difference present between all four samples after sonication is their height distribution (Figure S1E). There does not seem to be any correlation between the I/cB ratio and fibril height, as PH20 and PH24 samples have the largest height difference, yet similar shape I/cB ratio distribution dependencies on the total ThT concentration. No correlation is also present in the case of fluorescence EEM positions. This leads to the idea that fibril morphology is not the main determining factor for ThT binding characteristics.
In the case of secondary structure, the relation between each structural motif and dye binding/fluorescence has to be considered. The deuterated carboxyl group, related to the loss of salt bridges (46) during insulin aggregation, does not appear to be a significant factor, as there is a massive difference in both I/cB and EEM parameters between the two insulin conformations that have a peak at 1729 cm–1 in FTIR spectra. The turn/loop motives do not pertain to significant variations for them to be a factor, which leaves hydrogen bond strength in the β-sheet structure as the main suspect. The highest I/cB ratio at low ThT concentrations is observed in the AC sample, where fibrils have the largest amount of stronger hydrogen bonds (as indicated by the major 1628 cm–1 band in the second derivative spectrum). The PH20 sample also has a peak at 1628 cm–1, but in this case, the 1641 cm–1 band is relatively larger than in the case of AC. This sample has a significantly lower I/cB ratio than AC, however, which may indicate that the weaker hydrogen bonds in the fibril’s structure create binding positions with higher ThT binding affinity and lower fluorescence quantum yield. The PH24 FTIR spectrum has a single β-sheet-related band, and, coincidentally, the I/cB ratio experiences the least amount of variation throughout the entire ThT concentration range. The PH74 FTIR spectrum is the most peculiar of all, displaying mostly weaker hydrogen bonds within the β-sheet structure but also some stronger ones. Its corresponding I/cB ratio and EEM positions also experience the most drastic changes throughout the whole ThT concentration range. These observations hint at a possibility that weaker hydrogen bonds in the fibril’s structure lead to binding positions with higher ThT binding affinity and lower fluorescence quantum yield, while stronger ones result in binding position with lower ThT binding affinity and higher quantum yield.
When we combine AFM with FTIR and I/cB with EEM positions, it is clear that both pairs of methods are highly efficient at differentiating insulin fibrils prepared under different environmental conditions. Considering that a ThT binding examination has comparable efficiency to both AFM and FTIR, we have to discuss the advantages that this method has. In the case of FTIR, to acquire a high-quality spectrum and detect minor differences, a high concentration of fibrils has to be used. For AFM, the method of sample deposition can influence the overall fibril distribution (fibril clump formation, washing away smaller fibrils/oligomers, different aggregate/mica association propensities). Conversely, fibril–ThT fluorescence I/cB ratio and EEM position profile outlines, as well as bound ThT absorbance spectra, can be acquired by scanning relatively low fibril concentration samples at four ThT concentrations (0.1, 1, 10, and 100 μM). This method also uses fibril resuspension into MilliQ water, which negates the effect that the initial solution’s ionic strength or additives may have on ThT binding. The aggregates are also sonicated to both homogenize the sample, as well as disrupt any superstructural organization that may form during aggregation.
Despite insulin being a model for the study of amyloid aggregation and not considered as a neurodegenerative disease-associated protein, its ability to form multiple distinct fibril conformations serves as a means to display this method’s effectiveness. As of this time, there have been reports showing that different strains of disease-related proteins, such as α-synuclein (33) or prion proteins, (34) also possess different ThT binding modes, resulting in distinct fluorescence profiles. This means that ThT binding could serve in conjunction with other commonly used methods to both identify and differentiate the large variety of conformations that amyloid fibrils can obtain.

Conclusions

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ThT molecule interactions with insulin amyloid fibrils, prepared under different conditions, display a wide variety of bound ThT quantum yields, as well as distinct binding modes. Such interactions can be used to differentiate between fibrils to a similar extent as other widely used methods, such as AFM or FTIR. As this ThT binding assay requires minimal amounts of fibrils and is not affected by the initial aggregation solution or sample deposition, it can be applied as either an alternative or supplemental method in amyloid fibril research.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biomac.0c01178.

  • Atomic force microscopy images of insulin fibrils, prepared in AC, PH20, PH24, and PH74 conditions after multiple rounds of resuspension into MilliQ H2O and sonication (Figure S1); AC, PH20, PH24, and PH74 fibril length distributions before and after resuspension into MilliQ H2O and sonication (Figure S2); comparison of total (bound + free) ThT concentration calculated from sample absorbance data and total ThT added to the sample (Figure S3); AC, PH20, and PH24 fibril surface height along the fibril axis (Figure S4); and absorbance spectra of free and fibril-bound ThT and fluorescence intensity of all four types of fibrils in the presence of 1 μM ThT (Figure S5) (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Mantas Ziaunys - Institute of Biotechnology, Life Sciences Center, Vilnius University, Sauletekio al. 7, Vilnius LT-10257, Lithuania;  Orcidhttp://orcid.org/0000-0002-8368-6188
    • Andrius Sakalauskas - Institute of Biotechnology, Life Sciences Center, Vilnius University, Sauletekio al. 7, Vilnius LT-10257, Lithuania
  • Author Contributions

    M.Z. and V.S. designed the studies. M.Z. and A.S. undertook the experimental work. M.Z. and V.S. analyzed the data and prepared the manuscript.

  • Funding

    This research was funded by Vilnius University, Grant No. MSF-JM-3.

  • Notes
    The authors declare no competing financial interest.

