Thioflavin T interaction with amyloid fibrils as an instrument for their studying

Benzthiazole dye thioflavin T (ThT) is widely used to study the formation and structure of amyloid fibrils. Nevertheless, till now there is no common opinion concerning molecular mechanisms of ThT binding to amyloid fibrils and the reasons of dramatic increase in its fluorescence quantum yield on incorporation into amyloid fibrils. Our data prove that ThT molecules incorporate in the amyloid fibrils in the monomeric form and there is no ground to suppose the formation of ThT dimers, eximers, or micells. It was shown that the increase in the quantum yield of ThT incorporated in amyloid fibrils was caused by restriction of benzthiazole and aminobenzene rings torsion fluctuations relative to each other. The use of equilibrium microdialysis allowed determining the absorption spectrum, the number of binding modes of ThT with insulin amyloid fibrils and for each mode determining the binding constants and the number of binding sites for each mode.     

Cytotoxicity of albebetin oligomers depends on cross-beta-sheet formation

Prefibrillar cytotoxicity was suggested as a common amyloid characteristic. We showed two types of albebetin prefibrillar oligomers are formed during incubation at pH 7.3. Initial round-shaped oligomers consist of 10-15 molecules determined by atomic force microscopy, do not bind thioflavin-T and do not affect viability of granular neurons and SH-SY5Y cells. They are converted into ca. 30-40-mers possessing cross-beta-sheet and reducing viability of neuronal cells. Neither monomers nor fibrils possess cytotoxicity. We suggest that oligomeric size is important for stabilising cross-beta-sheet core critical for cytotoxicity. As albebetin was used as a carrier-protein for drug delivery, examination of amyloidogenicity is required prior polypeptide biomedical applications.     

Different propensity to form amyloid fibrils by two homologous proteins-Human stefins A and B: searching for an explanation

By using ThT fluorescence, X-ray diffraction, and atomic force microscopy (AFM), it has been shown that human stefins A and B (subfamily A of cystatins) form amyloid fibrils. Both protein fibrils show the 4.7 A and 10 A reflections characteristic for cross beta-structure. Similar height of approximately 3 nm and longitudinal repeat of 25-27 nm were observed by AFM for both protein fibrils. Fibrils with a double height of 5.6 nm were only observed with stefin A. The fibril's width for stefin A fibrils, as observed by transmission electron microscopy (TEM), was in the same range as previously reported for stefin B (Zerovnik et al., Biochem Biophys Acta 2002;1594:1-5). The conditions needed to undergo fibrillation differ, though. The amyloid fibrils start to form at pH 5 for stefin B, whereas in stefin A, preheated sample has to be acidified to pH < 2.5. In both cases, adding TFE, seeding, and alignment in a strong magnetic field accelerate the fibril growth. Visual analysis of the three-dimensional structures of monomers and domain-swapped dimers suggests that major differences in stability of both homologues stem from arrangement of specific salt bridges, which fix alpha-helix (and the alpha-loop) to beta-sheet in stefin A monomeric and dimeric forms.     

Detection and characterization of aggregates, prefibrillar amyloidogenic oligomers, and protofibrils using fluorescence spectroscopy

Transthyretin (TTR) is a protein linked to a number of different amyloid diseases including senile systemic amyloidosis and familial amyloidotic polyneuropathy. The transient nature of oligomeric intermediates of misfolded TTR that later mature into fibrillar aggregates makes them hard to study, and methods to study these species are sparse. In this work we explore a novel pathway for generation of prefibrillar aggregates of TTR, which provides important insight into TTR misfolding. Prefibrillar amyloidogenic oligomers and protofibrils of misfolded TTR were generated in vitro through induction of the molten globule type A-state from acid unfolded TTR through the addition of NaCl. The aggregation process produced fairly monodisperse oligomers (300-500 kD) within 2 h that matured after 20 h into larger spherical clusters (30-50 nm in diameter) and protofibrils as shown by transmission electron microscopy. Further maturation of the aggregates showed shrinkage of the spheres as the fibrils grew in length, suggesting a conformational change of the spheres into more rigid structures. The structural and physicochemical characteristics of the aggregates were investigated using fluorescence, circular dichroism, chemical cross-linking, and transmission electron microscopy. The fluorescent dyes 1-anilinonaphthalene-8-sulfonate (ANS), 4-4-bis-1-phenylamino-8-naphthalene sulfonate (Bis-ANS), 4-(dicyanovinyl)-julolidine (DCVJ), and thioflavin T (ThT) were employed in both static and kinetic assays to characterize these oligomeric and protofibrillar states using both steady-state and time-resolved fluorescence techniques. DCVJ, a molecular rotor, was employed for the first time for studies of an amyloidogenic process and is shown useful for detection of the early steps of the oligomerization process. DCVJ bound to the early prefibrillar oligomers (300-500 kD) with an apparent dissociation constant of 1.6 muM, which was slightly better than for ThT (6.8 muM). Time-resolved fluorescence anisotropy decay of ANS was shown to be a useful tool for giving further structural and kinetic information of the oligomeric aggregates. ThT dramatically increases its fluorescence quantum yield when bound to amyloid fibrils; however, the mechanism behind this property is unknown. Data from this work suggest that unbound ThT is also intrinsically quenched and functions similarly to a molecular rotor, which in combination with its environmental dependence provides a blue shift to the characteristic 482 nm wavelength when bound to amyloid fibrils.     

