Interference of low‐molecular substances with the thioflavin‐T fluorescence assay of amyloid fibrils

Abnormal fibrillization of amyloidogenic peptides/proteins has been linked to various neurodegenerative diseases such as Alzheimer's and Parkinson's disease as well as with type‐II diabetes mellitus. The kinetics of protein fibrillization is commonly studied by using a fluorescent dye Thioflavin T (ThT) that binds to protein fibrils and exerts increased fluorescence intensity in bound state. Recently, it has been demonstrated that several low‐molecular weight compounds like Basic Blue 41, Basic Blue 12, Azure C, and Tannic acid interfere with the fluorescence of ThT bound to Alzheimers' amyloid‐β fibrils and cause false positive results during the screening of fibrillization inhibitors. In the current study, we demonstrated that the same selected substances also decrease the fluorescence signal of ThT bound to insulin fibrils already at submicromolar or micromolar concentrations. Kinetic experiments show that unlike to true inhibitors, these compounds did neither decrease the fibrillization rate nor increase the lag‐period. Absence of soluble insulin in the end of the experiment confirmed that these compounds do not disaggregate the insulin fibrils and, thus, are not fibrillization inhibitors at concentrations studied. Our results show that interference with ThT test is a general phenomenon and more attention has to be paid to interpretation of kinetic results of protein fibrillization obtained by using fluorescent dyes. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Flavone Derivatives as Inhibitors of Insulin Amyloid-Like Fibril Formation

Several natural and synthetic flavone derivatives have been reported to inhibit formation of amyloid fibrils or to remodel existing fibrils. These studies suggest that the numbers and positions of hydroxyl groups on the flavone rings determine their effectiveness as amyloid inhibitors. In many studies the primary method for determining the effectiveness of inhibition is measuring Thioflavin T (ThT) fluorescence. This method demonstrably results in a number of false positives for inhibition. We studied the effects of 265 commercially available flavone derivatives on insulin fibril formation. We enhanced the effectiveness of ThT fluorescence measurements by fitting kinetic curves to obtain halftime of aggregation (t ₅₀). Maximal values of ThT fluorescence varied two fold or more in one third of all cases, but this did not correlate with changes in t ₅₀. Changes in t ₅₀ values were more accurate measures of inhibition of amyloid formation. We showed that without a change in an assay, but just by observing complete kinetic curves it is possible to eliminate numbers of false positive and sometimes even false negative results. Examining the data from all 265 flavones we confirmed previous observations that identified the importance of hydroxyl groups for inhibition. Our evidence suggests the importance of hydroxyl groups at locations 5, 6, 7, and 4’, and the absence of a hydroxyl group at location 3, for inhibiting amyloid formation. However, the main conclusion is that the positions are not additive. The structures and their effects must be thought of in the context of the whole molecule.     

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.     

Conformational changes during amyloid fibril formation of pancreatic thiol proteinase inhibitor: effect of copper and zinc

Pancreatic thiol proteinase inhibitor (PTPI), a variant of cystatin superfamily of cysteine protease inhibitors, has been isolated from pancreas of Capra hircus. In the present study, we examined the effects of acid denaturation and a co-solvent on PTPI with a focus on protein conformational changes and amyloid fibril formation. The results demonstrate that PTPI can form amyloid like fibrils. Acid denaturation as studied by CD and fluorescence spectroscopy showed that PTPI populates three partly unfolded species, a native like state at pH 3.0, a structured molten globule at pH 1.0 and partly unfolded species at pH 2.0, from each of which amyloid like fibrils grow as assessed by Thioflavin T (ThT) spectroscopy. Effect of trifluoroethanol (TFE) on acid induced states of PTPI was analyzed. TFE stabilized each of the three acid-induced intermediates at predenaturational concentrations (10%) and accelerated fibril formation. Morphology of the protein species at the beginning and end of reactions was observed using transmission electron microscopy. Solvent conditions were decisive for final fibril morphology. Biometals, Cu²⁺ and Zn²⁺ produced a concentration dependent decline in ThT fluorescence suggesting deaggregation of the fibrils. When added prior to amyloid fibril initiation 50 μM Cu²⁺ or 10 μM Zn²⁺ prevented any amyloid aggregation. Implications for therapeutics in view of Cu²⁺ and Zn²⁺ as essential micronutrients are suggested.     