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    Knowles, Tuomas P. J.; Vendruscolo, Michele; Dobson, Christopher M.
    Nature Reviews Molecular Cell Biology (2014), 15 (6), 384-396CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
    A review. The phenomenon of protein aggregation and amyloid formation has become the subject of rapidly increasing research activities across a wide range of scientific disciplines. Such activities have been stimulated by the assocn. of amyloid deposition with a range of debilitating medical disorders, from Alzheimer's disease to type II diabetes, many of which are major threats to human health and welfare in the modern world. It has become clear, however, that the ability to form the amyloid state is more general than previously imagined, and that its study can provide unique insights into the nature of the functional forms of peptides and proteins, as well as understanding the means by which protein homeostasis can be maintained and protein metastasis avoided.
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  3. 3
    Baker, K. R.; Rice, L. The Amyloidoses: Clinical Features, Diagnosis And Treatment. Methodist DeBakey Cardiovasc. J. 2012, 8, 3  DOI: 10.14797/mdcj-8-3-3
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    3
    The amyloidoses: clinical features, diagnosis and treatment
    Baker Kelty R; Rice Lawrence
    Methodist DeBakey cardiovascular journal (2012), 8 (3), 3-7 ISSN:.
    Amyloidosis is a rare disorder in which insoluble amyloid proteins are deposited in body organs, causing abnormal protein build-up in tissues and eventually leading to organ dysfunction and death. It affects less than 200,000 people in the United States, classifying it as a rare disease according to the National Institutes of Health. Definitive determination of the underlying protein is critical since prognosis and treatment of amyloidosis can vary widely depending on the responsible protein. The following paper describes the various types and clinical features of amyloidosis and provides an overview of current diagnostic tools and therapies.
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  4. 4
    Isik, A. T. Late Onset Alzheimer’s Disease in Older People. Clin. Interventions Aging 2010, 5, 307,  DOI: 10.2147/CIA.S11718
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    4
    Late onset Alzheimer's disease in older people
    Isik Ahmet Turan
    Clinical interventions in aging (2010), 5 (), 307-11 ISSN:.
    Dementia has become a common diagnosis in aging populations, and the numbers will increase in the forthcoming years. Alzheimer's disease (AD) is the most common form of dementia in the elderly, accounting for 50%-56% of cases at autopsy and in clinical series. Nowadays, the number of people affected by AD is rapidly increasing, and more than 35 million people worldwide have AD, a condition characterized by deterioration of memory and other cognitive domains, and leading to death 3-9 years after diagnosis. The number of patients with AD, the most common cause of disability in the elderly, is set to rise dramatically. Therefore, it is important for clinicians to recognize early signs and symptoms of dementia and to note potentially modifiable risk factors and early disease markers.
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  5. 5
    Hebert, L. E.; Weuve, J.; Scherr, P. A.; Evans, D. A. Alzheimer Disease in the United States (2010–2050) Estimated Using the 2010 Census. Neurology 2013, 80, 1778– 1783,  DOI: 10.1212/WNL.0b013e31828726f5
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    Alzheimer disease in the United States (2010-2050) estimated using the 2010 census
    Hebert Liesi E; Weuve Jennifer; Scherr Paul A; Evans Denis A
    Neurology (2013), 80 (19), 1778-83 ISSN:.
    OBJECTIVES: To provide updated estimates of Alzheimer disease (AD) dementia prevalence in the United States from 2010 through 2050. METHODS: Probabilities of AD dementia incidence were calculated from a longitudinal, population-based study including substantial numbers of both black and white participants. Incidence probabilities for single year of age, race, and level of education were calculated using weighted logistic regression and AD dementia diagnosis from 2,577 detailed clinical evaluations of 1,913 people obtained from stratified random samples of previously disease-free individuals in a population of 10,800. These were combined with US mortality, education, and new US Census Bureau estimates of current and future population to estimate current and future numbers of people with AD dementia in the United States. RESULTS: We estimated that in 2010, there were 4.7 million individuals aged 65 years or older with AD dementia (95% confidence interval [CI] = 4.0-5.5). Of these, 0.7 million (95% CI = 0.4-0.9) were between 65 and 74 years, 2.3 million were between 75 and 84 years (95% CI = 1.7-2.9), and 1.8 million were 85 years or older (95% CI = 1.4-2.2). The total number of people with AD dementia in 2050 is projected to be 13.8 million, with 7.0 million aged 85 years or older. CONCLUSION: The number of people in the United States with AD dementia will increase dramatically in the next 40 years unless preventive measures are developed.
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  6. 6
    Arthur, K. C.; Calvo, A.; Price, T. R.; Geiger, J. T.; Chiò, A.; Traynor, B. J. Projected Increase in Amyotrophic Lateral Sclerosis from 2015 to 2040. Nat. Commun. 2016, 7, 12408  DOI: 10.1038/ncomms12408
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    Projected increase in amyotrophic lateral sclerosis from 2015 to 2040
    Arthur, Karissa C.; Calvo, Andrea; Price, T. Ryan; Geiger, Joshua T.; Chio, Adriano; Traynor, Bryan J.
    Nature Communications (2016), 7 (), 12408CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
    Although amyotrophic lateral sclerosis (ALS) is relatively rare, the socioeconomic significance of the disease is extensive. It is therefore vital to project the epidemiol. trend of ALS. To date, there have been few published studies attempting to est. the no. and distribution of ALS cases in the upcoming years. Here we show that the no. of ALS cases across the globe will increase from 222,801 in 2015 to 376,674 in 2040, representing an increase of 69%. This increase is predominantly due to ageing of the population, particularly among developing nations. This projection is likely an underestimate due to improving healthcare and economic conditions. The results should be used to inform healthcare policy to more efficiently allocate healthcare resources.
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  7. 7
    Chatani, E.; Yamamoto, N. Recent Progress on Understanding the Mechanisms of Amyloid Nucleation. Biophys. Rev. 2018, 10, 527– 534,  DOI: 10.1007/s12551-017-0353-8
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    7
    Recent progress on understanding the mechanisms of amyloid nucleation
    Chatani, Eri; Yamamoto, Naoki
    Biophysical Reviews (2018), 10 (2), 527-534CODEN: BRIECG; ISSN:1867-2450. (Springer)
    A review. Amyloid fibrils are supramol. protein assemblies with a fibrous morphol. and cross-β structure. The formation of amyloid fibrils typically follows a nucleation-dependent polymn. mechanism, in which a one-step nucleation scheme has widely been accepted. However, a variety of oligomers have been identified in early stages of fibrillation, and a nucleated conformational conversion (NCC) mechanism, in which oligomers serve as a precursor of amyloid nucleation and convert to amyloid nuclei, has been proposed. This development has raised the need to consider more complicated multi-step nucleation processes in addn. to the simplest one-step process, and evidence for the direct involvement of oligomers as nucleation precursors has been obtained both exptl. and theor. Interestingly, the NCC mechanism has some analogy with the two-step nucleation mechanism proposed for inorg. and org. crystals and protein crystals, although a more dramatic conformational conversion of proteins should be considered in amyloid nucleation. Clarifying the properties of the nucleation precursors of amyloid fibrils in detail, in comparison with those of crystals, will allow a better understanding of the nucleation of amyloid fibrils and pave the way to develop techniques to regulate it.
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  8. 8
    Meisl, G.; Kirkegaard, J. B.; Arosio, P.; Michaels, T. C. T.; Vendruscolo, M.; Dobson, C. M.; Linse, S.; Knowles, T. P. J. Molecular Mechanisms of Protein Aggregation from Global Fitting of Kinetic Models. Nat. Protoc. 2016, 11, 252– 272,  DOI: 10.1038/nprot.2016.010
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    Molecular mechanisms of protein aggregation from global fitting of kinetic models
    Meisl, Georg; Kirkegaard, Julius B.; Arosio, Paolo; Michaels, Thomas C. T.; Vendruscolo, Michele; Dobson, Christopher M.; Linse, Sara; Knowles, Tuomas P. J.
    Nature Protocols (2016), 11 (2), 252-272CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
    The elucidation of the mol. mechanisms by which sol. proteins convert into their amyloid forms is a fundamental prerequisite for understanding and controlling disorders that are linked to protein aggregation, such as Alzheimer's and Parkinson's diseases. However, because of the complexity assocd. with aggregation reaction networks, the anal. of kinetic data of protein aggregation to obtain the underlying mechanisms represents a complex task. Here we describe a framework, using quant. kinetic assays and global fitting, to det. and to verify a mol. mechanism for aggregation reactions that is compatible with exptl. kinetic data. We implement this approach in a web-based software, AmyloFit. Our procedure starts from the results of kinetic expts. that measure the concn. of aggregate mass as a function of time. We illustrate the approach with results from the aggregation of the β-amyloid (Aβ) peptides measured using thioflavin T, but the method is suitable for data from any similar kinetic expt. measuring the accumulation of aggregate mass as a function of time; the input data are in the form of a tab-sepd. text file. We also outline general exptl. strategies and practical considerations for obtaining kinetic data of sufficient quality to draw detailed mechanistic conclusions, and the procedure starts with instructions for extensive data quality control. For the core part of the anal., we provide an online platform (http://www.amylofit.ch.cam.ac.uk) that enables robust global anal. of kinetic data without the need for extensive programming or detailed math. knowledge. The software automates repetitive tasks and guides users through the key steps of kinetic anal.: detn. of constraints to be placed on the aggregation mechanism based on the concn. dependence of the aggregation reaction, choosing from several fundamental models describing assembly into linear aggregates and fitting the chosen models using an advanced minimization algorithm to yield the reaction orders and rate consts. Finally, we outline how to use this approach to investigate which targets potential inhibitors of amyloid formation bind to and where in the reaction mechanism they act. The protocol, from processing data to detg. mechanisms, can be completed in <1 d.
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  9. 9
    Fitzpatrick, A. W. P.; Debelouchina, G. T.; Bayro, M. J.; Clare, D. K.; Caporini, M. A.; Bajaj, V. S.; Jaroniec, C. P.; Wang, L.; Ladizhansky, V.; Muller, S. A.; MacPhee, C. E.; Waudby, C. A.; Mott, H. R.; De Simone, A.; Knowles, T. P. J.; Saibil, H. R.; Vendruscolo, M.; Orlova, E. V.; Griffin, R. G.; Dobson, C. M. Atomic Structure and Hierarchical Assembly of a Cross-β Amyloid Fibril. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 5468– 5473,  DOI: 10.1073/pnas.1219476110
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    Atomic structure and hierarchical assembly of a cross-β amyloid fibril
    Fitzpatrick, Anthony W. P.; Debelouchina, Galia T.; Bayro, Marvin J.; Clare, Daniel K.; Caporini, Marc A.; Bajaj, Vikram S.; Jaroniec, Christopher P.; Wang, Luchun; Ladizhansky, Vladimir; Mueller, Shirley A.; MacPhee, Cait E.; Waudby, Christopher A.; Mott, Helen R.; De Simone, Alfonso; Knowles, Tuomas P. J.; Saibil, Helen R.; Vendruscolo, Michele; Orlova, Elena V.; Griffin, Robert G.; Dobson, Christopher M.
    Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (14), 5468-5473, S5468/1-S5468/32CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of β-sheet filaments. These complex aggregates have remarkable chem. and phys. properties, and the conversion of normally sol. functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-β amyloid fibrils have proved to be recalcitrant to detailed structural anal. By combining structural constraints from a series of exptl. techniques spanning five orders of magnitude in length scale - including magic angle spinning NMR spectroscopy, x-ray fiber diffraction, cryoelectron microscopy, scanning TEM, and at. force microscopy - we report the at.-resoln. (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent β-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils.
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  10. 10
    Fändrich, M.; Nyström, S.; Nilsson, K. P. R.; Böckmann, A.; LeVine, H.; Hammarström, P. Amyloid Fibril Polymorphism: A Challenge for Molecular Imaging and Therapy. J. Intern. Med. 2018, 283, 218– 237,  DOI: 10.1111/joim.12732
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    Amyloid fibril polymorphism: a challenge for molecular imaging and therapy
    Fandrich M; Nystrom S; Nilsson K P R; Hammarstrom P; Bockmann A; LeVine H 3rd; LeVine H 3rd
    Journal of internal medicine (2018), 283 (3), 218-237 ISSN:.
    The accumulation of misfolded proteins (MPs), both unique and common, for different diseases is central for many chronic degenerative diseases. In certain patients, MP accumulation is systemic (e.g. TTR amyloid), and in others, this is localized to a specific cell type (e.g. Alzheimer's disease). In neurodegenerative diseases, NDs, it is noticeable that the accumulation of MP progressively spreads throughout the nervous system. Our main hypothesis of this article is that MPs are not only markers but also active carriers of pathogenicity. Here, we discuss studies from comprehensive molecular approaches aimed at understanding MP conformational variations (polymorphism) and their bearing on spreading of MPs, MP toxicity, as well as MP targeting in imaging and therapy. Neurodegenerative disease (ND) represents a major and growing societal challenge, with millions of people worldwide suffering from Alzheimer's or Parkinson's diseases alone. For all NDs, current treatment is palliative without addressing the primary cause and is not curative. Over recent years, particularly the shape-shifting properties of misfolded proteins and their spreading pathways have been intensively researched. The difficulty in addressing ND has prompted most major pharma companies to severely downsize their nervous system disorder research. Increased academic research is pivotal for filling this void and to translate basic research into tools for medical professionals. Recent discoveries of targeting drug design against MPs and improved model systems to study structure, pathology spreading and toxicity strongly encourage future studies along these lines to provide an opportunity for selective imaging, prognostic diagnosis and therapy.
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    Cummings, J.; Lee, G.; Ritter, A.; Sabbagh, M.; Zhong, K. Alzheimer’s Disease Drug Development Pipeline: 2019. Alzheimer’s Dementia: Transl. Res. Clin. Interventions 2019, 5, 272– 293,  DOI: 10.1016/j.trci.2019.05.008
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    Šneideris, T.; Baranauskienė, L.; Cannon, J. G.; Rutkienė, R.; Meškys, R.; Smirnovas, V. Looking for a Generic Inhibitor of Amyloid-like Fibril Formation among Flavone Derivatives. PeerJ 2015, 3, e1271  DOI: 10.7717/peerj.1271
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    Srinivasan, E.; Rajasekaran, R. Probing the Inhibitory Activity of Epigallocatechin-Gallate on Toxic Aggregates of Mutant (L84F) SOD1 Protein through Geometry Based Sampling and Steered Molecular Dynamics. J. Mol. Graphics Model. 2017, 74, 288– 295,  DOI: 10.1016/j.jmgm.2017.04.019
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    Probing the inhibitory activity of epigallocatechin-gallate on toxic aggregates of mutant (L84F) SOD1 protein through geometry based sampling and steered molecular dynamics
    Srinivasan, E.; Rajasekaran, R.
    Journal of Molecular Graphics & Modelling (2017), 74 (), 288-295CODEN: JMGMFI; ISSN:1093-3263. (Elsevier Ltd.)
    Amyloid formation and protein aggregation are considered to be at the core of the disease pathol. for the various neurodegenerative disorders such as Amyotrophic lateral sclerosis (ALS). Considerable exptl. reports have suggested that epigallocatechin-gallate (EGCG), a natural polyphenol from the green tea inhibits the amyloid formation in multiple neurodegenerative disease. Mutations in SOD1 protein are considered to a key factor that contributes towards the rapid disease progression and the pathogenesis in both, the sporadic and familial form. In our study, we computationally examd. the inhibitory action of EGCG against the native and the mutant SOD1 through mol. docking, steered mol. dynamics and conformational sampling methods From the outcome, we could conjecture that the protein destabilization and increased β-sheet propensity that occurred due to mutation were regained upon the binding of EGCG. Moreover, the concepts of the free energy landscape anal. are introduced to establish the visual appearance of protein aggregation upon mutation. Altogether, we come to know that the binding of EGCG on mutant SOD1 has reduced the formation of the toxic aggregates upon mutation. Hence, our study could be an initiative in deciphering the inhibitory action of EGCG against the aggregated mutant SOD1, which could be a therapeutic potential against the treatment for the incurable neurodegenerative disorder (ALS) affecting the mankind.
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    Goyal, D.; Shuaib, S.; Mann, S.; Goyal, B. Rationally Designed Peptides and Peptidomimetics as Inhibitors of Amyloid-β (Aβ) Aggregation: Potential Therapeutics of Alzheimer’s Disease. ACS Comb. Sci. 2017, 19, 55– 80,  DOI: 10.1021/acscombsci.6b00116
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    14
    Rationally Designed Peptides and Peptidomimetics as Inhibitors of Amyloid-β (Aβ) Aggregation: Potential Therapeutics of Alzheimer's Disease
    Goyal, Deepti; Shuaib, Suniba; Mann, Sukhmani; Goyal, Bhupesh
    ACS Combinatorial Science (2017), 19 (2), 55-80CODEN: ACSCCC; ISSN:2156-8944. (American Chemical Society)
    A review. Alzheimer's disease (AD) is a progressive neurodegenerative disease with no clin. accepted treatment to cure or halt its progression. The worldwide effort to develop peptide-based inhibitors of amyloid-β (Aβ) aggregation can be considered an unplanned combinatorial expt. An understanding of what has been done and achieved may advance the understanding of AD pathol. and the discovery of effective therapeutic agents. The authors review here the history of such peptide-based inhibitors, including those based on the Aβ sequence and those not derived from that sequence, contg. both natural and unnatural amino acid building blocks. Peptide-based aggregation inhibitors hold significant promise for future AD therapy owing to their high selectivity, effectiveness, low toxicity, good tolerance, low accumulation in tissues, high chem. and biol. diversity, possibility of rational design and highly developed methods for analyzing their mode of action, proteolytic stability (modified peptides) and blood-brain barrier (BBB) permeability.