Amyloid nucleation and hierarchical assembly of Ure2p fibrils. Role of asparagine/glutamine repeat and nonrepeat regions of the prion domains

The yeast prion protein Ure2 forms amyloid-like filaments in vivo and in vitro. This ability depends on the N-terminal prion domain, which contains Asn/Gln repeats, a motif thought to cause human disease by forming stable protein aggregates. The Asn/Gln region of the Ure2p prion domain extends to residue 89, but residues 15-42 represent an island of "normal" random sequence, which is highly conserved in related species and is relatively hydrophobic. We compare the time course of structural changes monitored by thioflavin T (ThT) binding fluorescence and atomic force microscopy for Ure2 and a series of prion domain mutants under a range of conditions. Atomic force microscopy height images at successive time points during a single growth experiment showed the sequential appearance of at least four fibril types that could be readily differentiated by height (5, 8, 12, or 9 nm), morphology (twisted or smooth), and/or time of appearance (early or late in the plateau phase of ThT binding). The Ure2 dimer (h = 2.6 +/- 0.5 nm) and granular particles corresponding to higher order oligomers (h = 4-12 nm) could also be detected. The mutants 15Ure2 and Delta 15-42Ure2 showed the same time-dependent variation in fibril types but with an increased lag time detected by ThT binding compared with wild-type Ure2. In addition, Delta 15-42Ure2 showed reduced binding to ThT. The results imply a role of the conserved region in both amyloid nucleation and formation of the binding surface recognized by ThT. Further, Ure2 amyloid formation is a multistep process via a series of fibrillar intermediates.     

Human amylin oligomer growth and fibril elongation define two distinct phases in amyloid formation

Human amylin (hA), a 37-amino-acid polypeptide, is one of a number of peptides with the ability to form amyloid fibrils and cause disease. It is the main constituent of the pancreatic amyloid deposits associated with type 2 diabetes. Increasing interest in early assembly intermediates rather than the mature fibrils as the cytotoxic agent has led to this study in which the smallest hA oligomers have been captured by atomic force microscopy. These are 2.3 +/- 1.9 nm in height, 23 +/- 14 nm in length, and consist of an estimated 16 hA molecules. Oligomers first grow to a height of about 6 nm before they begin to significantly elongate into fibrils. Congo red inhibits elongation but not the growth in height of hA oligomers. Two distinct phases have thus been identified in hA fibrillogenesis: lateral growth of oligomers followed by longitudinal growth into mature fibrils. These observations suggest that mature fibrils are assembled directly via longitudinal growth of full-width oligomers, making assembly by lateral association of protofibrils appear less likely.     

Pitfalls associated with the use of Thioflavin-T to monitor anti-fibrillogenic activity

Thioflavin-T (ThT) is a cationic benzothiazole dye that displays enhanced fluorescence upon binding to amyloid fibrils. This property makes ThT the current reagent of choice for the quantification of amyloid fibrils. Herein, we investigate the main pitfalls associated with the use of ThT-based assays to monitor the fibrillation of α-synuclein (α-syn), a protein linked to Parkinson’s disease and other α-synucleinopathies. We demonstrated for the first time that ThT interacts with α-syn disordered monomer and accelerates the protein fibrillation in vitro. As a consequence, misleading conclusions may arise from the use of ThT-based real-time assays in the evaluation of anti-fibrillogenic compounds. Interestingly, NMR experiments indicated that C-terminal domain of α-syn is the main region perturbed by ThT interaction, similarly to that found for the pesticide paraquat, a well-documented accelerator of α-syn fibrillation. Moreover, we demonstrated that certain potent inhibitors of α-syn fibrillation, such as oxidized catecholamines and polyphenols, undergo spontaneous oxidation in aqueous solution, generating compounds that strongly quench ThT fluorescence. In light of these findings, we alert for possible artifacts associated to the measure of the anti-fibrillogenic activity based only on ThT fluorescence approach.     