Interactions between amyloidophilic dyes and their relevance to studies of amyloid inhibitors

Amyloid fibrils are filamentous aggregates of peptides and proteins implicated in a range of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. It has been known almost since their discovery that these β-sheet-rich proteinacious assemblies bind a range of specific dyes that, combined with other biophysical techniques, are convenient probes of the process of amyloid fibril formation. Two prominent examples of such dyes are Congo red (CR) and Thioflavin T (ThT). It has been reported that in addition to having a diagnostic role, CR is an inhibitor of the formation of amyloid structures, and these two properties have both been explained in terms of the same specific noncovalent interactions between the fibrils and the dye molecules. In this article, we show by means of quartz-crystal microbalance measurements that the binding of both ThT and CR to amyloid fibrils formed by the peptide whose aggregation is associated with Alzheimer's disease, Aβ(1-42), can be directly observed, and that the presence of CR interferes with the binding of ThT. Light scattering and fluorescence measurements confirm that an interaction exists between these dyes that can interfere with their ability to reflect accurately the quantity of amyloid material present in a given sample. Furthermore, we show that CR does not inhibit the process of amyloid fibril elongation, and therefore demonstrate the ability of the quartz-crystal microbalance method not only to detect and study the binding of small molecules to amyloid fibrils, but also to elucidate the mode of action of potential inhibitors.     

Interaction of Thioflavin T with amyloid fibrils of apolipoprotein A-I N-terminal fragment: Resonance energy transfer study

Apolipoprotein A-I is amenable to a number of specific mutations associated with hereditary systemic amyloidoses. Amyloidogenic properties of apoA-I are determined mainly by its N-terminal fragment. In the present study Förster resonance energy transfer between tryptophan as a donor and Thioflavin T as an acceptor was employed to obtain structural information on the amyloid fibrils formed by apoA-I variant 1-83/G26R/W@8. Analysis of the dye-fibril binding data provided evidence for the presence of two types of ThT binding sites with similar stoichiometries (bound dye to monomeric protein molar ratio ∼10), but different association constants (∼6 and 0.1 μM⁻¹) and ThT quantum yields in fibril-associated state (0.08 and 0.05, respectively). A β-strand–loop–β-strand structural model of 1-83/G26R/W@8 apoA-I fibrils has been proposed, with potential ThT binding sites located in the solvent-exposed grooves of the N-terminal β-sheet layer. Reasoning from the expanded FRET analysis allowing for heterogeneity of ThT binding centers and fibril polymorphism, the most probable locations of high- and low-affinity ThT binding sites were attributed to the grooves T16_Y18 and D20_L22, respectively.     

The binding of thioflavin T and its neutral analog BTA-1 to protofibrils of the Alzheimer's disease Abeta(16-22) peptide probed by molecular dynamics simulations

Thioflavin T (ThT) is a fluorescent dye commonly used to stain amyloid plaques, but the binding sites of this dye onto fibrils are poorly characterized. We present molecular dynamics simulations of the binding of ThT and its neutral analog BTA-1 [2-(4'-methylaminophenyl)benzothiazole] to model protofibrils of the Alzheimer's disease Abeta(16-22) (amyloid beta) peptide. Our simulations reveal two binding modes located at the grooves of the beta-sheet surfaces and at the ends of the beta-sheet. These simulations provide new insight into recent experimental work and allow us to characterize the high-capacity, micromolar-affinity site seen in experiment as binding to the beta-sheet surface grooves and the low-capacity, nanomolar-affinity site seen as binding to the beta-sheet extremities of the fibril. The structure-activity relationship upon mutating charged ThT to neutral BTA-1 in terms of increased lipophilicity and binding affinity was studied, with calculated solvation free energies and binding energies found to be in qualitative agreement with the experimental measurements.     

Isolation and identification of dieldrin-degrading Pseudonocardia sp. strain KSF27 using a soil-charcoal perfusion method with aldrin trans-diol as a structural analog of dieldrin