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  15. 15
    Byeon, S. R.; Lee, J. H.; Sohn, J.-H.; Kim, D. C.; Shin, K. J.; Yoo, K. H.; Mook-Jung, I.; Lee, W. K.; Kim, D. J. Bis-Styrylpyridine and Bis-Styrylbenzene Derivatives as Inhibitors for Aβ Fibril Formation. Bioorg. Med. Chem. Lett. 2007, 17, 1466– 1470,  DOI: 10.1016/j.bmcl.2006.10.090
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    15
    Bis-styrylpyridine and bis-styrylbenzene derivatives as inhibitors for Aβ fibril formation
    Byeon, Seong Rim; Lee, Ji Hoon; Sohn, Ji-Hoon; Kim, Dong Chan; Shin, Kye Jung; Yoo, Kyung Ho; Mook-Jung, Inhee; Lee, Won Koo; Kim, Dong Jin
    Bioorganic & Medicinal Chemistry Letters (2007), 17 (5), 1466-1470CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)
    New bis-styrylpyridine and bis-styrylbenzene derivs. were designed and synthesized. These 34 compds. were evaluated by Aβ fibril formation inhibitory assay using thioflavin T as a dye (named ThT assay). Most of them showed excellent inhibitory activities for Aβ fibril formation at IC50 of 0.1-2.7 μM which is comparable to curcumin (IC50 of 0.8 μM). Among them, nine compds. were screened for their cytotoxicities on HT-22 cell by MTT assay at 1, 10, and 50 μM. In particular, I-7 (I) and II-2 exhibited the best combination of inhibitory activity and compd. cytotoxicity.
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    Konar, M.; Bag, S.; Roy, P.; Dasgupta, S. Gallic Acid Induced Dose Dependent Inhibition of Lysozyme Fibrillation. Int. J. Biol. Macromol. 2017, 103, 1224– 1231,  DOI: 10.1016/j.ijbiomac.2017.05.158
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    16
    Gallic acid induced dose dependent inhibition of lysozyme fibrillation
    Konar, Mouli; Bag, Sudipta; Roy, Pritam; Dasgupta, Swagata
    International Journal of Biological Macromolecules (2017), 103 (), 1224-1231CODEN: IJBMDR; ISSN:0141-8130. (Elsevier B.V.)
    Amyloidosis is primarily characterized by the deposition of misfolded protein aggregates. Although the natural polyphenols have long been known as effective amyloid inhibitors, the mechanistic details of their inhibitory actions still remain unclear. Our present study explores the inhibition mechanism of polyphenols by studying the anti-amyloidogenic property of gallic acid (GA), the smallest structural unit of tea polyphenols, on hen egg white lysozyme (HEWL) at physiol. pH. Using various spectroscopic techniques such as UV-vis, fluorescence, CD and dynamic light scattering, and microscopic techniques such as TEM and FESEM, it has been shown that GA potentially inhibits the self-aggregation process in a concn. dependent manner. Gel electrophoresis studies suggest that the o-dihydroxy moiety of GA is oxidized into the quinone moiety and H2O2 in the system under the exptl. conditions. The quinone binds near the hydrophobic region of HEWL and restricts hydrophobic exposure. Cyclic voltammetry studies reveal that the Met residues of HEWL are oxidized by H2O2 to highly polar sulfoxide-modified side chains. The partially unfolded intermediates formed under the denaturing conditions employed remain in contact with the solvent thus preventing further aggregation.
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  17. 17
    Ruggeri, F. S.; Šneideris, T.; Vendruscolo, M.; Knowles, T. P. J. Atomic Force Microscopy for Single Molecule Characterisation of Protein Aggregation. Arch. Biochem. Biophys. 2019, 664, 134– 148,  DOI: 10.1016/j.abb.2019.02.001
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    17
    Atomic force microscopy for single molecule characterisation of protein aggregation
    Ruggeri, Francesco Simone; Sneideris, Tomas; Vendruscolo, Michele; Knowles, Tuomas P. J.
    Archives of Biochemistry and Biophysics (2019), 664 (), 134-148CODEN: ABBIA4; ISSN:0003-9861. (Elsevier B.V.)
    The development of at. force microscopy (AFM) has opened up a wide range of novel opportunities in nanoscience and new modalities of observation in complex biol. systems. AFM imaging has been widely employed to resolve the complex and heterogeneous conformational states involved in protein aggregation at the single mol. scale and shed light onto the mol. basis of a variety of human pathologies, including neurodegenerative disorders. The study of individual macromols. at nanoscale, however, remains challenging, esp. when fully quant. information is required. In this review, we first discuss the principles of AFM with a special emphasis on the fundamental factors defining its sensitivity and accuracy. We then review the fundamental parameters and approaches to work at the limit of AFM resoln. in order to perform single mol. statistical anal. of biomols. and nanoscale protein aggregates. This single mol. statistical approach has proved to be powerful to unravel the mol. and hierarchical assembly of the misfolded species present transiently during protein aggregation, to visualise their dynamics at the nanoscale, as well to study the structural properties of amyloid-inspired functional nanomaterials.
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    Podestà, A.; Tiana, G.; Milani, P.; Manno, M. Early Events in Insulin Fibrillization Studied by Time-Lapse Atomic Force Microscopy. Biophys. J. 2006, 90, 589– 597,  DOI: 10.1529/biophysj.105.068833
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    Early events in insulin fibrillization studied by time-lapse atomic force microscopy
    Podesta, Alessandro; Tiana, Guido; Milani, Paolo; Manno, Mauro
    Biophysical Journal (2006), 90 (2), 589-597CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    The importance of understanding the mechanism of protein aggregation into insol. amyloid fibrils lies not only in its medical consequences, but also in its more basic properties of self-organization. The discovery that a large no. of uncorrelated proteins can form, under proper conditions, structurally similar fibrils has suggested that the underlying mechanism is a general feature of polypeptide chains. The authors address the early events preceding amyloid fibril formation in solns. of zinc-free human insulin incubated at low pH and high temp. Here, the authors show by time-lapse at. force microscopy that a steady-state distribution of protein oligomers with a quasiexponential tail is reached within a few minutes after heating. This metastable phase lasts for a few hours, until fibrillar aggregates are observable. Although for such complex systems different aggregation mechanisms can occur simultaneously, the authors' results indicate that the prefibrillar phase is mainly controlled by a simple coagulation-evapn. kinetic mechanism, in which concn. acts as a crit. parameter. These exptl. facts, along with the kinetic model used, suggest a crit. role for thermal concn. fluctuations in the process of fibril nucleation.
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  19. 19
    Streets, A. M.; Sourigues, Y.; Kopito, R. R.; Melki, R.; Quake, S. R. Simultaneous Measurement of Amyloid Fibril Formation by Dynamic Light Scattering and Fluorescence Reveals Complex Aggregation Kinetics. PLoS One 2013, 8, e54541  DOI: 10.1371/journal.pone.0054541
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    19
    Simultaneous measurement of amyloid fibril formation by dynamic light scattering and fluorescence reveals complex aggregation kinetics
    Streets, Aaron M.; Sourigues, Yannick; Kopito, Ron R.; Melki, Ronald; Quake, Stephen R.
    PLoS One (2013), 8 (1), e54541CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    An app. that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit assocd. with Huntington's disease, in vitro. Huntington's disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer's and Parkinson's disease. These pathologies are all related by the propensity of their assocd. protein or polypeptide to form insol., β-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides assocd. with these diseases, the resulting amyloids display strikingly similar phys. structure, an observation which suggests a phys. basis for amyloid fibril formation. Thioflavin T fluorescence reports β-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation.
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  20. 20
    Zandomeneghi, G.; Krebs, M. R. H.; McCammon, M. G.; Fändrich, M. FTIR Reveals Structural Differences between Native β-Sheet Proteins and Amyloid Fibrils. Protein Sci. 2004, 13, 3314– 3321,  DOI: 10.1110/ps.041024904
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    FTIR reveals structural differences between native β-sheet proteins and amyloid fibrils
    Zandomeneghi, Giorgia; Krebs, Mark R. H.; McCammon, Margaret G.; Faendrich, Marcus
    Protein Science (2004), 13 (12), 3314-3321CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)
    The presence of β-sheets in the core of amyloid fibrils raised questions as to whether or not β-sheet-contg. proteins, such as transthyretin, are predisposed to form such fibrils. However, we show here that the mol. structure of amyloid fibrils differs more generally from the β-sheets in native proteins. This difference is evident from the amide I region of the IR spectrum and relates to the distribution of the .vphi./ψ dihedral angles within the Ramachandran plot, the av. no. of strands per sheet, and possibly, the β-sheet twist. These data imply that amyloid fibril formation from native β-sheet proteins can involve a substantial structural reorganization.
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  21. 21
    Malmos, K. G.; Blancas-Mejia, L. M.; Weber, B.; Buchner, J.; Ramirez-Alvarado, M.; Naiki, H.; Otzen, D. ThT 101: A Primer on the Use of Thioflavin T to Investigate Amyloid Formation. Amyloid 2017, 24, 1– 16,  DOI: 10.1080/13506129.2017.1304905
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    Picken, M. M.; Herrera, G. A. Thioflavin T Stain: An Easier and More Sensitive Method for Amyloid Detection. In Amyloid and Related Disorders; Picken, M. M.; Herrera, G. A.; Dogan, A., Eds.; Humana Press: Totowa, NJ, 2012; pp 187– 189.
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    Wetzel, R.; Chemuru, S.; Misra, P.; Kodali, R.; Mukherjee, S.; Kar, K. An Aggregate Weight-Normalized Thioflavin-T Measurement Scale for Characterizing Polymorphic Amyloids and Assembly Intermediates. Methods Mol. Biol. 2018, 1777, 121– 144,  DOI: 10.1007/978-1-4939-7811-3_6
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    23
    An aggregate weight-normalized thioflavin-T measurement scale for characterizing polymorphic amyloids and assembly intermediates
    Wetzel, Ronald; Chemuru, Saketh; Misra, Pinaki; Kodali, Ravi; Mukherjee, Smita; Kar, Karunakar
    Methods in Molecular Biology (New York, NY, United States) (2018), 1777 (Peptide Self-Assembly), 121-144CODEN: MMBIED; ISSN:1940-6029. (Springer)
    The red shift in the fluorescence excitation spectra of thioflavin dyes upon binding to fibrils has been a boon to the amyloid field, offering simple and effective methods for the qual. detection of amyloid in tissue samples and for quantitation of particular fibril prepns. with gravimetric linearity. The quant. aspect of the thioflavin T (ThT) response, however, comes with an important caveat that bestows both significant limitations and great untapped power. It is now well established that amyloid fibrils of different proteins, as well as polymorphic fibrils of the same protein, can exhibit vastly different ThT fluorescence intensities for the same wt. concn. of aggregates. Furthermore, the aggregated intermediates commonly obsd. in amyloid assembly reactions can exhibit aggregate wt.-normalized (AWN) ThT fluorescence intensities that vary from essentially zero through a wide range of intermediate values before reaching the intensity of homogeneous, mature amyloid. These features make it very difficult to quant. interpret, without addnl. data, the time-dependent development of ThT fluorescence intensity in an assembly reaction. In this chapter, we describe a method for coupling ex situ ThT fluorescence detns. with an anal. HPLC supported sedimentation assay (also described in detail) that can provide significant new insights into amyloid assembly reactions. The time dependent aggregation data provided by the sedimentation assay reveals a time course of aggregation that is largely independent of aggregate properties. In addn., the combination of these data with ThT measurements of the same reaction time points reveals important aspects of av. aggregate structure at each time point. Examples of the use and potential value of AWN-ThT measurements during amyloid assembly Aβ and polyglutamine peptides are provided.
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  24. 24
    Groenning, M. Binding Mode of Thioflavin T and Other Molecular Probes in the Context of Amyloid Fibrils—Current Status. J. Chem. Biol. 2010, 3, 1– 18,  DOI: 10.1007/s12154-009-0027-5
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    24
    Binding mode of Thioflavin T and other molecular probes in the context of amyloid fibrils-current status
    Groenning Minna
    Journal of chemical biology (2010), 3 (1), 1-18 ISSN:1864-6158.
    Because understanding amyloid fibrillation in molecular detail is essential for development of strategies to control amyloid formation and overcome neurodegenerative disorders, increased understanding of present molecular probes as well as development of new probes are of utmost importance. To date, the binding modes of these molecular probes to amyloid fibrils are by no means adequately described or understood, and the large number of studies on Thioflavin T (ThT) and Congo Red (CR) binding have resulted in models that are incomplete and conflicting. Different types of binding sites are likely to be present in amyloid fibrils with differences in binding modes. ThT may bind in channels running parallel to the long axis of the fibril. In the channels, ThT may bind in either a monomeric or dimeric form of which the molecular conformation is likely to be planar. CR may bind in grooves formed along the β-sheets as a planar molecule in either a monomeric or supramolecular form.
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  25. 25
    Lockhart, A.; Ye, L.; Judd, D. B.; Merritt, A. T.; Lowe, P. N.; Morgenstern, J. L.; Hong, G.; Gee, A. D.; Brown, J. Evidence for the Presence of Three Distinct Binding Sites for the Thioflavin T Class of Alzheimer’s Disease PET Imaging Agents on β-Amyloid Peptide Fibrils. J. Biol. Chem. 2005, 280, 7677– 7684,  DOI: 10.1074/jbc.M412056200
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    25
    Evidence for the Presence of Three Distinct Binding Sites for the Thioflavin T Class of Alzheimer's Disease PET Imaging Agents on β-Amyloid Peptide Fibrils
    Lockhart, Andrew; Ye, Liang; Judd, Duncan B.; Merritt, Andy T.; Lowe, Peter N.; Morgenstern, Jennifer L.; Hong, Guizhu; Gee, Antony D.; Brown, John
    Journal of Biological Chemistry (2005), 280 (9), 7677-7684CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Imaging the progression of Alzheimer's disease would greatly facilitate the discovery of therapeutics, and a wide range of ligands are currently under development for the detection of β-amyloid peptide (Aβ)-contg. plaques by using positron emission tomog. Here we report an in-depth characterization of the binding of seven previously described ligands to in vitro generated Aβ-(1-40) polymers. All of the compds. were derived from the benzothiazole compd. thioflavin T and include 2-[4'-(methylamino)phenyl]benzothiazole and 2-(4'-dimethylamino-)phenyl-imidazo[1,2-a]-pyridine derivs., 2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole and 2-[4'-(4''-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and a benzofuran compd. (5-bromo-2-(4-dimethylaminophenyl)benzofuran). By using a range of fluorescent and radioligand binding assays, we find that these compds. display a more complex binding pattern than described previously and are consistent with three classes of binding sites on the Aβ fibrils. All of the compds. bound with very high affinity (low nM Kd) to a low capacity site (BS3) (1 ligand-binding site per ∼300 Aβ-(1-40) monomers) consistent with the previously recognized binding site for these compds. on the fibrils. However, the compds. also bound with high affinity (Kd ∼100 nM) to either one of two addnl. binding sites on the Aβ-(1-40) polymer. The properties of these sites, BS1 and BS2, suggest they are adjacent or partially overlapping and have a higher capacity than BS3, occurring every ∼35 or every ∼4 monomers of Aβ-(1-40)-peptide, resp. Compds. appear to display selectivity for BS2 based on the presence of a halogen substitution (2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole, 2-[4'-(4''-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and 5-bromo-2-(4-dimethylaminophenyl)benzofuran) on their arom. ring system. The presence of addnl. ligand-binding sites presents potential new targets for ligand development and may allow a more complete modeling of the current positron emission tomog. data.
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  26. 26
    Ziaunys, M.; Smirnovas, V. Additional Thioflavin-T Binding Mode in Insulin Fibril Inner Core Region. J. Phys. Chem. B 2019, 123, 8727– 8732,  DOI: 10.1021/acs.jpcb.9b08652
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    26
    Additional Thioflavin-T Binding Mode in Insulin Fibril Inner Core Region
    Ziaunys, Mantas; Smirnovas, Vytautas
    Journal of Physical Chemistry B (2019), 123 (41), 8727-8732CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
    Amyloidogenic protein aggregation into fibrils is linked to several neurodegenerative disorders, such as Alzheimer's or Parkinson's disease. An amyloid specific fluorescent dye thioflavin-T (ThT) is often used to track the formation of these fibrils in vitro. Despite its wide application, it is still unknown how many types of ThT binding modes to amyloids exist, with multiple studies indicating varying nos. In this work, we examine the binding of ThT to insulin fibrils generated at pH 2.4 and reveal a possible inner core binding mode which is not accessible to the dye mol. after aggregation occurs.
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  27. 27
    Sulatskaya, A. I.; Kuznetsova, I. M.; Belousov, M. V.; Bondarev, S. A.; Zhouravleva, G. A.; Turoverov, K. K. Stoichiometry and Affinity of Thioflavin T Binding to Sup35p Amyloid Fibrils. PLoS One 2016, 11, e0156314  DOI: 10.1371/journal.pone.0156314
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    27
    Stoichiometry and affinity of thioflavin T binding to Sup35p amyloid fibrils
    Sulatskaya, Anna I.; Kuznetsova, Irina M.; Belousov, Mikhail V.; Bondarev, Stanislav A.; Zhouravleva, Galina A.; Turoverov, Konstantin K.
    PLoS One (2016), 11 (5), e0156314/1-e0156314/14CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    In this work two modes of binding of the fluorescent probe thioflavin T to yeast prion protein Sup35p amyloid fibrils were revealed by absorption spectrometry of solns. prepd. by equil. microdialysis. These binding modes exhibited significant differences in binding affinity and stoichiometry. Moreover, the absorption spectrum and the molar extinction coeff. of the dye bound in each mode were detd. The fluorescence quantum yield of the dye bound in each mode was detd. via a spectrofluorimetric study of the same solns. in which the recorded fluorescence intensity was cor. for the primary inner filter effect. As previously predicted, the existence of one of the detected binding modes may be due to the incorporation of the dye into the grooves along the fiber axis perpendicular to the β-sheets of the fibrils. It was assumed that the second type of binding with higher affinity may be due to the existence of ThT binding sites that are localized to areas where amyloid fibrils are clustered.