Binding mode of Thioflavin T and other molecular probes in the context of amyloid fibrils—current status

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.     

Amyloid protofilaments from the calcium-binding protein equine lysozyme: formation of ring and linear structures depends on pH and metal ion concentration

The calcium-binding equine lysozyme has been found to undergo conversion into amyloid fibrils during incubation in solution at acidic pH. At pH 4.5 and 57 degrees C, where equine lysozyme forms a partially unfolded molten globule state, the protein forms protofilaments with a width of ca. 2 nm. In the absence of Ca(2+) the protofilaments are present as annular structures with a diameter of 40-50 nm. In the presence of 10 mM CaCl(2) the protofilaments of equine lysozyme are straight or curved; they can assemble into thicker threads, but they do not appear to undergo circularisation. At pH 2.0, where the protein is more destabilised compared to pH 4.5, fibril formation occurs at 37 degrees C and 57 degrees C. At pH 2.0, both ring-shaped and linear protofilaments are formed, in which periodic repeats of ca 35 nm can be distinguished clearly. The rings constitute about 10% of all fibrillar species under these conditions and they are characterised by a larger diameter of 70-80 nm. All the structures bind Congo red and thioflavine T in a manner similar to fibrils associated with a variety of amyloid diseases. At pH 2.0, fibril formation is accompanied by some acidic hydrolysis, producing specific fragmentation of the protein, leading to the accumulation of two peptides in particular, consisting of residues 1-80 and 54-125. At the initial stages of incubation, however, full-length equine lysozyme represents the dominant species within the fibrils. We propose that the ring-shaped structures observed here, and in the case of disease-associated proteins such as alpha-synuclein, could be a second generic type of amyloid structure in addition to the more common linear fibrils.     

Fibrillation of human insulin A and B chains

Human insulin, which consists of disulfide cross-linked A and B polypeptide chains, readily forms amyloid fibrils under slightly destabilizing conditions. We examined whether the isolated A and B chain peptides of human insulin would form fibrils at neutral and acidic pH. Although insulin exhibits a pH-dependent lag phase in fibrillation, the A chain formed fibrils without a lag at both pHs. In contrast, the B chain exhibited complex concentration-dependent fibrillation behavior at acidic pH. At higher concentrations, e.g., >0.2 mg/mL, the B chains preferentially and rapidly formed stable protofilaments rather than mature fibrils upon incubation at 37 degrees C. Surprisingly, these protofilaments did not convert into mature fibrils. At lower B chain concentrations, however, mature fibrils were formed. The explanation for the concentration dependence of B chain fibrillation is as follows. The B chains exist as soluble oligomers at acidic pH, have a beta-sheet rich conformation as determined by CD, and bind ANS strongly, and these oligomers rapidly form dead-end protofilaments. However, under conditions in which the B chain monomer is present, such as low B chain concentration (<0.2 mg/mL) or in the presence of low concentrations of GuHCl, which dissociates the soluble oligomers, mature fibrils were formed. Thus, both A and B chain peptides can form amyloid fibrils, and both are likely to be involved in the interactions leading to the fibrillation of intact insulin.     

Direct observation of amyloid fibril growth monitored by thioflavin T fluorescence

Real-time monitoring of fibril growth is essential to clarify the mechanism of amyloid fibril formation. Thioflavin T (ThT) is a reagent known to become strongly fluorescent upon binding to amyloid fibrils. Here, we show that, by monitoring ThT fluorescence with total internal reflection fluorescence microscopy (TIRFM), amyloid fibrils of beta2-microgobulin (beta2-m) can be visualized without requiring covalent fluorescence labeling. One of the advantages of TIRFM would be that we selectively monitor fibrils lying along the slide glass, so that we can obtain the exact length of fibrils. This method was used to follow the kinetics of seed-dependent beta2-m fibril extension. The extension was unidirectional with various rates, suggesting the heterogeneity of the amyloid structures. Since ThT binding is common to all amyloid fibrils, the present method will have general applicability for the analysis of amyloid fibrils. We confirmed this with the octapeptide corresponding to the C terminus derived from human medin and the Alzheimer's amyloid beta-peptide.     