We isolated a novel aerobic dieldrin-degrading bacterium from an enrichment culture in a soil–charcoal perfusion system. Enrichment culture using a soil–charcoal perfusion system was an effective way to obtain microorganisms that degrade recalcitrant compounds. The soil–charcoal perfusion was performed using aldrin trans-diol, which was a metabolite of dieldrin. Aldrin trans-diol had higher bioavailability (2.5 mg/l) than dieldrin (0.1–0.25 mg/l), therefore it is possible for microorganisms to utilize it as a substrate in soil. After 100 days of circulation and three exchanges of the medium, the enriched charcoal was harvested and a bacterium isolated. The isolate was designated as strain KSF27 and was found to be closely related to Pseudonocardia spp. as determined by 16S rRNA sequencing analysis. Strain KSF27 degraded aldrin trans-diol by 0.05 μmol/l from an initial concentration of 25.5 μmol/l. The metabolite of aldrin trans-diol was detected by HPLC/MS and determined to be aldrindicarboxylic acid based on retention time and the MS fragment. Moreover, strain KSF27 degraded dieldrin from 14.06 μmol/l to 2.01 μmol/l over a 10-day incubation at 30 °C. This strain degraded dieldrin and other persistent organochlorine pesticides, such as α-endosulfan, β-endosulfan, endosulfan sulfate, heptachlor, heptachlor epoxide and chlordecone.     

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.     

On the Origin of the Stronger Binding of PIB over Thioflavin T to Protofibrils of the Alzheimer Amyloid-β Peptide: A Molecular Dynamics Study

Pittsburgh compound B (PIB) is a neutral derivative of the fluorescent dye Thioflavin T (ThT), which displays enhanced hydrophobicity and binding affinity to amyloid fibrils. We present molecular dynamics simulations of binding of PIB and ThT to a common cross-β-subunit of the Alzheimer Amyloid-β peptide (Aβ). Our simulations of binding to Aβ_9-40 protofibrils show that PIB, like ThT, selectively binds to the hydrophobic or aromatic surface grooves on the β-sheet surface along the fibril axis. The lack of two methyl groups and charge in PIB not only improves its hydrophobicity but also leads to a deeper insertion of PIB compared to ThT into the surface grooves. This significantly increases the steric, aromatic, and hydrophobic interactions, and hence leads to stronger binding. Simulations on protofibrils consisting of the more-toxic Aβ_17-42 revealed an additional binding mode in which PIB and ThT insert into the channel that forms in the loop region of the protofibril, sandwiched between two sheet layers. Our simulations indicate that the rotation between the two ring parts of the dyes is significantly more restricted when the dyes are bound to the surface of the cross-β-subunits or to the channel inside the Aβ_17-42 cross-β-subunit, compared with free solution. The specific conformations of the dyes are influenced by small chemical modifications (ThT versus PIB) and by the environment in which the dye is placed.     

Amyloid fibril formation is suppressed in microgravity

In the future, humans may live in space because of global pollution and weather fluctuations. In microgravity, convection does not occur, which may change the amyloidogenicity of proteins. However, the effect of gravity on amyloid fibril formation is unclear and remains to be elucidated. Here, we analyzed the effect of microgravity on amyloid fibril formation of amyloidogenic proteins including insulin, amyloid β₄₂ (Aβ₄₂), and transthyretin (TTR). We produced microgravity (10^?3 g) by using the gravity controller Gravite. Human insulin, Aβ₄₂, and human wild-type TTR (TTRwt) were incubated at pH 3.0, 7.0, and 3.5 at 37 °C, respectively, in 1 g on the ground or in microgravity. We measured amyloidogenicity via the thioflavin T (ThT) method and cell-based 1-fluoro-2,5-bis[(E)-3-carboxy-4-hydroxystyryl]benzene (FSB) assay. ThT fluorescence intensity and cell-based FSB assay results for human insulin samples were decreased in microgravity compared with results in 1 g. Aβ₄₂ samples did not differ in ThT fluorescence intensity in microgravity and in 1 g on the ground. However, in the cell-based FSB assay, the staining intensity was reduced in microgravity compared with that on 1 g. Human TTRwt tended to form fewer amyloid fibrils in ThT fluorescence intensity and cell-based FSB assays in microgravity than in 1 g. Human insulin and Aβ₄₂ showed decreased amyloid fibril formation in microgravity compared with that in 1 g. Human TTRwt tended to form fewer amyloid fibrils in microgravity. Our experiments suggest that the earth's gravity may be an accelerating factor for amyloid fibril formation.     

A Peptide Derived from the HIV-1 gp120 Coreceptor-Binding Region Promotes Formation of PAP248-286 Amyloid Fibrils to Enhance HIV-1 Infection

BackgroundSemen is a major vehicle for HIV transmission. Prostatic acid phosphatase (PAP) fragments, such as PAP248-286, in human semen can form amyloid fibrils to enhance HIV infection. Other endogenous or exogenous factors present during sexual intercourse have also been reported to promote the formation of seminal amyloid fibrils.