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  28. 28
    Groenning, M.; Norrman, M.; Flink, J. M.; van de Weert, M.; Bukrinsky, J. T.; Schluckebier, G.; Frokjaer, S. Binding Mode of Thioflavin T in Insulin Amyloid Fibrils. J. Struct. Biol. 2007, 159, 483– 497,  DOI: 10.1016/j.jsb.2007.06.004
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    28
    Binding mode of Thioflavin T in insulin amyloid fibrils
    Groenning, Minna; Norrman, Mathias; Flink, James M.; van de Weert, Marco; Bukrinsky, Jens T.; Schluckebier, Gerd; Frokjaer, Sven
    Journal of Structural Biology (2007), 159 (3), 483-497CODEN: JSBIEM; ISSN:1047-8477. (Elsevier)
    Amyloid fibrils share various common structural features and their presence can be detected by Thioflavin T (ThT). In this paper, the binding mode of ThT to insulin amyloid fibrils was examd. Scatchard anal. and isothermal titrn. calorimetry (ITC) showed at least two binding site populations. The binding site population with the strongest binding was responsible for the characteristic ThT fluorescence. This binding had a capacity of about 0.1 mol of ThT bound per mol of insulin in fibril form. The binding capacity was unaffected by pH, but the affinity was lowest at low pH. Notably, presence of a third binding process prior to the other processes was suggested by ITC. Binding of ThT resulted in only minor changes in the fibril structure according to the x-ray diffraction patterns, where a slightly more dominant equatorial reflection at 16 Å relative to the intersheet distance of 11 Å was obsd. No change in the interstrand distance of 4.8 Å was obsd. On the basis of these results, the authors propose that ThT binds in cavities running parallel to the fibril axis, e.g., between the protofilaments forming the fibrils. Such cavities have been proposed previously in insulin fibrils and several other amyloid fibril models.
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  29. 29
    Kuznetsova, I. M.; Sulatskaya, A. I.; Uversky, V. N.; Turoverov, K. K. Analyzing Thioflavin T Binding to Amyloid Fibrils by an Equilibrium Microdialysis-Based Technique. PLoS One 2012, 7, e30724  DOI: 10.1371/journal.pone.0030724
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    29
    Analyzing thioflavin T binding to amyloid fibrils by an equilibrium microdialysis-based technique
    Kuznetsova, Irina M.; Sulatskaya, Anna I.; Uversky, Vladimir N.; Turoverov, Konstantin K.
    PLoS One (2012), 7 (2), e30724CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    A new approach for the detn. of the amyloid fibril - thioflavin T (ThT) binding parameters (the no. of binding modes, stoichiometry, and binding consts. of each mode) is proposed. This approach is based on the absorption spectroscopy detn. of the concn. of free and bound to fibril dye in solns., which are prepd. by equil. microdialysis. Furthermore, the proposed approach allowed us, for the first time, to det. the absorption spectrum, molar extinction coeff., and fluorescence quantum yield of the ThT bound to fibril by each binding modes. This approach is universal and can be used for detg. the binding parameters of any dye interaction with a receptor, such as ANS binding to proteins in the molten globule state or to protein amorphous aggregates.
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  30. 30
    Kawai, R.; Araki, M.; Yoshimura, M.; Kamiya, N.; Ono, M.; Saji, H.; Okuno, Y. Core Binding Site of a Thioflavin-T-Derived Imaging Probe on Amyloid β Fibrils Predicted by Computational Methods. ACS Chem. Neurosci. 2018, 9, 957– 966,  DOI: 10.1021/acschemneuro.7b00389
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    30
    Core Binding Site of a Thioflavin-T-Derived Imaging Probe on Amyloid β Fibrils Predicted by Computational Methods
    Kawai, Ryoko; Araki, Mitsugu; Yoshimura, Masashi; Kamiya, Narutoshi; Ono, Masahiro; Saji, Hideo; Okuno, Yasushi
    ACS Chemical Neuroscience (2018), 9 (5), 957-966CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)
    Development of new diagnostic imaging probes for Alzheimer's disease (AD), such as positron emission tomog. (PET) and single photon emission computed tomog. (SPECT) probes, has been strongly desired. In this study, the authors investigated the most accessible amyloid β (Aβ) binding site of [123I]IMPY, a Thioflavin-T-derived SPECT probe, using exptl. and computational methods. First, the authors performed a competitive inhibition assay with Orange-G, which recognizes the KLVFFA region in Aβ fibrils, suggesting that IMPY and Orange-G bind to different sites in Aβ fibrils. Next, the authors precisely predicted the IMPY binding site on a multiple-protofilament Aβ fibril model using computational approaches, consisting of mol. dynamics and docking simulations. The authors generated possible IMPY-binding structures using docking simulations to identify candidates for probe-binding sites. The binding free energy of IMPY with the Aβ fibril was calcd. by a free energy simulation method, MP-CAFEE. These computational results suggest that IMPY preferentially binds to an interfacial pocket located between two protofilaments and it is stabilized mainly through hydrophobic interactions. Finally, the computational approach was validated by comparing with the exptl. results. The present study demonstrates the possibility of computational approaches to screen new PET/SPECT probes for Aβ imaging.
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  31. 31
    Ivancic, V. A.; Ekanayake, O.; Lazo, N. D. Binding Modes of Thioflavin T on the Surface of Amyloid Fibrils Studied by NMR. ChemPhysChem 2016, 17, 2461– 2464,  DOI: 10.1002/cphc.201600246
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    31
    Binding Modes of Thioflavin T on the Surface of Amyloid Fibrils Studied by NMR
    Ivancic, Valerie A.; Ekanayake, Oshini; Lazo, Noel D.
    ChemPhysChem (2016), 17 (16), 2461-2464CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The mechanism for the interaction of thioflavin T (ThT) with amyloid fibrils at the mol. level is not known. Here, 1H NMR spectroscopy was used to det. the binding mode of ThT on the surface of fibrils from lysozyme and insulin. Relayed rotating-frame Overhauser enhancements in ThT were obsd., indicating that the orientation of ThT is orthogonal to the fibril surface. Importantly, the assembly state of ThT on both surfaces is different. On the surface of insulin fibrils, ThT is oligomeric, as indicated by rapid 1H spin-lattice relaxation rate in the rotating frame (R1ρ), presumably due to intermol. dipole-dipole interactions between ThT mols. In contrast, ThT on the surface of lysozyme fibrils is a monomer, as indicated by slower 1H R1ρ. These results shed new light into the mechanism for the enhancement of ThT fluorescence and may lead to more efficient detectors of amyloid assemblies, which have escaped detection by ThT in monomer form.
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  32. 32
    Mao, X.; Guo, Y.; Wang, C.; Zhang, M.; Ma, X.; Liu, L.; Niu, L.; Zeng, Q.; Yang, Y.; Wang, C. Binding Modes of Thioflavin T Molecules to Prion Peptide Assemblies Identified by Using Scanning Tunneling Microscopy. ACS Chem. Neurosci. 2011, 2, 281– 287,  DOI: 10.1021/cn200006h
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    32
    Binding Modes of Thioflavin T Molecules to Prion Peptide Assemblies Identified by Using Scanning Tunneling Microscopy
    Mao, Xiaobo; Guo, Yuanyuan; Wang, Chenxuan; Zhang, Min; Ma, Xiaojing; Liu, Lei; Niu, Lin; Zeng, Qingdao; Yang, Yanlian; Wang, Chen
    ACS Chemical Neuroscience (2011), 2 (6), 281-287CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)
    The widely used method to monitor the aggregation process of amyloid peptide is thioflavin T (ThT) assay, while the detailed mol. mechanism is still not clear. In this work, the authors report here the direct identification of the binding modes of ThT mols. with the prion peptide GNNQQNY by using scanning tunneling microscopy (STM). The assembly structures of GNNQQNY were first obsd. by STM on a graphite surface, and the introduction of ThT mols. to the surface facilitated the STM observations of the adsorption conformations of ThT with peptide strands. ThT mols. are apt to adsorb on the peptide assembly with β-sheet structure and oriented parallel with the peptide strands adopting four different binding modes. This effort could benefit the understanding of the mechanisms of the interactions between labeling species or inhibitory ligands and amyloid peptides, which is keenly needed for developing diagnostic and therapeutic approaches.
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  33. 33
    Sidhu, A.; Vaneyck, J.; Blum, C.; Segers-Nolten, I.; Subramaniam, V. Polymorph-Specific Distribution of Binding Sites Determines Thioflavin-T Fluorescence Intensity in α-Synuclein Fibrils. Amyloid 2018, 25, 189– 196,  DOI: 10.1080/13506129.2018.1517736
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    33
    Polymorph-specific distribution of binding sites determines thioflavin-T fluorescence intensity in α-synuclein fibrils
    Sidhu, Arshdeep; Vaneyck, Jonathan; Blum, Christian; Segers-Nolten, Ine; Subramaniam, Vinod
    Amyloid (2018), 25 (3), 189-196CODEN: AIJIET; ISSN:1350-6129. (Taylor & Francis Ltd.)
    Thioflavin-T (ThT) is the most commonly used fluorescent dye for following amyloid formation semi-quant. in vitro, specifically probing the fibrillar cross-β-sheet content. In recent years, structural polymorphism of amyloid fibrils has been shown to be an important aspect of amyloid formation, both in vitro and in neurodegenerative diseases. Therefore, understanding ThT-amyloid interactions in the context of structural polymorphism of amyloids is necessary for correct interpretation of ThT fluorescence data. Here we study the influence of fibril morphol. on ThT fluorescence and ThT binding sites, with two morphol. distinct but chem. identical α-synuclein polymorphs. In ThT fluorescence assays the two polymorphs show type-specific fluorescence intensity behavior although their β-sheet content has been shown to be similar. Further, fluorescence lifetime measurements of fibril-bound ThT reveal the presence of at least two qual. different ThT binding sites on the polymorphs. The relative distributions of the binding sites on the fibril surfaces appear to be morphol. dependent, thus detg. the obsd. polymorph-specific ThT fluorescence intensities. These results, highlighting the role of fibril morphol. in ThT-based amyloid studies, underline the relevance of polymorphs in ThT-amyloid interaction and can explain the variability often obsd. in ThT amyloid binding assays.
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    Ziaunys, M.; Sneideris, T.; Smirnovas, V. Formation of Distinct Prion Protein Amyloid Fibrils under Identical Experimental Conditions. Sci. Rep. 2020, 10, 4572  DOI: 10.1038/s41598-020-61663-2
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    Formation of distinct prion protein amyloid fibrils under identical experimental conditions
    Ziaunys, Mantas; Sneideris, Tomas; Smirnovas, Vytautas
    Scientific Reports (2020), 10 (1), 4572CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
    Protein aggregation into amyloid fibrils is linked to multiple neurodegenerative disorders, such as Alzheimer's, Parkinson's or Creutzfeldt-Jakob disease. A better understanding of the way these aggregates form is vital for the development of drugs. A large detriment to amyloid research is the ability of amyloidogenic proteins to spontaneously aggregate into multiple structurally distinct fibrils (strains) with different stability and seeding properties. In this work we show that prion proteins are capable of forming more than one type of fibril under the exact same conditions by assessing their Thioflavin T (ThT) binding ability, morphol., secondary structure, stability and seeding potential.
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    Sakalauskas, A.; Ziaunys, M.; Smirnovas, V. Concentration-Dependent Polymorphism of Insulin Amyloid Fibrils. PeerJ 2019, 7, e8208  DOI: 10.7717/peerj.8208
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    Sneideris, T.; Sakalauskas, A.; Sternke-Hoffmann, R.; Peduzzo, A.; Ziaunys, M.; Buell, A. K.; Smirnovas, V. The Environment Is a Key Factor in Determining the Anti-Amyloid Efficacy of EGCG. Biomolecules 2019, 9, 855  DOI: 10.3390/biom9120855
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    The environment is a key factor in determining the anti-amyloid efficacy of EGCG
    Sneideris, Tomas; Sakalauskas, Andrius; Sternke-Hoffmann, Rebecca; Peduzzo, Alessia; Ziaunys, Mantas; Buell, Alexander K.; Smirnovas, Vytautas
    Biomolecules (2019), 9 (12), 855CODEN: BIOMHC; ISSN:2218-273X. (MDPI AG)
    Millions of people around the world suffer from amyloid-related disorders, including Alzheimer's and Parkinson's diseases. Despite significant and sustained efforts, there are still no disease-modifying drugs available for the majority of amyloid-related disorders, and the overall failure rate in clin. trials is very high, even for compds. that show promising anti-amyloid activity in vitro. In this study, it demonstrate that even small changes in the chem. environment can strongly modulate the inhibitory effects of anti-amyloid compds. Using one of the best-established amyloid inhibitory compds., epigallocatechin-3-gallate (EGCG), as an example, and two amyloid-forming proteins, insulin and Parkinson's disease-related a-synuclein, we shed light on the previously unexplored sensitivity to soln. conditions of the action of this compd. on amyloid fibril formation. In the case of insulin, it show that the classification of EGCG as an amyloid inhibitor depends on the exptl. conditions select, on the method used for the evaluation of the efficacy, and on whether or not EGCG is allowed to oxidise before the expt. For a-synuclein, it show that a small change in pH value, from 7 to 6, transforms EGCG from an efficient inhibitor to completely ineffective, and we were able to explain this behavior by the increased stability of EGCG against oxidn. at pH 6.
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    Gupta, Y.; Singla, G.; Singla, R. Insulin-Derived Amyloidosis. Indian J. Endocrinol. Metab. 2015, 19, 174– 177,  DOI: 10.4103/2230-8210.146879
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    Insulin-derived amyloidosis
    Gupta Yashdeep; Singla Gaurav; Singla Rajiv
    Indian journal of endocrinology and metabolism (2015), 19 (1), 174-7 ISSN:2230-8210.
    UNLABELLED: Amyloidosis is the term for diseases caused by the extracellular deposition of insoluble polymeric protein fibrils in tissues and organs. Insulin-derived amyloidosis is a rare, yet significant complication of insulin therapy. Insulin-derived amyloidosis at injection site can cause poor glycemic control and increased insulin dose requirements because of the impairment in insulin absorption, which reverse on change of injection site and/or excision of the mass. This entity should be considered and assessed by histopathology and immunohistochemistry, in patients with firm/hard local site reactions, which do not regress after cessation of insulin injection at the affected site. SEARCH STRATEGY: PubMed was searched with terms "insulin amyloidosis". Full text of articles available in English was reviewed. Relevant cross references were also reviewed. Last search was made on October 15, 2014.
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    Foderà, V.; Cataldo, S.; Librizzi, F.; Pignataro, B.; Spiccia, P.; Leone, M. Self-Organization Pathways and Spatial Heterogeneity in Insulin Amyloid Fibril Formation. J. Phys. Chem. B 2009, 113, 10830– 10837,  DOI: 10.1021/jp810972y
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    Self-organization pathways and spatial heterogeneity in insulin amyloid fibril formation
    Fodera, Vito; Cataldo, Sebastiano; Librizzi, Fabio; Pignataro, Bruno; Spiccia, Paola; Leone, Maurizio
    Journal of Physical Chemistry B (2009), 113 (31), 10830-10837CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
    At high temp. and low pH, the protein hormone insulin is highly prone to form amyloid fibrils, and for this reason it is widely used as a model system to study fibril formation mechanisms. In this work, we focused on insulin aggregation mechanisms occurring in HCl solns. (pH 1.6) at 60 °C. By means of in situ Thioflavin T (ThT) staining, the kinetics profiles were characterized as a function of the protein concn., and two concurrent aggregation pathways were pointed out, being concn. dependent. In correspondence to these pathways, different morphologies of self-assembled protein mols. were detected by at. force microscopy images also evidencing the presence of secondary nucleation processes as a peculiar mechanism for insulin fibrillation. Moreover, combining ThT fluorescence and light scattering, the early stages of the process were analyzed in the low concn. regime, pointing out a pronounced spatial heterogeneity in the formation of the first stable fibrils in soln. and the onset of the secondary nucleation pathways.
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    Gong, H.; He, Z.; Peng, A.; Zhang, X.; Cheng, B.; Sun, Y.; Zheng, L.; Huang, K. Effects of Several Quinones on Insulin Aggregation. Sci. Rep. 2015, 4, 5648  DOI: 10.1038/srep05648
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    Wang, J.-B.; Wang, Y.-M.; Zeng, C.-M. Quercetin Inhibits Amyloid Fibrillation of Bovine Insulin and Destabilizes Preformed Fibrils. Biochem. Biophys. Res. Commun. 2011, 415, 675– 679,  DOI: 10.1016/j.bbrc.2011.10.135
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    Quercetin inhibits amyloid fibrillation of bovine insulin and destabilizes preformed fibrils
    Wang, Jian-Bo; Wang, Yi-Min; Zeng, Cheng-Ming
    Biochemical and Biophysical Research Communications (2011), 415 (4), 675-679CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)
    Growing interest and research efforts have recently been focused on elucidating the mol. mechanism of amyloid formation and the screening of effective inhibitors to interrupt amyloid structures. In the present study, the anti-amyloidogenic effects of quercetin were investigated in vitro using bovine insulin as a model protein. The results demonstrated that quercetin dose-dependently inhibited amyloid formation of insulin. Moreover, quercetin destabilized the preformed insulin fibrils and transformed the fibrils into amorphous aggregates. Hemolysis was obsd. when human erythrocytes were co-incubated with insulin fibrils. Quercetin inhibited fibril-induced hemolysis in a dose-dependent manner. SDS-PAGE showed that insulin fibrils induced the aggregation of cytoskeletal proteins of erythrocyte membranes and that quercetin attenuated this fibril-induced cytoskeletal aggregation. The results of the present work suggest that quercetin may serve as a lead structure for the design of novel anti-amyloidogenic drugs.
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    Jayamani, J.; Shanmugam, G. Gallic Acid, One of the Components in Many Plant Tissues, Is a Potential Inhibitor for Insulin Amyloid Fibril Formation. Eur. J. Med. Chem. 2014, 85, 352– 358,  DOI: 10.1016/j.ejmech.2014.07.111
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    Gallic acid, one of the components in many plant tissues, is a potential inhibitor for insulin amyloid fibril formation
    Jayamani, Jayaraman; Shanmugam, Ganesh
    European Journal of Medicinal Chemistry (2014), 85 (), 352-358CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