Fluorescence quantum yield of thioflavin T in rigid isotropic solution and incorporated into the amyloid fibrils

In this work, the fluorescence of thioflavin T (ThT) was studied in a wide range of viscosity and temperature. It was shown that ThT fluorescence quantum yield varies from 0.0001 in water at room temperature to 0.28 in rigid isotropic solution (T/η→0). The deviation of the fluorescence quantum yield from unity in rigid isotropic solution suggests that fluorescence quantum yield depends not only on the ultra-fast oscillation of ThT fragments relative to each other in an excited state as was suggested earlier, but also depends on the molecular configuration in the ground state. This means that the fluorescence quantum yield of the dye incorporated into amyloid fibrils must depend on its conformation, which, in turn, depends on the ThT environment. Therefore, the fluorescence quantum yield of ThT incorporated into amyloid fibrils can differ from that in the rigid isotropic solution. In particular, the fluorescence quantum yield of ThT incorporated into insulin fibrils was determined to be 0.43. Consequently, the ThT fluorescence quantum yield could be used to characterize the peculiarities of the fibrillar structure, which opens some new possibilities in the ThT use for structural characterization of the amyloid fibrils.     

RNA Aptamers Generated against Oligomeric Aβ40 Recognize Common Amyloid Aptatopes with Low Specificity but High Sensitivity

Aptamers are useful molecular recognition tools in research, diagnostics, and therapy. Despite promising results in other fields, aptamer use has remained scarce in amyloid research, including Alzheimer''s disease (AD). AD is a progressive neurodegenerative disease believed to be caused by neurotoxic amyloid β-protein (Aβ) oligomers. Aβ oligomers therefore are an attractive target for development of diagnostic and therapeutic reagents. We used covalently-stabilized oligomers of the 40-residue form of Aβ (Aβ40) for aptamer selection. Despite gradually increasing the stringency of selection conditions, the selected aptamers did not recognize Aβ40 oligomers but reacted with fibrils of Aβ40, Aβ42, and several other amyloidogenic proteins. Aptamer reactivity with amyloid fibrils showed some degree of protein-sequence dependency. Significant fibril binding also was found for the naïve library and could not be eliminated by counter-selection using Aβ40 fibrils, suggesting that aptamer binding to amyloid fibrils was RNA-sequence-independent. Aptamer binding depended on fibrillogenesis and showed a lag phase. Interestingly, aptamers detected fibril formation with ≥15-fold higher sensitivity than thioflavin T (ThT), revealing substantial β-sheet and fibril formation undetected by ThT. The data suggest that under physiologic conditions, aptamers for oligomeric forms of amyloidogenic proteins cannot be selected due to high, non-specific affinity of oligonucleotides for amyloid fibrils. Nevertheless, the high sensitivity, whereby aptamers detect β-sheet formation, suggests that they can serve as superior amyloid recognition tools.     

Study on the binding of Thioflavin T to beta-sheet-rich and non-beta-sheet cavities

Amyloid fibril formation plays a role in more than 20 diseases including Alzheimer's disease. In vitro detection of these fibrils is often performed using Thioflavin T (ThT), though the ThT binding mode is largely unknown. In the present study, spectral properties of ThT in binding environments representing beta-sheet-rich and non-beta-sheet cavities were examined. Acetylcholinesterase and gamma-cyclodextrin induced a characteristic ThT fluorescence similar to that with amyloid fibrils, whereas beta-cyclodextrin and the beta-sheet-rich transthyretin did not. The cavities of acetylcholinesterase and gamma-cyclodextrin were of similar diameter and only these cavities could accommodate two ThT ions according to molecular modelling. Binding stoichiometry studies also showed a possible binding of two ThT ions. Thus, the characteristic ThT fluorescence is induced in cavities with a diameter of 8-9A and a length able to accommodate the entire length of the ThT ion. The importance of a cavity diameter capable of binding two ThT ions, among others, indicates that an excimer formation is a plausible mechanism for the characteristic fluorescence. We propose a similar ThT binding mode in amyloid fibrils, where cavities of an appropriate size running parallel to the fibril axis have previously been proposed in several amyloid fibril models.     