    Proteins under stressful conditions can lead to the formation of an ordered self-assembled structure, referred to as amyloid fibrils, to which many neurodegenerative diseases such as Type II diabetes, Alzheimer's, Parkinson's, Huntington's, etc., are attributed. Inhibition of amyloid fibril formation using natural products is one of the main therapeutic strategies to prevent the progression of these diseases. Polyphenols are the mostly consumed as antioxidants in a human nutrition. Herein, we have studied the effect of a simple polyphenol, gallic acid (GA), one of the main components in plant tissues, esp. in tea leaves, on the insulin amyloid fibril formation. Different biophys. characterizations such as turbidity, at. force microscopy (AFM), Thioflavin T (ThT) assays, CD, and Fourier transform-IR spectroscopy have been used to analyze the inhibition of amyloid fibril formation. The occurrence of fibrils in an AFM image and ThT fluorescence enhancement confirms the formation of insulin amyloid fibrils when incubated under acidic pH 2 at 65 °C. In the presence of GA, absence of fibrils in AFM image and no change in the intensity of ThT fluorescence confirms the inhibition of insulin amyloid fibrils by GA. Spectroscopic results reveal that GA inhibits the conformational transition of α-helix → β-sheet, which is generally induced during the insulin fibril formation. It was found that the inhibitory effect of GA is concn. dependent and non-linear. Based on the obsd. results, we propose that GA interacts with native insulin, preventing nuclei formation, which is essential for fibril growth, thereby inhibiting the amyloid fibril formation. The present results thus demonstrate that GA can effectively inhibit insulin amyloid fibril formation in vitro.
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    Sneideris, T.; Darguzis, D.; Botyriute, A.; Grigaliunas, M.; Winter, R.; Smirnovas, V. PH-Driven Polymorphism of Insulin Amyloid-Like Fibrils. PLoS One 2015, 10, e0136602  DOI: 10.1371/journal.pone.0136602
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    pH-driven polymorphism of insulin amyloid- like fibrils
    Sneideris, Tomas; Darguzis, Domantas; Botyriute, Akvile; Grigaliunas, Martynas; Winter, Roland; Smirnovas, Vytautas
    PLoS One (2015), 10 (8), e0136602/1-e0136602/13CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Prions are infective proteins, which can self-assemble into different strain conformations, leading to different disease phenotypes. An increasing no. of studies suggest that prion-like self-propagation may be a common feature of amyloid-like structures. Thus it is important to unravel every possible factor leading to the formation of different amyloid strains. Here we report on the formation of two types of insulin amyloid-like fibrils with distinct IR spectroscopic features grown under slightly different pH conditions. Similar to prion strains, both insulin fibril types are able to self-propagate their conformational template under conditions, favoring spontaneous formation of different type fibrils. The low-pHinduced insulin amyloid strain is structurally very similar to previously reported strains formed either in the presence of 20% ethanol, or by modification of the amino acid sequence of insulin. A deeper anal. of literature data in the context of our current findings suggests a shift of the monomer-dimer equil. of insulin as a possible factor controlling the formation of different strains.
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    Dzwolak, W.; Smirnovas, V.; Jansen, R.; Winter, R. Insulin Forms Amyloid in a Strain-Dependent Manner: An FT-IR Spectroscopic Study. Protein Sci. 2004, 13, 1927– 1932,  DOI: 10.1110/ps.03607204
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    Insulin forms amyloid in a strain-dependent manner: An FT-IR spectroscopic study
    Dzwolak, Wojciech; Smirnovas, Vytautas; Jansen, Ralf; Winter, Roland
    Protein Science (2004), 13 (7), 1927-1932CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)
    The presence of 20% (vol./vol.) ethanol triggers growth of insulin amyloid with distinct IR spectroscopic features, compared with the fibrils obtained under ambient conditions. Here we report that the two insulin amyloid types behave in the prion strain-like manner regarding seeding specificity and ability of the self-propagating conformational template to overrule unfavorable environmental factors and maintain the initial folding pattern. The type of the original seed has been shown to prevail over cosolvent effects and dets. spectral position and width of the amide I' IR band of the heterogeneously seeded amyloid. These findings imply that "strains" may be a common generic trait of amyloids.
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    Iannuzzi, C.; Borriello, M.; Portaccio, M.; Irace, G.; Sirangelo, I. Insights into Insulin Fibril Assembly at Physiological and Acidic Ph and Related Amyloid Intrinsic Fluorescence. Int. J. Mol. Sci. 2017, 18, 2551  DOI: 10.3390/ijms18122551
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    Insights into insulin fibril assembly at physiological and acidic ph and related amyloid intrinsic fluorescence
    Iannuzzi, Clara; Borriello, Margherita; Portaccio, Marianna; Irace, Gaetano; Sirangelo, Ivana
    International Journal of Molecular Sciences (2017), 18 (12), 2551/1-2551/14CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
    Human insulin is a widely used model protein for the study of amyloid formation as both assocd. to insulin injection amyloidosis in type II diabetes and highly prone to form amyloid fibrils in vitro. In this study, we aim to gain new structural insights into insulin fibril formation under two different aggregating conditions at neutral and acidic pH, using a combination of fluorescence, CD, Fourier-transform IR spectroscopy, and transmission electron miscroscopy. We reveal that fibrils formed at neutral pH are morphol. different from those obtained at lower pH. Moreover, differences in FTIR spectra were also detected. In addn., only insulin fibrils formed at neutral pH showed the characteristic blue-green fluorescence generally assocd. to amyloid fibrils. So far, the mol. origin of this fluorescence phenomenon has not been clarified and different hypotheses have been proposed. In this respect, our data provide exptl. evidence that allow identifying the mol. origin of such intrinsic property.
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    Milhiet, P.-E.; Yamamoto, D.; Berthoumieu, O.; Dosset, P.; Le Grimellec, C.; Verdier, J.-M.; Marchal, S.; Ando, T. Deciphering the Structure, Growth and Assembly of Amyloid-Like Fibrils Using High-Speed Atomic Force Microscopy. PLoS One 2010, 5, e13240  DOI: 10.1371/journal.pone.0013240
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    Deciphering the structure, growth and assembly of amyloid-like fibrils using high-speed atomic force microscopy
    Milhiet Pierre-Emmanuel; Yamamoto Daisuke; Berthoumieu Olivia; Dosset Patrice; Le Grimellec Christian; Verdier Jean-Michel; Marchal Stephane; Ando Toshio
    PloS one (2010), 5 (10), e13240 ISSN:.
    Formation of fibrillar structures of proteins that deposit into aggregates has been suggested to play a key role in various neurodegenerative diseases. However mechanisms and dynamics of fibrillization remains to be elucidated. We have previously established that lithostathine, a protein overexpressed in the pre-clinical stages of Alzheimer's disease and present in the pathognomonic lesions associated with this disease, form fibrillar aggregates after its N-terminal truncation. In this paper we visualized, using high-speed atomic force microscopy (HS-AFM), growth and assembly of lithostathine protofibrils under physiological conditions with a time resolution of one image/s. Real-time imaging highlighted a very high velocity of elongation. Formation of fibrils via protofibril lateral association and stacking was also monitored revealing a zipper-like mechanism of association. We also demonstrate that, like other amyloid ss peptides, two lithostathine protofibrils can associate to form helical fibrils. Another striking finding is the propensity of the end of a growing protofibril or fibril to associate with the edge of a second fibril, forming false branching point. Taken together this study provides new clues about fibrillization mechanism of amyloid proteins.
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    Surmacz-Chwedoruk, W.; Nieznańska, H.; Wójcik, S.; Dzwolak, W. Cross-Seeding of Fibrils from Two Types of Insulin Induces New Amyloid Strains. Biochemistry 2012, 51, 9460– 9469,  DOI: 10.1021/bi301144d
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    Cross-Seeding of Fibrils from Two Types of Insulin Induces New Amyloid Strains
    Surmacz-Chwedoruk, Weronika; Nieznanska, Hanna; Wojcik, Slawomir; Dzwolak, Wojciech
    Biochemistry (2012), 51 (47), 9460-9469CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The irreversibility and autocatalytic character of amyloidogenesis and the polymorphism of amyloid fibrils underlie the phenomenon of self-propagating strains, wherein the mother seed, rather than the seeding environment, dets. the properties of daughter fibrils. Here we study the formation of amyloid fibrils from bovine insulin and the recombinant LysB31-ArgB32 human insulin analog. The two polypeptides are similar enough to cross-seed but, upon spontaneous aggregation, form amyloid fibrils with distinct spectral features in the IR amide I' band region. When bovine insulin is cross-seeded with the analog amyloid (and vice versa), the shape, absorption max., and even fine fingerprint features of the amide I' band are passed from the mother to daughter fibrils with a high degree of fidelity. Although the differences in primary structure between bovine insulin and the LysB31-ArgB32 analog of human insulin lie outside of the polypeptide's crit. amyloidogenic regions, they affect the secondary structure of fibrils, possibly the formation of intermol. salt bridges, and the susceptibility to dissection and denaturation with DMSO. All these phenotypic features of mother fibrils are imprinted in daughter amyloid upon cross-seeding. Anal. of noncooperative DMSO-induced denaturation of daughter fibrils suggests that the self-propagating polymorphism underlying the emergence of new amyloid strains is encoded on the level of secondary structure. Our findings have been discussed in the context of polymorphism of fibrils, amyloid strains, and possible implications for mechanisms of amyloidogenesis.
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    Barth, A. Infrared Spectroscopy of Proteins. Biochim. Biophys. Acta 2007, 1767, 1073– 1101,  DOI: 10.1016/j.bbabio.2007.06.004
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    Infrared spectroscopy of proteins
    Barth, Andreas
    Biochimica et Biophysica Acta, Bioenergetics (2007), 1767 (9), 1073-1101CODEN: BBBEB4; ISSN:0005-2728. (Elsevier Ltd.)
    A review. This review discusses the application of IR spectroscopy to the study of proteins. The focus is on the mid-IR spectral region and the study of protein reactions by reaction-induced IR difference spectroscopy.
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    Lindberg, D. J.; Wenger, A.; Sundin, E.; Wesén, E.; Westerlund, F.; Esbjörner, E. K. Binding of Thioflavin-T to Amyloid Fibrils Leads to Fluorescence Self-Quenching and Fibril Compaction. Biochemistry 2017, 56, 2170– 2174,  DOI: 10.1021/acs.biochem.7b00035
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    Binding of thioflavin-T to amyloid fibrils leads to fluorescence self-quenching and fibril compaction
    Lindberg, David J.; Wenger, Anna; Sundin, Elin; Wesen, Emelie; Westerlund, Fredrik; Esbjoerner, Elin K.
    Biochemistry (2017), 56 (16), 2170-2174CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Thioflavin-T binds to and detects amyloid fibrils via fluorescence enhancement. Here, using a combination of linear dichroism and fluorescence spectroscopies, we report that the relation between the emission intensity and binding of thioflavin-T to insulin fibrils is nonlinear and discuss this in relation to its use in kinetic assays. We demonstrate, from fluorescence lifetime recordings, that the nonlinearity is due to thioflavin-T being sensitive to self-quenching. In addn., thioflavin-T can induce fibril compaction, but not alter fibril structure. This work underscores the photophys. complexity of thioflavin-T and the necessity of calibrating the linear range of its emission response for quant. in vitro studies.
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    Ran, C.; Zhao, W.; Moir, R. D.; Moore, A. Non-Conjugated Small Molecule FRET for Differentiating Monomers from Higher Molecular Weight Amyloid Beta Species. PLoS One 2011, 6, e19362  DOI: 10.1371/journal.pone.0019362
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    Non-conjugated small molecule FRET for differentiating monomers from higher molecular weight amyloid beta species
    Ran, Chongzhao; Zhao, Wei; Moir, Robert D.; Moore, Anna
    PLoS One (2011), 6 (4), e19362CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Systematic differentiation of amyloid (Aβ) species could be important for diagnosis of Alzheimer's disease (AD). In spite of significant progress, controversies remain regarding which species are the primary contributors to the AD pathol., and which species could be used as the best biomarkers for its diagnosis. These controversies are partially caused by the lack of reliable methods to differentiate the complicated subtypes of Aβ species. Particularly, differentiation of Aβ monomers from toxic higher mol. wt. species (HrMW) would be beneficial for drug screening, diagnosis, and mol. mechanism studies. However, fast and cheap methods for these specific aims are still lacking. We demonstrated the feasibility of a non-conjugated FRET (Forster resonance energy transfer) technique that utilized amyloid beta (Aβ) species as intrinsic platforms for the FRET pair assembly. Mixing two structurally similar curcumin derivs. that served as the small mol. FRET pair with Aβ40 aggregates resulted in a FRET signal, while no signal was detected when using Aβ40 monomer soln. Lastly, this FRET technique enabled us to quantify the concns. of Aβ monomers and high mol. wt. species in soln. We believe that this FRET technique could potentially be used as a tool for screening for inhibitors of Aβ aggregation. We also suggest that this concept could be generalized to other misfolded proteins/peptides implicated in various pathologies including amyloid in diabetes, prion in bovine spongiform encephalopathy, tau protein in AD, and α-synuclein in Parkinson disease.
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    Ziaunys, M.; Mikalauskaite, K.; Smirnovas, V. Amyloidophilic Molecule Interactions on the Surface of Insulin Fibrils: Cooperative Binding and Fluorescence Quenching. Sci. Rep. 2019, 9, 20303  DOI: 10.1038/s41598-019-56788-y
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    Amyloidophilic Molecule Interactions on the Surface of Insulin Fibrils: Cooperative Binding and Fluorescence Quenching
    Ziaunys, Mantas; Mikalauskaite, Kamile; Smirnovas, Vytautas
    Scientific Reports (2019), 9 (1), 20303CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
    Protein aggregation into insol. fibrillar aggregates is linked to several neurodegenerative disorders, such as Alzheimer's or Parkinson's disease. Commonly used methods to study aggregation inhibition or fibril destabilization by potential drugs include spectroscopic measurements of amyloidophilic dye mol. fluorescence or absorbance changes. In this work we show the cross-interactions of five different dye mols. on the surface of insulin amyloid fibrils, resulting in cooperative binding and fluorescence quenching.
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  10. Shubham Patil, Pratham Lotia. Peptidomimetics: A Synthetic Tool to Treat Alzheimer’s Disease. The Bombay Technologist 2023, https://doi.org/10.36664/bt/2022/v69i1/172472
  11. Manjeet Kumar, Magdalena I. Ivanova, Ayyalusamy Ramamoorthy. Non-micellar ganglioside GM1 induces an instantaneous conformational change in Aβ42 leading to the modulation of the peptide amyloid-fibril pathway. Biophysical Chemistry 2023, 301 , 107091. https://doi.org/10.1016/j.bpc.2023.107091
  12. Xing-Yu Liu, Shuai-Chen Du, Shu-Lan Li, Feng-Lei Jiang, Peng Jiang, Yi Liu. Inhibition mechanism of human insulin fibrillation by Bodipy carbon polymer dots and photothermal defibrillation effect of Bodipy carbon polymer dots modified by ThT. Biophysical Chemistry 2023, 297 , 107009. https://doi.org/10.1016/j.bpc.2023.107009
  13. Victoria T. Reichelderfer, Andres F. Chaparro Sosa, Joel L. Kaar, Daniel K. Schwartz. Tuning the surface charge of phospholipid bilayers inhibits insulin fibrilization. Colloids and Surfaces B: Biointerfaces 2022, 220 , 112904. https://doi.org/10.1016/j.colsurfb.2022.112904
  14. Mantas Ziaunys, Vytautas Smirnovas. Exploring Epigallocatechin-3-Gallate Autoxidation Products: Specific Incubation Times Required for Emergence of Anti-Amyloid Properties. Antioxidants 2022, 11 (10) , 1887. https://doi.org/10.3390/antiox11101887
  15. Dibakar Sarkar, Narayan Chandra Maity, Gourav Shome, Kyriakos Gabriel Varnava, Vijayalekshmi Sarojini, Subramanian Vivekanandan, Nirakar Sahoo, Sourav Kumar, Atin Kumar Mandal, Ranjit Biswas, Anirban Bhunia. Mechanistic insight into functionally different human islet polypeptide (hIAPP) amyloid: the intrinsic role of the C-terminal structural motifs. Physical Chemistry Chemical Physics 2022, 24 (36) , 22250-22262. https://doi.org/10.1039/D2CP01650H
  16. Kamile Mikalauskaite, Mantas Ziaunys, Vytautas Smirnovas. Lysozyme Amyloid Fibril Structural Variability Dependence on Initial Protein Folding State. International Journal of Molecular Sciences 2022, 23 (10) , 5421. https://doi.org/10.3390/ijms23105421
  17. Mantas Ziaunys, Andrius Sakalauskas, Kamile Mikalauskaite, Vytautas Smirnovas. Rapid restructurization of conformationally-distinct alpha-synuclein amyloid fibrils at an elevated temperature. PeerJ 2022, 10 , e14137. https://doi.org/10.7717/peerj.14137
  18. Mantas Ziaunys, Andrius Sakalauskas, Kamile Mikalauskaite, Vytautas Smirnovas. Polymorphism of Alpha-Synuclein Amyloid Fibrils Depends on Ionic Strength and Protein Concentration. International Journal of Molecular Sciences 2021, 22 (22) , 12382. https://doi.org/10.3390/ijms222212382
  19. Jēkabs Fridmanis, Zigmantas Toleikis, Tomas Sneideris, Mantas Ziaunys, Raitis Bobrovs, Vytautas Smirnovas, Kristaps Jaudzems. Aggregation Condition–Structure Relationship of Mouse Prion Protein Fibrils. International Journal of Molecular Sciences 2021, 22 (17) , 9635. https://doi.org/10.3390/ijms22179635
  20. Raziyeh Sharafdini, Hamid Mosaddeghi. Inhibition of Insulin Amyloid Fibrillation by Salvianolic Acids and Calix[ n ]arenes: Molecular Docking Insight. Journal of Computational Biophysics and Chemistry 2021, 20 (05) , 539-555. https://doi.org/10.1142/S2737416521500332
  21. Mantas Ziaunys, Andrius Sakalauskas, Kamile Mikalauskaite, Ruta Snieckute, Vytautas Smirnovas. Temperature-Dependent Structural Variability of Prion Protein Amyloid Fibrils. International Journal of Molecular Sciences 2021, 22 (10) , 5075. https://doi.org/10.3390/ijms22105075
  22. Mantas Ziaunys, Andrius Sakalauskas, Kamile Mikalauskaite, Vytautas Smirnovas. Exploring the occurrence of thioflavin-T-positive insulin amyloid aggregation intermediates. PeerJ 2021, 9 , e10918. https://doi.org/10.7717/peerj.10918
  23. Mantas Ziaunys, Kamile Mikalauskaite, Andrius Sakalauskas, Vytautas Smirnovas. Interplay between epigallocatechin-3-gallate and ionic strength during amyloid aggregation. PeerJ 2021, 9 , e12381. https://doi.org/10.7717/peerj.12381
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Cite this: Biomacromolecules 2020, 21, 12, 4989–4997
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https://doi.org/10.1021/acs.biomac.0c01178
Published November 17, 2020

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  • Abstract

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    Figure 1

    Figure 1. Atomic force microscopy images and fibril height and width distributions of insulin samples prepared under different conditions. Insulin fibrils were prepared under AC (A), PH20 (B), PH24 (C), and PH74 (D) conditions. Fibril height (E) and width (F) distribution (n = 50), where box plots indicate the interquartile range and error bars are 1 standard deviation.

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    Figure 2

    Figure 2. Insulin sample’s FTIR spectra (A), second derivatives (B), and spectrum positions associated with β-sheets, turns, loops, deuterated carboxyl groups, and each spectrum’s band’s width at its half-height (table inserted).

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    Figure 3

    Figure 3. Insulin sample’s ThT fluorescence intensity and bound ThT concentration ratios (I/cB) at different total dye concentrations. Insulin fibrils were prepared under AC (A), PH20 (B), PH24 (C) and PH74 (D) conditions.

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    Figure 4

    Figure 4. Insulin sample’s ThT fluorescence EEM intensity center of mass positions at different total ThT concentrations. Insulin fibrils were prepared under AC (A), PH20 (B), PH24 (C), and PH74 (D) conditions.

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      Amyloidosis is a rare disorder in which insoluble amyloid proteins are deposited in body organs, causing abnormal protein build-up in tissues and eventually leading to organ dysfunction and death. It affects less than 200,000 people in the United States, classifying it as a rare disease according to the National Institutes of Health. Definitive determination of the underlying protein is critical since prognosis and treatment of amyloidosis can vary widely depending on the responsible protein. The following paper describes the various types and clinical features of amyloidosis and provides an overview of current diagnostic tools and therapies.
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      Dementia has become a common diagnosis in aging populations, and the numbers will increase in the forthcoming years. Alzheimer's disease (AD) is the most common form of dementia in the elderly, accounting for 50%-56% of cases at autopsy and in clinical series. Nowadays, the number of people affected by AD is rapidly increasing, and more than 35 million people worldwide have AD, a condition characterized by deterioration of memory and other cognitive domains, and leading to death 3-9 years after diagnosis. The number of patients with AD, the most common cause of disability in the elderly, is set to rise dramatically. Therefore, it is important for clinicians to recognize early signs and symptoms of dementia and to note potentially modifiable risk factors and early disease markers.
      >> More from SciFinder ®
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      Neurology (2013), 80 (19), 1778-83 ISSN:.
      OBJECTIVES: To provide updated estimates of Alzheimer disease (AD) dementia prevalence in the United States from 2010 through 2050. METHODS: Probabilities of AD dementia incidence were calculated from a longitudinal, population-based study including substantial numbers of both black and white participants. Incidence probabilities for single year of age, race, and level of education were calculated using weighted logistic regression and AD dementia diagnosis from 2,577 detailed clinical evaluations of 1,913 people obtained from stratified random samples of previously disease-free individuals in a population of 10,800. These were combined with US mortality, education, and new US Census Bureau estimates of current and future population to estimate current and future numbers of people with AD dementia in the United States. RESULTS: We estimated that in 2010, there were 4.7 million individuals aged 65 years or older with AD dementia (95% confidence interval [CI] = 4.0-5.5). Of these, 0.7 million (95% CI = 0.4-0.9) were between 65 and 74 years, 2.3 million were between 75 and 84 years (95% CI = 1.7-2.9), and 1.8 million were 85 years or older (95% CI = 1.4-2.2). The total number of people with AD dementia in 2050 is projected to be 13.8 million, with 7.0 million aged 85 years or older. CONCLUSION: The number of people in the United States with AD dementia will increase dramatically in the next 40 years unless preventive measures are developed.
      >> More from SciFinder ®
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      Although amyotrophic lateral sclerosis (ALS) is relatively rare, the socioeconomic significance of the disease is extensive. It is therefore vital to project the epidemiol. trend of ALS. To date, there have been few published studies attempting to est. the no. and distribution of ALS cases in the upcoming years. Here we show that the no. of ALS cases across the globe will increase from 222,801 in 2015 to 376,674 in 2040, representing an increase of 69%. This increase is predominantly due to ageing of the population, particularly among developing nations. This projection is likely an underestimate due to improving healthcare and economic conditions. The results should be used to inform healthcare policy to more efficiently allocate healthcare resources.
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      A review. Amyloid fibrils are supramol. protein assemblies with a fibrous morphol. and cross-β structure. The formation of amyloid fibrils typically follows a nucleation-dependent polymn. mechanism, in which a one-step nucleation scheme has widely been accepted. However, a variety of oligomers have been identified in early stages of fibrillation, and a nucleated conformational conversion (NCC) mechanism, in which oligomers serve as a precursor of amyloid nucleation and convert to amyloid nuclei, has been proposed. This development has raised the need to consider more complicated multi-step nucleation processes in addn. to the simplest one-step process, and evidence for the direct involvement of oligomers as nucleation precursors has been obtained both exptl. and theor. Interestingly, the NCC mechanism has some analogy with the two-step nucleation mechanism proposed for inorg. and org. crystals and protein crystals, although a more dramatic conformational conversion of proteins should be considered in amyloid nucleation. Clarifying the properties of the nucleation precursors of amyloid fibrils in detail, in comparison with those of crystals, will allow a better understanding of the nucleation of amyloid fibrils and pave the way to develop techniques to regulate it.
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      Meisl, G.; Kirkegaard, J. B.; Arosio, P.; Michaels, T. C. T.; Vendruscolo, M.; Dobson, C. M.; Linse, S.; Knowles, T. P. J. Molecular Mechanisms of Protein Aggregation from Global Fitting of Kinetic Models. Nat. Protoc. 2016, 11, 252– 272,  DOI: 10.1038/nprot.2016.010
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      Molecular mechanisms of protein aggregation from global fitting of kinetic models
      Meisl, Georg; Kirkegaard, Julius B.; Arosio, Paolo; Michaels, Thomas C. T.; Vendruscolo, Michele; Dobson, Christopher M.; Linse, Sara; Knowles, Tuomas P. J.
      Nature Protocols (2016), 11 (2), 252-272CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)
      The elucidation of the mol. mechanisms by which sol. proteins convert into their amyloid forms is a fundamental prerequisite for understanding and controlling disorders that are linked to protein aggregation, such as Alzheimer's and Parkinson's diseases. However, because of the complexity assocd. with aggregation reaction networks, the anal. of kinetic data of protein aggregation to obtain the underlying mechanisms represents a complex task. Here we describe a framework, using quant. kinetic assays and global fitting, to det. and to verify a mol. mechanism for aggregation reactions that is compatible with exptl. kinetic data. We implement this approach in a web-based software, AmyloFit. Our procedure starts from the results of kinetic expts. that measure the concn. of aggregate mass as a function of time. We illustrate the approach with results from the aggregation of the β-amyloid (Aβ) peptides measured using thioflavin T, but the method is suitable for data from any similar kinetic expt. measuring the accumulation of aggregate mass as a function of time; the input data are in the form of a tab-sepd. text file. We also outline general exptl. strategies and practical considerations for obtaining kinetic data of sufficient quality to draw detailed mechanistic conclusions, and the procedure starts with instructions for extensive data quality control. For the core part of the anal., we provide an online platform (http://www.amylofit.ch.cam.ac.uk) that enables robust global anal. of kinetic data without the need for extensive programming or detailed math. knowledge. The software automates repetitive tasks and guides users through the key steps of kinetic anal.: detn. of constraints to be placed on the aggregation mechanism based on the concn. dependence of the aggregation reaction, choosing from several fundamental models describing assembly into linear aggregates and fitting the chosen models using an advanced minimization algorithm to yield the reaction orders and rate consts. Finally, we outline how to use this approach to investigate which targets potential inhibitors of amyloid formation bind to and where in the reaction mechanism they act. The protocol, from processing data to detg. mechanisms, can be completed in <1 d.
      >> More from SciFinder ®
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    9. 9
      Fitzpatrick, A. W. P.; Debelouchina, G. T.; Bayro, M. J.; Clare, D. K.; Caporini, M. A.; Bajaj, V. S.; Jaroniec, C. P.; Wang, L.; Ladizhansky, V.; Muller, S. A.; MacPhee, C. E.; Waudby, C. A.; Mott, H. R.; De Simone, A.; Knowles, T. P. J.; Saibil, H. R.; Vendruscolo, M.; Orlova, E. V.; Griffin, R. G.; Dobson, C. M. Atomic Structure and Hierarchical Assembly of a Cross-β Amyloid Fibril. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 5468– 5473,  DOI: 10.1073/pnas.1219476110
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      Atomic structure and hierarchical assembly of a cross-β amyloid fibril
      Fitzpatrick, Anthony W. P.; Debelouchina, Galia T.; Bayro, Marvin J.; Clare, Daniel K.; Caporini, Marc A.; Bajaj, Vikram S.; Jaroniec, Christopher P.; Wang, Luchun; Ladizhansky, Vladimir; Mueller, Shirley A.; MacPhee, Cait E.; Waudby, Christopher A.; Mott, Helen R.; De Simone, Alfonso; Knowles, Tuomas P. J.; Saibil, Helen R.; Vendruscolo, Michele; Orlova, Elena V.; Griffin, Robert G.; Dobson, Christopher M.
      Proceedings of the National Academy of Sciences of the United States of America (2013), 110 (14), 5468-5473, S5468/1-S5468/32CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of β-sheet filaments. These complex aggregates have remarkable chem. and phys. properties, and the conversion of normally sol. functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-β amyloid fibrils have proved to be recalcitrant to detailed structural anal. By combining structural constraints from a series of exptl. techniques spanning five orders of magnitude in length scale - including magic angle spinning NMR spectroscopy, x-ray fiber diffraction, cryoelectron microscopy, scanning TEM, and at. force microscopy - we report the at.-resoln. (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent β-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils.
      >> More from SciFinder ®
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      Fändrich, M.; Nyström, S.; Nilsson, K. P. R.; Böckmann, A.; LeVine, H.; Hammarström, P. Amyloid Fibril Polymorphism: A Challenge for Molecular Imaging and Therapy. J. Intern. Med. 2018, 283, 218– 237,  DOI: 10.1111/joim.12732
      10
      Amyloid fibril polymorphism: a challenge for molecular imaging and therapy
      Fandrich M; Nystrom S; Nilsson K P R; Hammarstrom P; Bockmann A; LeVine H 3rd; LeVine H 3rd
      Journal of internal medicine (2018), 283 (3), 218-237 ISSN:.
      The accumulation of misfolded proteins (MPs), both unique and common, for different diseases is central for many chronic degenerative diseases. In certain patients, MP accumulation is systemic (e.g. TTR amyloid), and in others, this is localized to a specific cell type (e.g. Alzheimer's disease). In neurodegenerative diseases, NDs, it is noticeable that the accumulation of MP progressively spreads throughout the nervous system. Our main hypothesis of this article is that MPs are not only markers but also active carriers of pathogenicity. Here, we discuss studies from comprehensive molecular approaches aimed at understanding MP conformational variations (polymorphism) and their bearing on spreading of MPs, MP toxicity, as well as MP targeting in imaging and therapy. Neurodegenerative disease (ND) represents a major and growing societal challenge, with millions of people worldwide suffering from Alzheimer's or Parkinson's diseases alone. For all NDs, current treatment is palliative without addressing the primary cause and is not curative. Over recent years, particularly the shape-shifting properties of misfolded proteins and their spreading pathways have been intensively researched. The difficulty in addressing ND has prompted most major pharma companies to severely downsize their nervous system disorder research. Increased academic research is pivotal for filling this void and to translate basic research into tools for medical professionals. Recent discoveries of targeting drug design against MPs and improved model systems to study structure, pathology spreading and toxicity strongly encourage future studies along these lines to provide an opportunity for selective imaging, prognostic diagnosis and therapy.
      >> More from SciFinder ®
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      Cummings, J.; Lee, G.; Ritter, A.; Sabbagh, M.; Zhong, K. Alzheimer’s Disease Drug Development Pipeline: 2019. Alzheimer’s Dementia: Transl. Res. Clin. Interventions 2019, 5, 272– 293,  DOI: 10.1016/j.trci.2019.05.008
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      Šneideris, T.; Baranauskienė, L.; Cannon, J. G.; Rutkienė, R.; Meškys, R.; Smirnovas, V. Looking for a Generic Inhibitor of Amyloid-like Fibril Formation among Flavone Derivatives. PeerJ 2015, 3, e1271  DOI: 10.7717/peerj.1271
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      Srinivasan, E.; Rajasekaran, R. Probing the Inhibitory Activity of Epigallocatechin-Gallate on Toxic Aggregates of Mutant (L84F) SOD1 Protein through Geometry Based Sampling and Steered Molecular Dynamics. J. Mol. Graphics Model. 2017, 74, 288– 295,  DOI: 10.1016/j.jmgm.2017.04.019
      13
      Probing the inhibitory activity of epigallocatechin-gallate on toxic aggregates of mutant (L84F) SOD1 protein through geometry based sampling and steered molecular dynamics
      Srinivasan, E.; Rajasekaran, R.
      Journal of Molecular Graphics & Modelling (2017), 74 (), 288-295CODEN: JMGMFI; ISSN:1093-3263. (Elsevier Ltd.)
      Amyloid formation and protein aggregation are considered to be at the core of the disease pathol. for the various neurodegenerative disorders such as Amyotrophic lateral sclerosis (ALS). Considerable exptl. reports have suggested that epigallocatechin-gallate (EGCG), a natural polyphenol from the green tea inhibits the amyloid formation in multiple neurodegenerative disease. Mutations in SOD1 protein are considered to a key factor that contributes towards the rapid disease progression and the pathogenesis in both, the sporadic and familial form. In our study, we computationally examd. the inhibitory action of EGCG against the native and the mutant SOD1 through mol. docking, steered mol. dynamics and conformational sampling methods From the outcome, we could conjecture that the protein destabilization and increased β-sheet propensity that occurred due to mutation were regained upon the binding of EGCG. Moreover, the concepts of the free energy landscape anal. are introduced to establish the visual appearance of protein aggregation upon mutation. Altogether, we come to know that the binding of EGCG on mutant SOD1 has reduced the formation of the toxic aggregates upon mutation. Hence, our study could be an initiative in deciphering the inhibitory action of EGCG against the aggregated mutant SOD1, which could be a therapeutic potential against the treatment for the incurable neurodegenerative disorder (ALS) affecting the mankind.
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    14. 14
      Goyal, D.; Shuaib, S.; Mann, S.; Goyal, B. Rationally Designed Peptides and Peptidomimetics as Inhibitors of Amyloid-β (Aβ) Aggregation: Potential Therapeutics of Alzheimer’s Disease. ACS Comb. Sci. 2017, 19, 55– 80,  DOI: 10.1021/acscombsci.6b00116
      14
      Rationally Designed Peptides and Peptidomimetics as Inhibitors of Amyloid-β (Aβ) Aggregation: Potential Therapeutics of Alzheimer's Disease
      Goyal, Deepti; Shuaib, Suniba; Mann, Sukhmani; Goyal, Bhupesh
      ACS Combinatorial Science (2017), 19 (2), 55-80CODEN: ACSCCC; ISSN:2156-8944. (American Chemical Society)
      A review. Alzheimer's disease (AD) is a progressive neurodegenerative disease with no clin. accepted treatment to cure or halt its progression. The worldwide effort to develop peptide-based inhibitors of amyloid-β (Aβ) aggregation can be considered an unplanned combinatorial expt. An understanding of what has been done and achieved may advance the understanding of AD pathol. and the discovery of effective therapeutic agents. The authors review here the history of such peptide-based inhibitors, including those based on the Aβ sequence and those not derived from that sequence, contg. both natural and unnatural amino acid building blocks. Peptide-based aggregation inhibitors hold significant promise for future AD therapy owing to their high selectivity, effectiveness, low toxicity, good tolerance, low accumulation in tissues, high chem. and biol. diversity, possibility of rational design and highly developed methods for analyzing their mode of action, proteolytic stability (modified peptides) and blood-brain barrier (BBB) permeability.
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    15. 15
      Byeon, S. R.; Lee, J. H.; Sohn, J.-H.; Kim, D. C.; Shin, K. J.; Yoo, K. H.; Mook-Jung, I.; Lee, W. K.; Kim, D. J. Bis-Styrylpyridine and Bis-Styrylbenzene Derivatives as Inhibitors for Aβ Fibril Formation. Bioorg. Med. Chem. Lett. 2007, 17, 1466– 1470,  DOI: 10.1016/j.bmcl.2006.10.090
      15
      Bis-styrylpyridine and bis-styrylbenzene derivatives as inhibitors for Aβ fibril formation
      Byeon, Seong Rim; Lee, Ji Hoon; Sohn, Ji-Hoon; Kim, Dong Chan; Shin, Kye Jung; Yoo, Kyung Ho; Mook-Jung, Inhee; Lee, Won Koo; Kim, Dong Jin
      Bioorganic & Medicinal Chemistry Letters (2007), 17 (5), 1466-1470CODEN: BMCLE8; ISSN:0960-894X. (Elsevier Ltd.)
      New bis-styrylpyridine and bis-styrylbenzene derivs. were designed and synthesized. These 34 compds. were evaluated by Aβ fibril formation inhibitory assay using thioflavin T as a dye (named ThT assay). Most of them showed excellent inhibitory activities for Aβ fibril formation at IC50 of 0.1-2.7 μM which is comparable to curcumin (IC50 of 0.8 μM). Among them, nine compds. were screened for their cytotoxicities on HT-22 cell by MTT assay at 1, 10, and 50 μM. In particular, I-7 (I) and II-2 exhibited the best combination of inhibitory activity and compd. cytotoxicity.
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    16. 16
      Konar, M.; Bag, S.; Roy, P.; Dasgupta, S. Gallic Acid Induced Dose Dependent Inhibition of Lysozyme Fibrillation. Int. J. Biol. Macromol. 2017, 103, 1224– 1231,  DOI: 10.1016/j.ijbiomac.2017.05.158
      16
      Gallic acid induced dose dependent inhibition of lysozyme fibrillation
      Konar, Mouli; Bag, Sudipta; Roy, Pritam; Dasgupta, Swagata
      International Journal of Biological Macromolecules (2017), 103 (), 1224-1231CODEN: IJBMDR; ISSN:0141-8130. (Elsevier B.V.)
      Amyloidosis is primarily characterized by the deposition of misfolded protein aggregates. Although the natural polyphenols have long been known as effective amyloid inhibitors, the mechanistic details of their inhibitory actions still remain unclear. Our present study explores the inhibition mechanism of polyphenols by studying the anti-amyloidogenic property of gallic acid (GA), the smallest structural unit of tea polyphenols, on hen egg white lysozyme (HEWL) at physiol. pH. Using various spectroscopic techniques such as UV-vis, fluorescence, CD and dynamic light scattering, and microscopic techniques such as TEM and FESEM, it has been shown that GA potentially inhibits the self-aggregation process in a concn. dependent manner. Gel electrophoresis studies suggest that the o-dihydroxy moiety of GA is oxidized into the quinone moiety and H2O2 in the system under the exptl. conditions. The quinone binds near the hydrophobic region of HEWL and restricts hydrophobic exposure. Cyclic voltammetry studies reveal that the Met residues of HEWL are oxidized by H2O2 to highly polar sulfoxide-modified side chains. The partially unfolded intermediates formed under the denaturing conditions employed remain in contact with the solvent thus preventing further aggregation.
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    17. 17
      Ruggeri, F. S.; Šneideris, T.; Vendruscolo, M.; Knowles, T. P. J. Atomic Force Microscopy for Single Molecule Characterisation of Protein Aggregation. Arch. Biochem. Biophys. 2019, 664, 134– 148,  DOI: 10.1016/j.abb.2019.02.001
      17
      Atomic force microscopy for single molecule characterisation of protein aggregation
      Ruggeri, Francesco Simone; Sneideris, Tomas; Vendruscolo, Michele; Knowles, Tuomas P. J.
      Archives of Biochemistry and Biophysics (2019), 664 (), 134-148CODEN: ABBIA4; ISSN:0003-9861. (Elsevier B.V.)
      The development of at. force microscopy (AFM) has opened up a wide range of novel opportunities in nanoscience and new modalities of observation in complex biol. systems. AFM imaging has been widely employed to resolve the complex and heterogeneous conformational states involved in protein aggregation at the single mol. scale and shed light onto the mol. basis of a variety of human pathologies, including neurodegenerative disorders. The study of individual macromols. at nanoscale, however, remains challenging, esp. when fully quant. information is required. In this review, we first discuss the principles of AFM with a special emphasis on the fundamental factors defining its sensitivity and accuracy. We then review the fundamental parameters and approaches to work at the limit of AFM resoln. in order to perform single mol. statistical anal. of biomols. and nanoscale protein aggregates. This single mol. statistical approach has proved to be powerful to unravel the mol. and hierarchical assembly of the misfolded species present transiently during protein aggregation, to visualise their dynamics at the nanoscale, as well to study the structural properties of amyloid-inspired functional nanomaterials.
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    18. 18
      Podestà, A.; Tiana, G.; Milani, P.; Manno, M. Early Events in Insulin Fibrillization Studied by Time-Lapse Atomic Force Microscopy. Biophys. J. 2006, 90, 589– 597,  DOI: 10.1529/biophysj.105.068833
      18
      Early events in insulin fibrillization studied by time-lapse atomic force microscopy
      Podesta, Alessandro; Tiana, Guido; Milani, Paolo; Manno, Mauro
      Biophysical Journal (2006), 90 (2), 589-597CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      The importance of understanding the mechanism of protein aggregation into insol. amyloid fibrils lies not only in its medical consequences, but also in its more basic properties of self-organization. The discovery that a large no. of uncorrelated proteins can form, under proper conditions, structurally similar fibrils has suggested that the underlying mechanism is a general feature of polypeptide chains. The authors address the early events preceding amyloid fibril formation in solns. of zinc-free human insulin incubated at low pH and high temp. Here, the authors show by time-lapse at. force microscopy that a steady-state distribution of protein oligomers with a quasiexponential tail is reached within a few minutes after heating. This metastable phase lasts for a few hours, until fibrillar aggregates are observable. Although for such complex systems different aggregation mechanisms can occur simultaneously, the authors' results indicate that the prefibrillar phase is mainly controlled by a simple coagulation-evapn. kinetic mechanism, in which concn. acts as a crit. parameter. These exptl. facts, along with the kinetic model used, suggest a crit. role for thermal concn. fluctuations in the process of fibril nucleation.