The circularization of amyloid fibrils formed by apolipoprotein C-II

Amyloid fibrils have historically been characterized by diagnostic dye-binding assays, their fibrillar morphology, and a "cross-beta" x-ray diffraction pattern. Whereas the latter demonstrates that amyloid fibrils have a common beta-sheet core structure, they display a substantial degree of morphological variation. One striking example is the remarkable ability of human apolipoprotein C-II amyloid fibrils to circularize and form closed rings. Here we explore in detail the structure of apoC-II amyloid fibrils using electron microscopy, atomic force microscopy, and x-ray diffraction studies. Our results suggest a model for apoC-II fibrils as ribbons approximately 2.1-nm thick and 13-nm wide with a helical repeat distance of 53 nm +/- 12 nm. We propose that the ribbons are highly flexible with a persistence length of 36 nm. We use these observed biophysical properties to model the apoC-II amyloid fibrils either as wormlike chains or using a random-walk approach, and confirm that the probability of ring formation is critically dependent on the fibril flexibility. More generally, the ability of apoC-II fibrils to form rings also highlights the degree to which the common cross-beta superstructure can, as a function of the protein constituent, give rise to great variation in the physical properties of amyloid fibrils.     

Existence of Different Structural Intermediates on the Fibrillation Pathway of Human Serum Albumin

The fibrillation propensity of the multidomain protein human serum albumin (HSA) was analyzed under different solution conditions. The aggregation kinetics, protein conformational changes upon self-assembly, and structure of the different intermediates on the fibrillation pathway were determined by means of thioflavin T (ThT) fluorescence and Congo Red absorbance; far- and near-ultraviolet circular dichroism; tryptophan fluorescence; Fourier transform infrared spectroscopy; x-ray diffraction; and transmission electron, scanning electron, atomic force, and microscopies. HSA fibrillation extends over several days of incubation without the presence of a lag phase, except for HSA samples incubated at acidic pH and room temperature in the absence of electrolyte. The absence of a lag phase occurs if the initial aggregation is a downhill process that does not require a highly organized and unstable nucleus. The fibrillation process is accompanied by a progressive increase in the β-sheet (up to 26%) and unordered conformation at the expense of α-helical conformation, as revealed by ThT fluorescence and circular dichroism and Fourier transform infrared spectroscopies, but changes in the secondary structure contents depend on solution conditions. These changes also involve the presence of different structural intermediates in the aggregation pathway, such as oligomeric clusters (globules), bead-like structures, and ring-shaped aggregates. We suggest that fibril formation may take place through the role of association-competent oligomeric intermediates, resulting in a kinetic pathway via clustering of these oligomeric species to yield protofibrils and then fibrils. The resultant fibrils are elongated but curly, and differ in length depending on solution conditions. Under acidic conditions, circular fibrils are commonly observed if the fibrils are sufficiently flexible and long enough for the ends to find themselves regularly in close proximity to each other. These fibrils can be formed by an antiparallel arrangement of β-strands forming the β-sheet structure of the HSA fibrils as the most probable configuration. Very long incubation times lead to a more complex morphological variability of amyloid mature fibrils (i.e., long straight fibrils, flat-ribbon structures, laterally connected fibers, etc.). We also observed that mature straight fibrils can also grow by protein oligomers tending to align within the immediate vicinity of the fibers. This filament + monomers/oligomers scenario is an alternative pathway to the otherwise dominant filament + filament manner of the protein fibril's lateral growth. Conformational preferences for a certain pathway to become active may exist, and the influence of environmental conditions such as pH, temperature, and salt must be considered.     

Spectral properties of thioflavin T in solvents with different dielectric properties and in a fibril-incorporated form