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    19. 19
      Streets, A. M.; Sourigues, Y.; Kopito, R. R.; Melki, R.; Quake, S. R. Simultaneous Measurement of Amyloid Fibril Formation by Dynamic Light Scattering and Fluorescence Reveals Complex Aggregation Kinetics. PLoS One 2013, 8, e54541  DOI: 10.1371/journal.pone.0054541
      19
      Simultaneous measurement of amyloid fibril formation by dynamic light scattering and fluorescence reveals complex aggregation kinetics
      Streets, Aaron M.; Sourigues, Yannick; Kopito, Ron R.; Melki, Ronald; Quake, Stephen R.
      PLoS One (2013), 8 (1), e54541CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      An app. that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit assocd. with Huntington's disease, in vitro. Huntington's disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer's and Parkinson's disease. These pathologies are all related by the propensity of their assocd. protein or polypeptide to form insol., β-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides assocd. with these diseases, the resulting amyloids display strikingly similar phys. structure, an observation which suggests a phys. basis for amyloid fibril formation. Thioflavin T fluorescence reports β-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation.
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    20. 20
      Zandomeneghi, G.; Krebs, M. R. H.; McCammon, M. G.; Fändrich, M. FTIR Reveals Structural Differences between Native β-Sheet Proteins and Amyloid Fibrils. Protein Sci. 2004, 13, 3314– 3321,  DOI: 10.1110/ps.041024904
      20
      FTIR reveals structural differences between native β-sheet proteins and amyloid fibrils
      Zandomeneghi, Giorgia; Krebs, Mark R. H.; McCammon, Margaret G.; Faendrich, Marcus
      Protein Science (2004), 13 (12), 3314-3321CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)
      The presence of β-sheets in the core of amyloid fibrils raised questions as to whether or not β-sheet-contg. proteins, such as transthyretin, are predisposed to form such fibrils. However, we show here that the mol. structure of amyloid fibrils differs more generally from the β-sheets in native proteins. This difference is evident from the amide I region of the IR spectrum and relates to the distribution of the .vphi./ψ dihedral angles within the Ramachandran plot, the av. no. of strands per sheet, and possibly, the β-sheet twist. These data imply that amyloid fibril formation from native β-sheet proteins can involve a substantial structural reorganization.
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    21. 21
      Malmos, K. G.; Blancas-Mejia, L. M.; Weber, B.; Buchner, J.; Ramirez-Alvarado, M.; Naiki, H.; Otzen, D. ThT 101: A Primer on the Use of Thioflavin T to Investigate Amyloid Formation. Amyloid 2017, 24, 1– 16,  DOI: 10.1080/13506129.2017.1304905
      There is no corresponding record for this reference.
    22. 22
      Picken, M. M.; Herrera, G. A. Thioflavin T Stain: An Easier and More Sensitive Method for Amyloid Detection. In Amyloid and Related Disorders; Picken, M. M.; Herrera, G. A.; Dogan, A., Eds.; Humana Press: Totowa, NJ, 2012; pp 187– 189.
      There is no corresponding record for this reference.
    23. 23
      Wetzel, R.; Chemuru, S.; Misra, P.; Kodali, R.; Mukherjee, S.; Kar, K. An Aggregate Weight-Normalized Thioflavin-T Measurement Scale for Characterizing Polymorphic Amyloids and Assembly Intermediates. Methods Mol. Biol. 2018, 1777, 121– 144,  DOI: 10.1007/978-1-4939-7811-3_6
      23
      An aggregate weight-normalized thioflavin-T measurement scale for characterizing polymorphic amyloids and assembly intermediates
      Wetzel, Ronald; Chemuru, Saketh; Misra, Pinaki; Kodali, Ravi; Mukherjee, Smita; Kar, Karunakar
      Methods in Molecular Biology (New York, NY, United States) (2018), 1777 (Peptide Self-Assembly), 121-144CODEN: MMBIED; ISSN:1940-6029. (Springer)
      The red shift in the fluorescence excitation spectra of thioflavin dyes upon binding to fibrils has been a boon to the amyloid field, offering simple and effective methods for the qual. detection of amyloid in tissue samples and for quantitation of particular fibril prepns. with gravimetric linearity. The quant. aspect of the thioflavin T (ThT) response, however, comes with an important caveat that bestows both significant limitations and great untapped power. It is now well established that amyloid fibrils of different proteins, as well as polymorphic fibrils of the same protein, can exhibit vastly different ThT fluorescence intensities for the same wt. concn. of aggregates. Furthermore, the aggregated intermediates commonly obsd. in amyloid assembly reactions can exhibit aggregate wt.-normalized (AWN) ThT fluorescence intensities that vary from essentially zero through a wide range of intermediate values before reaching the intensity of homogeneous, mature amyloid. These features make it very difficult to quant. interpret, without addnl. data, the time-dependent development of ThT fluorescence intensity in an assembly reaction. In this chapter, we describe a method for coupling ex situ ThT fluorescence detns. with an anal. HPLC supported sedimentation assay (also described in detail) that can provide significant new insights into amyloid assembly reactions. The time dependent aggregation data provided by the sedimentation assay reveals a time course of aggregation that is largely independent of aggregate properties. In addn., the combination of these data with ThT measurements of the same reaction time points reveals important aspects of av. aggregate structure at each time point. Examples of the use and potential value of AWN-ThT measurements during amyloid assembly Aβ and polyglutamine peptides are provided.
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    24. 24
      Groenning, M. Binding Mode of Thioflavin T and Other Molecular Probes in the Context of Amyloid Fibrils—Current Status. J. Chem. Biol. 2010, 3, 1– 18,  DOI: 10.1007/s12154-009-0027-5
      24
      Binding mode of Thioflavin T and other molecular probes in the context of amyloid fibrils-current status
      Groenning Minna
      Journal of chemical biology (2010), 3 (1), 1-18 ISSN:1864-6158.
      Because understanding amyloid fibrillation in molecular detail is essential for development of strategies to control amyloid formation and overcome neurodegenerative disorders, increased understanding of present molecular probes as well as development of new probes are of utmost importance. To date, the binding modes of these molecular probes to amyloid fibrils are by no means adequately described or understood, and the large number of studies on Thioflavin T (ThT) and Congo Red (CR) binding have resulted in models that are incomplete and conflicting. Different types of binding sites are likely to be present in amyloid fibrils with differences in binding modes. ThT may bind in channels running parallel to the long axis of the fibril. In the channels, ThT may bind in either a monomeric or dimeric form of which the molecular conformation is likely to be planar. CR may bind in grooves formed along the β-sheets as a planar molecule in either a monomeric or supramolecular form.
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    25. 25
      Lockhart, A.; Ye, L.; Judd, D. B.; Merritt, A. T.; Lowe, P. N.; Morgenstern, J. L.; Hong, G.; Gee, A. D.; Brown, J. Evidence for the Presence of Three Distinct Binding Sites for the Thioflavin T Class of Alzheimer’s Disease PET Imaging Agents on β-Amyloid Peptide Fibrils. J. Biol. Chem. 2005, 280, 7677– 7684,  DOI: 10.1074/jbc.M412056200
      25
      Evidence for the Presence of Three Distinct Binding Sites for the Thioflavin T Class of Alzheimer's Disease PET Imaging Agents on β-Amyloid Peptide Fibrils
      Lockhart, Andrew; Ye, Liang; Judd, Duncan B.; Merritt, Andy T.; Lowe, Peter N.; Morgenstern, Jennifer L.; Hong, Guizhu; Gee, Antony D.; Brown, John
      Journal of Biological Chemistry (2005), 280 (9), 7677-7684CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Imaging the progression of Alzheimer's disease would greatly facilitate the discovery of therapeutics, and a wide range of ligands are currently under development for the detection of β-amyloid peptide (Aβ)-contg. plaques by using positron emission tomog. Here we report an in-depth characterization of the binding of seven previously described ligands to in vitro generated Aβ-(1-40) polymers. All of the compds. were derived from the benzothiazole compd. thioflavin T and include 2-[4'-(methylamino)phenyl]benzothiazole and 2-(4'-dimethylamino-)phenyl-imidazo[1,2-a]-pyridine derivs., 2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole and 2-[4'-(4''-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and a benzofuran compd. (5-bromo-2-(4-dimethylaminophenyl)benzofuran). By using a range of fluorescent and radioligand binding assays, we find that these compds. display a more complex binding pattern than described previously and are consistent with three classes of binding sites on the Aβ fibrils. All of the compds. bound with very high affinity (low nM Kd) to a low capacity site (BS3) (1 ligand-binding site per ∼300 Aβ-(1-40) monomers) consistent with the previously recognized binding site for these compds. on the fibrils. However, the compds. also bound with high affinity (Kd ∼100 nM) to either one of two addnl. binding sites on the Aβ-(1-40) polymer. The properties of these sites, BS1 and BS2, suggest they are adjacent or partially overlapping and have a higher capacity than BS3, occurring every ∼35 or every ∼4 monomers of Aβ-(1-40)-peptide, resp. Compds. appear to display selectivity for BS2 based on the presence of a halogen substitution (2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole, 2-[4'-(4''-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and 5-bromo-2-(4-dimethylaminophenyl)benzofuran) on their arom. ring system. The presence of addnl. ligand-binding sites presents potential new targets for ligand development and may allow a more complete modeling of the current positron emission tomog. data.
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    26. 26
      Ziaunys, M.; Smirnovas, V. Additional Thioflavin-T Binding Mode in Insulin Fibril Inner Core Region. J. Phys. Chem. B 2019, 123, 8727– 8732,  DOI: 10.1021/acs.jpcb.9b08652
      26
      Additional Thioflavin-T Binding Mode in Insulin Fibril Inner Core Region
      Ziaunys, Mantas; Smirnovas, Vytautas
      Journal of Physical Chemistry B (2019), 123 (41), 8727-8732CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      Amyloidogenic protein aggregation into fibrils is linked to several neurodegenerative disorders, such as Alzheimer's or Parkinson's disease. An amyloid specific fluorescent dye thioflavin-T (ThT) is often used to track the formation of these fibrils in vitro. Despite its wide application, it is still unknown how many types of ThT binding modes to amyloids exist, with multiple studies indicating varying nos. In this work, we examine the binding of ThT to insulin fibrils generated at pH 2.4 and reveal a possible inner core binding mode which is not accessible to the dye mol. after aggregation occurs.
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    27. 27
      Sulatskaya, A. I.; Kuznetsova, I. M.; Belousov, M. V.; Bondarev, S. A.; Zhouravleva, G. A.; Turoverov, K. K. Stoichiometry and Affinity of Thioflavin T Binding to Sup35p Amyloid Fibrils. PLoS One 2016, 11, e0156314  DOI: 10.1371/journal.pone.0156314
      27
      Stoichiometry and affinity of thioflavin T binding to Sup35p amyloid fibrils
      Sulatskaya, Anna I.; Kuznetsova, Irina M.; Belousov, Mikhail V.; Bondarev, Stanislav A.; Zhouravleva, Galina A.; Turoverov, Konstantin K.
      PLoS One (2016), 11 (5), e0156314/1-e0156314/14CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      In this work two modes of binding of the fluorescent probe thioflavin T to yeast prion protein Sup35p amyloid fibrils were revealed by absorption spectrometry of solns. prepd. by equil. microdialysis. These binding modes exhibited significant differences in binding affinity and stoichiometry. Moreover, the absorption spectrum and the molar extinction coeff. of the dye bound in each mode were detd. The fluorescence quantum yield of the dye bound in each mode was detd. via a spectrofluorimetric study of the same solns. in which the recorded fluorescence intensity was cor. for the primary inner filter effect. As previously predicted, the existence of one of the detected binding modes may be due to the incorporation of the dye into the grooves along the fiber axis perpendicular to the β-sheets of the fibrils. It was assumed that the second type of binding with higher affinity may be due to the existence of ThT binding sites that are localized to areas where amyloid fibrils are clustered.
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    28. 28
      Groenning, M.; Norrman, M.; Flink, J. M.; van de Weert, M.; Bukrinsky, J. T.; Schluckebier, G.; Frokjaer, S. Binding Mode of Thioflavin T in Insulin Amyloid Fibrils. J. Struct. Biol. 2007, 159, 483– 497,  DOI: 10.1016/j.jsb.2007.06.004
      28
      Binding mode of Thioflavin T in insulin amyloid fibrils
      Groenning, Minna; Norrman, Mathias; Flink, James M.; van de Weert, Marco; Bukrinsky, Jens T.; Schluckebier, Gerd; Frokjaer, Sven
      Journal of Structural Biology (2007), 159 (3), 483-497CODEN: JSBIEM; ISSN:1047-8477. (Elsevier)
      Amyloid fibrils share various common structural features and their presence can be detected by Thioflavin T (ThT). In this paper, the binding mode of ThT to insulin amyloid fibrils was examd. Scatchard anal. and isothermal titrn. calorimetry (ITC) showed at least two binding site populations. The binding site population with the strongest binding was responsible for the characteristic ThT fluorescence. This binding had a capacity of about 0.1 mol of ThT bound per mol of insulin in fibril form. The binding capacity was unaffected by pH, but the affinity was lowest at low pH. Notably, presence of a third binding process prior to the other processes was suggested by ITC. Binding of ThT resulted in only minor changes in the fibril structure according to the x-ray diffraction patterns, where a slightly more dominant equatorial reflection at 16 Å relative to the intersheet distance of 11 Å was obsd. No change in the interstrand distance of 4.8 Å was obsd. On the basis of these results, the authors propose that ThT binds in cavities running parallel to the fibril axis, e.g., between the protofilaments forming the fibrils. Such cavities have been proposed previously in insulin fibrils and several other amyloid fibril models.
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    29. 29
      Kuznetsova, I. M.; Sulatskaya, A. I.; Uversky, V. N.; Turoverov, K. K. Analyzing Thioflavin T Binding to Amyloid Fibrils by an Equilibrium Microdialysis-Based Technique. PLoS One 2012, 7, e30724  DOI: 10.1371/journal.pone.0030724
      29
      Analyzing thioflavin T binding to amyloid fibrils by an equilibrium microdialysis-based technique
      Kuznetsova, Irina M.; Sulatskaya, Anna I.; Uversky, Vladimir N.; Turoverov, Konstantin K.
      PLoS One (2012), 7 (2), e30724CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      A new approach for the detn. of the amyloid fibril - thioflavin T (ThT) binding parameters (the no. of binding modes, stoichiometry, and binding consts. of each mode) is proposed. This approach is based on the absorption spectroscopy detn. of the concn. of free and bound to fibril dye in solns., which are prepd. by equil. microdialysis. Furthermore, the proposed approach allowed us, for the first time, to det. the absorption spectrum, molar extinction coeff., and fluorescence quantum yield of the ThT bound to fibril by each binding modes. This approach is universal and can be used for detg. the binding parameters of any dye interaction with a receptor, such as ANS binding to proteins in the molten globule state or to protein amorphous aggregates.
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    30. 30
      Kawai, R.; Araki, M.; Yoshimura, M.; Kamiya, N.; Ono, M.; Saji, H.; Okuno, Y. Core Binding Site of a Thioflavin-T-Derived Imaging Probe on Amyloid β Fibrils Predicted by Computational Methods. ACS Chem. Neurosci. 2018, 9, 957– 966,  DOI: 10.1021/acschemneuro.7b00389
      30
      Core Binding Site of a Thioflavin-T-Derived Imaging Probe on Amyloid β Fibrils Predicted by Computational Methods
      Kawai, Ryoko; Araki, Mitsugu; Yoshimura, Masashi; Kamiya, Narutoshi; Ono, Masahiro; Saji, Hideo; Okuno, Yasushi
      ACS Chemical Neuroscience (2018), 9 (5), 957-966CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)
      Development of new diagnostic imaging probes for Alzheimer's disease (AD), such as positron emission tomog. (PET) and single photon emission computed tomog. (SPECT) probes, has been strongly desired. In this study, the authors investigated the most accessible amyloid β (Aβ) binding site of [123I]IMPY, a Thioflavin-T-derived SPECT probe, using exptl. and computational methods. First, the authors performed a competitive inhibition assay with Orange-G, which recognizes the KLVFFA region in Aβ fibrils, suggesting that IMPY and Orange-G bind to different sites in Aβ fibrils. Next, the authors precisely predicted the IMPY binding site on a multiple-protofilament Aβ fibril model using computational approaches, consisting of mol. dynamics and docking simulations. The authors generated possible IMPY-binding structures using docking simulations to identify candidates for probe-binding sites. The binding free energy of IMPY with the Aβ fibril was calcd. by a free energy simulation method, MP-CAFEE. These computational results suggest that IMPY preferentially binds to an interfacial pocket located between two protofilaments and it is stabilized mainly through hydrophobic interactions. Finally, the computational approach was validated by comparing with the exptl. results. The present study demonstrates the possibility of computational approaches to screen new PET/SPECT probes for Aβ imaging.
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    31. 31
      Ivancic, V. A.; Ekanayake, O.; Lazo, N. D. Binding Modes of Thioflavin T on the Surface of Amyloid Fibrils Studied by NMR. ChemPhysChem 2016, 17, 2461– 2464,  DOI: 10.1002/cphc.201600246
      31
      Binding Modes of Thioflavin T on the Surface of Amyloid Fibrils Studied by NMR
      Ivancic, Valerie A.; Ekanayake, Oshini; Lazo, Noel D.
      ChemPhysChem (2016), 17 (16), 2461-2464CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The mechanism for the interaction of thioflavin T (ThT) with amyloid fibrils at the mol. level is not known. Here, 1H NMR spectroscopy was used to det. the binding mode of ThT on the surface of fibrils from lysozyme and insulin. Relayed rotating-frame Overhauser enhancements in ThT were obsd., indicating that the orientation of ThT is orthogonal to the fibril surface. Importantly, the assembly state of ThT on both surfaces is different. On the surface of insulin fibrils, ThT is oligomeric, as indicated by rapid 1H spin-lattice relaxation rate in the rotating frame (R1ρ), presumably due to intermol. dipole-dipole interactions between ThT mols. In contrast, ThT on the surface of lysozyme fibrils is a monomer, as indicated by slower 1H R1ρ. These results shed new light into the mechanism for the enhancement of ThT fluorescence and may lead to more efficient detectors of amyloid assemblies, which have escaped detection by ThT in monomer form.
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    32. 32
      Mao, X.; Guo, Y.; Wang, C.; Zhang, M.; Ma, X.; Liu, L.; Niu, L.; Zeng, Q.; Yang, Y.; Wang, C. Binding Modes of Thioflavin T Molecules to Prion Peptide Assemblies Identified by Using Scanning Tunneling Microscopy. ACS Chem. Neurosci. 2011, 2, 281– 287,  DOI: 10.1021/cn200006h
      32
      Binding Modes of Thioflavin T Molecules to Prion Peptide Assemblies Identified by Using Scanning Tunneling Microscopy
      Mao, Xiaobo; Guo, Yuanyuan; Wang, Chenxuan; Zhang, Min; Ma, Xiaojing; Liu, Lei; Niu, Lin; Zeng, Qingdao; Yang, Yanlian; Wang, Chen
      ACS Chemical Neuroscience (2011), 2 (6), 281-287CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)
      The widely used method to monitor the aggregation process of amyloid peptide is thioflavin T (ThT) assay, while the detailed mol. mechanism is still not clear. In this work, the authors report here the direct identification of the binding modes of ThT mols. with the prion peptide GNNQQNY by using scanning tunneling microscopy (STM). The assembly structures of GNNQQNY were first obsd. by STM on a graphite surface, and the introduction of ThT mols. to the surface facilitated the STM observations of the adsorption conformations of ThT with peptide strands. ThT mols. are apt to adsorb on the peptide assembly with β-sheet structure and oriented parallel with the peptide strands adopting four different binding modes. This effort could benefit the understanding of the mechanisms of the interactions between labeling species or inhibitory ligands and amyloid peptides, which is keenly needed for developing diagnostic and therapeutic approaches.
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    33. 33
      Sidhu, A.; Vaneyck, J.; Blum, C.; Segers-Nolten, I.; Subramaniam, V. Polymorph-Specific Distribution of Binding Sites Determines Thioflavin-T Fluorescence Intensity in α-Synuclein Fibrils. Amyloid 2018, 25, 189– 196,  DOI: 10.1080/13506129.2018.1517736
      33
      Polymorph-specific distribution of binding sites determines thioflavin-T fluorescence intensity in α-synuclein fibrils
      Sidhu, Arshdeep; Vaneyck, Jonathan; Blum, Christian; Segers-Nolten, Ine; Subramaniam, Vinod
      Amyloid (2018), 25 (3), 189-196CODEN: AIJIET; ISSN:1350-6129. (Taylor & Francis Ltd.)
      Thioflavin-T (ThT) is the most commonly used fluorescent dye for following amyloid formation semi-quant. in vitro, specifically probing the fibrillar cross-β-sheet content. In recent years, structural polymorphism of amyloid fibrils has been shown to be an important aspect of amyloid formation, both in vitro and in neurodegenerative diseases. Therefore, understanding ThT-amyloid interactions in the context of structural polymorphism of amyloids is necessary for correct interpretation of ThT fluorescence data. Here we study the influence of fibril morphol. on ThT fluorescence and ThT binding sites, with two morphol. distinct but chem. identical α-synuclein polymorphs. In ThT fluorescence assays the two polymorphs show type-specific fluorescence intensity behavior although their β-sheet content has been shown to be similar. Further, fluorescence lifetime measurements of fibril-bound ThT reveal the presence of at least two qual. different ThT binding sites on the polymorphs. The relative distributions of the binding sites on the fibril surfaces appear to be morphol. dependent, thus detg. the obsd. polymorph-specific ThT fluorescence intensities. These results, highlighting the role of fibril morphol. in ThT-based amyloid studies, underline the relevance of polymorphs in ThT-amyloid interaction and can explain the variability often obsd. in ThT amyloid binding assays.
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    34. 34
      Ziaunys, M.; Sneideris, T.; Smirnovas, V. Formation of Distinct Prion Protein Amyloid Fibrils under Identical Experimental Conditions. Sci. Rep. 2020, 10, 4572  DOI: 10.1038/s41598-020-61663-2
      34
      Formation of distinct prion protein amyloid fibrils under identical experimental conditions
      Ziaunys, Mantas; Sneideris, Tomas; Smirnovas, Vytautas
      Scientific Reports (2020), 10 (1), 4572CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
      Protein aggregation into amyloid fibrils is linked to multiple neurodegenerative disorders, such as Alzheimer's, Parkinson's or Creutzfeldt-Jakob disease. A better understanding of the way these aggregates form is vital for the development of drugs. A large detriment to amyloid research is the ability of amyloidogenic proteins to spontaneously aggregate into multiple structurally distinct fibrils (strains) with different stability and seeding properties. In this work we show that prion proteins are capable of forming more than one type of fibril under the exact same conditions by assessing their Thioflavin T (ThT) binding ability, morphol., secondary structure, stability and seeding potential.