The increase in the solvent polarity induces a significant shift of the long-wavelength absorption band of the thioflavin T (ThT) to the shorter wavelengths. This is due to the fact that the positive charge of the ThT molecule (Z = +1e) is unequally and very differently distributed between the benzthiazole and aminobenzene rings in the ground and excited states. Therefore, ThT ground state is stabilized by the orientational interactions of the polar solvent dipoles with the positively charged ThT fragments, whereas the configuration of the solvation shell of the ThT molecule in the excited Franck-Condon state is likely far from being equilibrium. ThT absorption spectrum has the shortest (412 nm) and the longest (450 nm) wavelengths in water and in water being incorporated to the amyloid fibrils, respectively. Intriguingly, the position of the ThT fluorescence spectrum depends on the polarity of solvent to a significantly lesser degree than its absorption spectrum: being excited at 440 nm, ThT has emission with maxima at 493 and 478 nm in water and fibrils, respectively. This can be due to the fact that, in the excited state, the rotational oscillations of the ThT fragments relative to each other prevent establishing equilibrium with the solvent and fluorescence occurs from the partially equilibrium excited stated to the partially equilibrium ground state. For the fibril-incorporated ThT, the maximum of the fluorescence excitation spectrum coincides with the maximum of the long wavelength absorption band (450 nm), whereas for ThT in aqueous and alcohol solutions, additional short-wavelength bands of fluorescence and fluorescence excitation spectra were described (Naiki et al. Anal. Biochem. 1989, 177, 244-249; Le Vine Methods Enzymol. 1999, 309, 274-284). These bands could result either from some fluorescent admixtures (including free benzthiazole and aminobenzene) or from the specific ThT conformers in which benzthiazole and aminobenzene rings, being oriented at phi angle close to 90 or 270 degrees, serve as independent chromophores. On the basis of the results of the quantum-chemical calculations, it is proposed that at phi = 90 degrees (270 degrees), the relatively low barrier (only 700 cm-1) of the internal rotation of the benzthiazole and aminobenzene rings relative to each other gives rise to a subpopulation of ThT molecules possessing a violated system of the pi-conjugated bonds of the benzthiazole and aminobenzene rings.     

Heterogeneous triangular structures of human islet amyloid polypeptide (amylin) with internal hydrophobic cavity and external wrapping morphology reveal the polymorphic nature of amyloid fibrils

The misfolding and self-assembly of human islet amyloid polypeptide (hIAPP or amylin) into amyloid fibrils is pathologically linked to type II diabetes. The polymorphic nature of both hIAPP oligomers and fibrils has been implicated for the molecular origin of hIAPP toxicity to islet β-cells, but little is known about the polymorphic structure and dynamics of these hIAPP oligomers/fibrils at the atomic level. Here, we model the polymorphism of full length hIAPP(1-37) oligomers based on experimental data from solid-state NMR, mass per length, and electron microscopy using all-atom molecular dynamics simulation with explicit solvent. As an alternative to steric zipper structures mostly presented in the 2-fold symmetrical fibrils, the most striking structural feature of our proposed hIAPP oligomers is the presence of 3-fold symmetry along the fibril growth axis, in which three β-sheet-layers wind around a hydrophobic core with different periodicities. These 3-fold triangular hIAPP structures dramatically differ in the details of the β-layer assembly and core-forming sequence at the cross section, but all display a high structural stability with favorable layer-to-layer interactions. The 3-fold hIAPP structures can also serve as templates to present triple-stranded helical fibrils via peptide elongation, with different widths from 8.7 to 9.9 nm, twists from 2.8° to 11.8°, and pitches from 14.5 to 61.1 nm, in reasonable agreement with available biophysical data. Because similar 3-fold Aβ oligomers are also observed by both NMR experiments and our previous simulations, the 3-fold structure could be a general conformation to a broad range of amyloid oligomers and fibrils. Most importantly, unlike the conventional stacking sandwich model, the proposed wrapping-cord structures can readily accommodate more than three β-layers via a two dimension conformation search by rotating and translating the β-layers to adopt different favorable packings, which can greatly enrich the polymorphism of amyloid oligomers and fibrils.     

A New Trend in the Experimental Methodology for the Analysis of the Thioflavin T Binding to Amyloid Fibrils

The studies on the determination of the characteristics of the amyloid fibril interaction with the dye were based on the analysis of the dependence of the ThT fluorescence intensity on its concentration in the solution containing the amyloid fibrils. In the present work, we revealed that this intuitive approach provided erroneous data. We propose a new approach which provides a means for characterizing the interaction of thioflavin T (ThT) with amyloid fibrils and for determining the binding stoichiometry and binding constants, absorption spectrum, molar extinction coefficient, and fluorescence quantum yield of the ThT bound to the sites of different binding modes of fibrils. The key point of this approach is sample preparation by equilibrium microdialysis. The efficiency of the proposed approach is demonstrated via the examination of the ThT binding to insulin and Aβ42 fibrils as well as to the native form of the Electrophorus electricus acetylcholinesterase. We show that the peculiarities of ThT interaction with amyloid fibrils depend on the amyloidogenic protein and on the binding mode. This approach is universal and can be used for the analysis of binding mechanism of any dye that interacts with its receptor. Therefore, the proposed approach represents an important addition to the existing arsenal of means for the diagnostics and therapy of the neurodegenerative diseases.