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    35. 35
      Sakalauskas, A.; Ziaunys, M.; Smirnovas, V. Concentration-Dependent Polymorphism of Insulin Amyloid Fibrils. PeerJ 2019, 7, e8208  DOI: 10.7717/peerj.8208
      There is no corresponding record for this reference.
    36. 36
      Sneideris, T.; Sakalauskas, A.; Sternke-Hoffmann, R.; Peduzzo, A.; Ziaunys, M.; Buell, A. K.; Smirnovas, V. The Environment Is a Key Factor in Determining the Anti-Amyloid Efficacy of EGCG. Biomolecules 2019, 9, 855  DOI: 10.3390/biom9120855
      36
      The environment is a key factor in determining the anti-amyloid efficacy of EGCG
      Sneideris, Tomas; Sakalauskas, Andrius; Sternke-Hoffmann, Rebecca; Peduzzo, Alessia; Ziaunys, Mantas; Buell, Alexander K.; Smirnovas, Vytautas
      Biomolecules (2019), 9 (12), 855CODEN: BIOMHC; ISSN:2218-273X. (MDPI AG)
      Millions of people around the world suffer from amyloid-related disorders, including Alzheimer's and Parkinson's diseases. Despite significant and sustained efforts, there are still no disease-modifying drugs available for the majority of amyloid-related disorders, and the overall failure rate in clin. trials is very high, even for compds. that show promising anti-amyloid activity in vitro. In this study, it demonstrate that even small changes in the chem. environment can strongly modulate the inhibitory effects of anti-amyloid compds. Using one of the best-established amyloid inhibitory compds., epigallocatechin-3-gallate (EGCG), as an example, and two amyloid-forming proteins, insulin and Parkinson's disease-related a-synuclein, we shed light on the previously unexplored sensitivity to soln. conditions of the action of this compd. on amyloid fibril formation. In the case of insulin, it show that the classification of EGCG as an amyloid inhibitor depends on the exptl. conditions select, on the method used for the evaluation of the efficacy, and on whether or not EGCG is allowed to oxidise before the expt. For a-synuclein, it show that a small change in pH value, from 7 to 6, transforms EGCG from an efficient inhibitor to completely ineffective, and we were able to explain this behavior by the increased stability of EGCG against oxidn. at pH 6.
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    37. 37
      Gupta, Y.; Singla, G.; Singla, R. Insulin-Derived Amyloidosis. Indian J. Endocrinol. Metab. 2015, 19, 174– 177,  DOI: 10.4103/2230-8210.146879
      37
      Insulin-derived amyloidosis
      Gupta Yashdeep; Singla Gaurav; Singla Rajiv
      Indian journal of endocrinology and metabolism (2015), 19 (1), 174-7 ISSN:2230-8210.
      UNLABELLED: Amyloidosis is the term for diseases caused by the extracellular deposition of insoluble polymeric protein fibrils in tissues and organs. Insulin-derived amyloidosis is a rare, yet significant complication of insulin therapy. Insulin-derived amyloidosis at injection site can cause poor glycemic control and increased insulin dose requirements because of the impairment in insulin absorption, which reverse on change of injection site and/or excision of the mass. This entity should be considered and assessed by histopathology and immunohistochemistry, in patients with firm/hard local site reactions, which do not regress after cessation of insulin injection at the affected site. SEARCH STRATEGY: PubMed was searched with terms "insulin amyloidosis". Full text of articles available in English was reviewed. Relevant cross references were also reviewed. Last search was made on October 15, 2014.
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    38. 38
      Foderà, V.; Cataldo, S.; Librizzi, F.; Pignataro, B.; Spiccia, P.; Leone, M. Self-Organization Pathways and Spatial Heterogeneity in Insulin Amyloid Fibril Formation. J. Phys. Chem. B 2009, 113, 10830– 10837,  DOI: 10.1021/jp810972y
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      Self-organization pathways and spatial heterogeneity in insulin amyloid fibril formation
      Fodera, Vito; Cataldo, Sebastiano; Librizzi, Fabio; Pignataro, Bruno; Spiccia, Paola; Leone, Maurizio
      Journal of Physical Chemistry B (2009), 113 (31), 10830-10837CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
      At high temp. and low pH, the protein hormone insulin is highly prone to form amyloid fibrils, and for this reason it is widely used as a model system to study fibril formation mechanisms. In this work, we focused on insulin aggregation mechanisms occurring in HCl solns. (pH 1.6) at 60 °C. By means of in situ Thioflavin T (ThT) staining, the kinetics profiles were characterized as a function of the protein concn., and two concurrent aggregation pathways were pointed out, being concn. dependent. In correspondence to these pathways, different morphologies of self-assembled protein mols. were detected by at. force microscopy images also evidencing the presence of secondary nucleation processes as a peculiar mechanism for insulin fibrillation. Moreover, combining ThT fluorescence and light scattering, the early stages of the process were analyzed in the low concn. regime, pointing out a pronounced spatial heterogeneity in the formation of the first stable fibrils in soln. and the onset of the secondary nucleation pathways.
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    39. 39
      Gong, H.; He, Z.; Peng, A.; Zhang, X.; Cheng, B.; Sun, Y.; Zheng, L.; Huang, K. Effects of Several Quinones on Insulin Aggregation. Sci. Rep. 2015, 4, 5648  DOI: 10.1038/srep05648
      There is no corresponding record for this reference.
    40. 40
      Wang, J.-B.; Wang, Y.-M.; Zeng, C.-M. Quercetin Inhibits Amyloid Fibrillation of Bovine Insulin and Destabilizes Preformed Fibrils. Biochem. Biophys. Res. Commun. 2011, 415, 675– 679,  DOI: 10.1016/j.bbrc.2011.10.135
      40
      Quercetin inhibits amyloid fibrillation of bovine insulin and destabilizes preformed fibrils
      Wang, Jian-Bo; Wang, Yi-Min; Zeng, Cheng-Ming
      Biochemical and Biophysical Research Communications (2011), 415 (4), 675-679CODEN: BBRCA9; ISSN:0006-291X. (Elsevier B.V.)
      Growing interest and research efforts have recently been focused on elucidating the mol. mechanism of amyloid formation and the screening of effective inhibitors to interrupt amyloid structures. In the present study, the anti-amyloidogenic effects of quercetin were investigated in vitro using bovine insulin as a model protein. The results demonstrated that quercetin dose-dependently inhibited amyloid formation of insulin. Moreover, quercetin destabilized the preformed insulin fibrils and transformed the fibrils into amorphous aggregates. Hemolysis was obsd. when human erythrocytes were co-incubated with insulin fibrils. Quercetin inhibited fibril-induced hemolysis in a dose-dependent manner. SDS-PAGE showed that insulin fibrils induced the aggregation of cytoskeletal proteins of erythrocyte membranes and that quercetin attenuated this fibril-induced cytoskeletal aggregation. The results of the present work suggest that quercetin may serve as a lead structure for the design of novel anti-amyloidogenic drugs.
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    41. 41
      Jayamani, J.; Shanmugam, G. Gallic Acid, One of the Components in Many Plant Tissues, Is a Potential Inhibitor for Insulin Amyloid Fibril Formation. Eur. J. Med. Chem. 2014, 85, 352– 358,  DOI: 10.1016/j.ejmech.2014.07.111
      41
      Gallic acid, one of the components in many plant tissues, is a potential inhibitor for insulin amyloid fibril formation
      Jayamani, Jayaraman; Shanmugam, Ganesh
      European Journal of Medicinal Chemistry (2014), 85 (), 352-358CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
      Proteins under stressful conditions can lead to the formation of an ordered self-assembled structure, referred to as amyloid fibrils, to which many neurodegenerative diseases such as Type II diabetes, Alzheimer's, Parkinson's, Huntington's, etc., are attributed. Inhibition of amyloid fibril formation using natural products is one of the main therapeutic strategies to prevent the progression of these diseases. Polyphenols are the mostly consumed as antioxidants in a human nutrition. Herein, we have studied the effect of a simple polyphenol, gallic acid (GA), one of the main components in plant tissues, esp. in tea leaves, on the insulin amyloid fibril formation. Different biophys. characterizations such as turbidity, at. force microscopy (AFM), Thioflavin T (ThT) assays, CD, and Fourier transform-IR spectroscopy have been used to analyze the inhibition of amyloid fibril formation. The occurrence of fibrils in an AFM image and ThT fluorescence enhancement confirms the formation of insulin amyloid fibrils when incubated under acidic pH 2 at 65 °C. In the presence of GA, absence of fibrils in AFM image and no change in the intensity of ThT fluorescence confirms the inhibition of insulin amyloid fibrils by GA. Spectroscopic results reveal that GA inhibits the conformational transition of α-helix → β-sheet, which is generally induced during the insulin fibril formation. It was found that the inhibitory effect of GA is concn. dependent and non-linear. Based on the obsd. results, we propose that GA interacts with native insulin, preventing nuclei formation, which is essential for fibril growth, thereby inhibiting the amyloid fibril formation. The present results thus demonstrate that GA can effectively inhibit insulin amyloid fibril formation in vitro.
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    42. 42
      Sneideris, T.; Darguzis, D.; Botyriute, A.; Grigaliunas, M.; Winter, R.; Smirnovas, V. PH-Driven Polymorphism of Insulin Amyloid-Like Fibrils. PLoS One 2015, 10, e0136602  DOI: 10.1371/journal.pone.0136602
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      pH-driven polymorphism of insulin amyloid- like fibrils
      Sneideris, Tomas; Darguzis, Domantas; Botyriute, Akvile; Grigaliunas, Martynas; Winter, Roland; Smirnovas, Vytautas
      PLoS One (2015), 10 (8), e0136602/1-e0136602/13CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Prions are infective proteins, which can self-assemble into different strain conformations, leading to different disease phenotypes. An increasing no. of studies suggest that prion-like self-propagation may be a common feature of amyloid-like structures. Thus it is important to unravel every possible factor leading to the formation of different amyloid strains. Here we report on the formation of two types of insulin amyloid-like fibrils with distinct IR spectroscopic features grown under slightly different pH conditions. Similar to prion strains, both insulin fibril types are able to self-propagate their conformational template under conditions, favoring spontaneous formation of different type fibrils. The low-pHinduced insulin amyloid strain is structurally very similar to previously reported strains formed either in the presence of 20% ethanol, or by modification of the amino acid sequence of insulin. A deeper anal. of literature data in the context of our current findings suggests a shift of the monomer-dimer equil. of insulin as a possible factor controlling the formation of different strains.
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    43. 43
      Dzwolak, W.; Smirnovas, V.; Jansen, R.; Winter, R. Insulin Forms Amyloid in a Strain-Dependent Manner: An FT-IR Spectroscopic Study. Protein Sci. 2004, 13, 1927– 1932,  DOI: 10.1110/ps.03607204
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      Insulin forms amyloid in a strain-dependent manner: An FT-IR spectroscopic study
      Dzwolak, Wojciech; Smirnovas, Vytautas; Jansen, Ralf; Winter, Roland
      Protein Science (2004), 13 (7), 1927-1932CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)
      The presence of 20% (vol./vol.) ethanol triggers growth of insulin amyloid with distinct IR spectroscopic features, compared with the fibrils obtained under ambient conditions. Here we report that the two insulin amyloid types behave in the prion strain-like manner regarding seeding specificity and ability of the self-propagating conformational template to overrule unfavorable environmental factors and maintain the initial folding pattern. The type of the original seed has been shown to prevail over cosolvent effects and dets. spectral position and width of the amide I' IR band of the heterogeneously seeded amyloid. These findings imply that "strains" may be a common generic trait of amyloids.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXlsFWktbc%253D&md5=3eae00f0c32f7d79bb951b7bb2d53b51
    44. 44
      Iannuzzi, C.; Borriello, M.; Portaccio, M.; Irace, G.; Sirangelo, I. Insights into Insulin Fibril Assembly at Physiological and Acidic Ph and Related Amyloid Intrinsic Fluorescence. Int. J. Mol. Sci. 2017, 18, 2551  DOI: 10.3390/ijms18122551
      44
      Insights into insulin fibril assembly at physiological and acidic ph and related amyloid intrinsic fluorescence
      Iannuzzi, Clara; Borriello, Margherita; Portaccio, Marianna; Irace, Gaetano; Sirangelo, Ivana
      International Journal of Molecular Sciences (2017), 18 (12), 2551/1-2551/14CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
      Human insulin is a widely used model protein for the study of amyloid formation as both assocd. to insulin injection amyloidosis in type II diabetes and highly prone to form amyloid fibrils in vitro. In this study, we aim to gain new structural insights into insulin fibril formation under two different aggregating conditions at neutral and acidic pH, using a combination of fluorescence, CD, Fourier-transform IR spectroscopy, and transmission electron miscroscopy. We reveal that fibrils formed at neutral pH are morphol. different from those obtained at lower pH. Moreover, differences in FTIR spectra were also detected. In addn., only insulin fibrils formed at neutral pH showed the characteristic blue-green fluorescence generally assocd. to amyloid fibrils. So far, the mol. origin of this fluorescence phenomenon has not been clarified and different hypotheses have been proposed. In this respect, our data provide exptl. evidence that allow identifying the mol. origin of such intrinsic property.
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    45. 45
      Milhiet, P.-E.; Yamamoto, D.; Berthoumieu, O.; Dosset, P.; Le Grimellec, C.; Verdier, J.-M.; Marchal, S.; Ando, T. Deciphering the Structure, Growth and Assembly of Amyloid-Like Fibrils Using High-Speed Atomic Force Microscopy. PLoS One 2010, 5, e13240  DOI: 10.1371/journal.pone.0013240
      45
      Deciphering the structure, growth and assembly of amyloid-like fibrils using high-speed atomic force microscopy
      Milhiet Pierre-Emmanuel; Yamamoto Daisuke; Berthoumieu Olivia; Dosset Patrice; Le Grimellec Christian; Verdier Jean-Michel; Marchal Stephane; Ando Toshio
      PloS one (2010), 5 (10), e13240 ISSN:.
      Formation of fibrillar structures of proteins that deposit into aggregates has been suggested to play a key role in various neurodegenerative diseases. However mechanisms and dynamics of fibrillization remains to be elucidated. We have previously established that lithostathine, a protein overexpressed in the pre-clinical stages of Alzheimer's disease and present in the pathognomonic lesions associated with this disease, form fibrillar aggregates after its N-terminal truncation. In this paper we visualized, using high-speed atomic force microscopy (HS-AFM), growth and assembly of lithostathine protofibrils under physiological conditions with a time resolution of one image/s. Real-time imaging highlighted a very high velocity of elongation. Formation of fibrils via protofibril lateral association and stacking was also monitored revealing a zipper-like mechanism of association. We also demonstrate that, like other amyloid ss peptides, two lithostathine protofibrils can associate to form helical fibrils. Another striking finding is the propensity of the end of a growing protofibril or fibril to associate with the edge of a second fibril, forming false branching point. Taken together this study provides new clues about fibrillization mechanism of amyloid proteins.
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    46. 46
      Surmacz-Chwedoruk, W.; Nieznańska, H.; Wójcik, S.; Dzwolak, W. Cross-Seeding of Fibrils from Two Types of Insulin Induces New Amyloid Strains. Biochemistry 2012, 51, 9460– 9469,  DOI: 10.1021/bi301144d
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      Cross-Seeding of Fibrils from Two Types of Insulin Induces New Amyloid Strains
      Surmacz-Chwedoruk, Weronika; Nieznanska, Hanna; Wojcik, Slawomir; Dzwolak, Wojciech
      Biochemistry (2012), 51 (47), 9460-9469CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The irreversibility and autocatalytic character of amyloidogenesis and the polymorphism of amyloid fibrils underlie the phenomenon of self-propagating strains, wherein the mother seed, rather than the seeding environment, dets. the properties of daughter fibrils. Here we study the formation of amyloid fibrils from bovine insulin and the recombinant LysB31-ArgB32 human insulin analog. The two polypeptides are similar enough to cross-seed but, upon spontaneous aggregation, form amyloid fibrils with distinct spectral features in the IR amide I' band region. When bovine insulin is cross-seeded with the analog amyloid (and vice versa), the shape, absorption max., and even fine fingerprint features of the amide I' band are passed from the mother to daughter fibrils with a high degree of fidelity. Although the differences in primary structure between bovine insulin and the LysB31-ArgB32 analog of human insulin lie outside of the polypeptide's crit. amyloidogenic regions, they affect the secondary structure of fibrils, possibly the formation of intermol. salt bridges, and the susceptibility to dissection and denaturation with DMSO. All these phenotypic features of mother fibrils are imprinted in daughter amyloid upon cross-seeding. Anal. of noncooperative DMSO-induced denaturation of daughter fibrils suggests that the self-propagating polymorphism underlying the emergence of new amyloid strains is encoded on the level of secondary structure. Our findings have been discussed in the context of polymorphism of fibrils, amyloid strains, and possible implications for mechanisms of amyloidogenesis.
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    47. 47
      Barth, A. Infrared Spectroscopy of Proteins. Biochim. Biophys. Acta 2007, 1767, 1073– 1101,  DOI: 10.1016/j.bbabio.2007.06.004
      47
      Infrared spectroscopy of proteins
      Barth, Andreas
      Biochimica et Biophysica Acta, Bioenergetics (2007), 1767 (9), 1073-1101CODEN: BBBEB4; ISSN:0005-2728. (Elsevier Ltd.)
      A review. This review discusses the application of IR spectroscopy to the study of proteins. The focus is on the mid-IR spectral region and the study of protein reactions by reaction-induced IR difference spectroscopy.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXpvFWmt7Y%253D&md5=5a6e0e38c2d6f692b81c31ab07aea4eb
    48. 48
      Lindberg, D. J.; Wenger, A.; Sundin, E.; Wesén, E.; Westerlund, F.; Esbjörner, E. K. Binding of Thioflavin-T to Amyloid Fibrils Leads to Fluorescence Self-Quenching and Fibril Compaction. Biochemistry 2017, 56, 2170– 2174,  DOI: 10.1021/acs.biochem.7b00035
      48
      Binding of thioflavin-T to amyloid fibrils leads to fluorescence self-quenching and fibril compaction
      Lindberg, David J.; Wenger, Anna; Sundin, Elin; Wesen, Emelie; Westerlund, Fredrik; Esbjoerner, Elin K.
      Biochemistry (2017), 56 (16), 2170-2174CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Thioflavin-T binds to and detects amyloid fibrils via fluorescence enhancement. Here, using a combination of linear dichroism and fluorescence spectroscopies, we report that the relation between the emission intensity and binding of thioflavin-T to insulin fibrils is nonlinear and discuss this in relation to its use in kinetic assays. We demonstrate, from fluorescence lifetime recordings, that the nonlinearity is due to thioflavin-T being sensitive to self-quenching. In addn., thioflavin-T can induce fibril compaction, but not alter fibril structure. This work underscores the photophys. complexity of thioflavin-T and the necessity of calibrating the linear range of its emission response for quant. in vitro studies.
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    49. 49
      Ran, C.; Zhao, W.; Moir, R. D.; Moore, A. Non-Conjugated Small Molecule FRET for Differentiating Monomers from Higher Molecular Weight Amyloid Beta Species. PLoS One 2011, 6, e19362  DOI: 10.1371/journal.pone.0019362
      49
      Non-conjugated small molecule FRET for differentiating monomers from higher molecular weight amyloid beta species
      Ran, Chongzhao; Zhao, Wei; Moir, Robert D.; Moore, Anna
      PLoS One (2011), 6 (4), e19362CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Systematic differentiation of amyloid (Aβ) species could be important for diagnosis of Alzheimer's disease (AD). In spite of significant progress, controversies remain regarding which species are the primary contributors to the AD pathol., and which species could be used as the best biomarkers for its diagnosis. These controversies are partially caused by the lack of reliable methods to differentiate the complicated subtypes of Aβ species. Particularly, differentiation of Aβ monomers from toxic higher mol. wt. species (HrMW) would be beneficial for drug screening, diagnosis, and mol. mechanism studies. However, fast and cheap methods for these specific aims are still lacking. We demonstrated the feasibility of a non-conjugated FRET (Forster resonance energy transfer) technique that utilized amyloid beta (Aβ) species as intrinsic platforms for the FRET pair assembly. Mixing two structurally similar curcumin derivs. that served as the small mol. FRET pair with Aβ40 aggregates resulted in a FRET signal, while no signal was detected when using Aβ40 monomer soln. Lastly, this FRET technique enabled us to quantify the concns. of Aβ monomers and high mol. wt. species in soln. We believe that this FRET technique could potentially be used as a tool for screening for inhibitors of Aβ aggregation. We also suggest that this concept could be generalized to other misfolded proteins/peptides implicated in various pathologies including amyloid in diabetes, prion in bovine spongiform encephalopathy, tau protein in AD, and α-synuclein in Parkinson disease.
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    50. 50
      Ziaunys, M.; Mikalauskaite, K.; Smirnovas, V. Amyloidophilic Molecule Interactions on the Surface of Insulin Fibrils: Cooperative Binding and Fluorescence Quenching. Sci. Rep. 2019, 9, 20303  DOI: 10.1038/s41598-019-56788-y
      50
      Amyloidophilic Molecule Interactions on the Surface of Insulin Fibrils: Cooperative Binding and Fluorescence Quenching
      Ziaunys, Mantas; Mikalauskaite, Kamile; Smirnovas, Vytautas
      Scientific Reports (2019), 9 (1), 20303CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
      Protein aggregation into insol. fibrillar aggregates is linked to several neurodegenerative disorders, such as Alzheimer's or Parkinson's disease. Commonly used methods to study aggregation inhibition or fibril destabilization by potential drugs include spectroscopic measurements of amyloidophilic dye mol. fluorescence or absorbance changes. In this work we show the cross-interactions of five different dye mols. on the surface of insulin amyloid fibrils, resulting in cooperative binding and fluorescence quenching.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biomac.0c01178.

    • Atomic force microscopy images of insulin fibrils, prepared in AC, PH20, PH24, and PH74 conditions after multiple rounds of resuspension into MilliQ H2O and sonication (Figure S1); AC, PH20, PH24, and PH74 fibril length distributions before and after resuspension into MilliQ H2O and sonication (Figure S2); comparison of total (bound + free) ThT concentration calculated from sample absorbance data and total ThT added to the sample (Figure S3); AC, PH20, and PH24 fibril surface height along the fibril axis (Figure S4); and absorbance spectra of free and fibril-bound ThT and fluorescence intensity of all four types of fibrils in the presence of 1 μM ThT (Figure S5) (PDF)


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