Methodology and Principal Findings

Here, we demonstrated that a synthetic 15-residue peptide derived from the HIV-1 gp120 coreceptor-binding region, designated enhancing peptide 2 (EP2), can rapidly self-assemble into nanofibers. These EP2-derivated nanofibers promptly accelerated the formation of semen amyloid fibrils by PAP248-286, as shown by Thioflavin T (ThT) and Congo red assays. The amyloid fibrils presented similar morphology, assessed via transmission electron microscopy (TEM), in the presence or absence of EP2. Circular dichroism (CD) spectroscopy revealed that EP2 accelerates PAP248-286 amyloid fibril formation by promoting the structural transition of PAP248-286 from a random coil into a cross-β-sheet. Newly formed semen amyloid fibrils effectively enhanced HIV-1 infection in TZM-bl cells and U87 cells by promoting the binding of HIV-1 virions to target cells.

Conclusions and Significance

Nanofibers composed of EP2 promote the formation of PAP248-286 amyloid fibrils and enhance HIV-1 infection.     

Binding Modes of Thioflavin-T to the Single-Layer β-Sheet of the Peptide Self-Assembly Mimics

Although the amyloid dye thioflavin-T (ThT) is among the most widely used tools in the study of amyloid fibrils, the mechanism by which ThT binds to fibrils and other β-rich peptide self-assemblies remains elusive. The development of the water-soluble peptide self-assembly mimic (PSAM) system has provided a set of ideal model proteins for experimentally exploring the properties and minimal dye-binding requirements of amyloid fibrils. PSAMs consist of a single-layer β-sheet (SLB) capped by two globular domains, which capture the flat, extended β-sheet features common among fibril-like surfaces. Recently, a PSAM that binds to ThT with amyloid-like affinity (low micromolar K_d) has been designed, and its crystal structure in the absence of bound ThT was determined. This PSAM thus provides a unique opportunity to examine the interactions of ThT with a β-rich structure. Here, we present molecular dynamics simulations of the binding of ThT to this PSAM β-sheet. We show that the primary binding site for ThT is along a shallow groove formed by adjacent Tyr and Leu residues on the β-sheet surface. These simulations provide an atomic-scale rationale for this PSAM's experimentally determined dye-binding properties. Together, our results suggest that an aromatic-hydrophobic groove spanning across four consecutive β-strands represents a minimal ThT binding site on amyloid fibrils. Grooves formed by aromatic-hydrophobic residues on amyloid fibril surfaces may therefore offer a generic mode of recognition for amyloid dyes.     

On the binding of Thioflavin-T to HET-s amyloid fibrils assembled at pH 2

Amyloid fibrils are ordered β-sheet protein or peptide polymers. The benzothiazole dye Thioflavin-T (ThT) shows a strong increase in fluorescence upon binding to amyloid fibrils and has hence become the most commonly used amyloid-specific dye. In spite of this widespread use, the mechanism underlying specific binding and fluorescence enhancement upon interaction with amyloid fibrils remains largely unknown. Recent contradictory reports have proposed radically different modes of binding. We have studied the interaction of ThT with fibrils of the prion forming domain of the fungal HET-s prion protein assembled at pH 2 in order to try to gain some insight into the general mechanism of ThT-binding and fluorescence. We found that ThT does not bind to HET-s(218–289) fibrils as a micelle as previously proposed in the case of insulin fibrils. We have measured binding kinetics, affinity and stoichiometry at pH values above and below the pI of the HET-s(218–289) fibrils and found that binding is dramatically affected by pH and ionic strength. Binding is poor at acidic pH, presumably as a result of repulsive electrostatic interaction between the positively charged ThT molecule and the fibril surface. Finally, we found that ThT acquires chiral properties when it is fibril-bound. These results are discussed in relation to the different ThT-binding modes that have been proposed.     

l-Arginine reduces thioflavin T fluorescence but not fibrillation of bovine serum albumin

This work examines the effects of l-arginine (l-Arg) on the aggregation and amyloid fibrillation of bovine serum albumin (BSA). We demonstrate that l-Arg dose-dependently reduces thioflavin T (ThT) fluorescence of BSA within the l-Arg concentration range used (0–1.4 M). However, as revealed by electron microscopy, size exclusion chromatography, and dynamic light scattering results, l-Arg does not prevent amyloid-like fibril formation by BSA. We conclude that l-Arg competes against ThT for binding sites on BSA amyloid-like fibrils, leading to biased results in ThT fluorescence measurements. Moreover, the use of ThT fluorescence assay to screen for potential inhibitors against amyloid fibrillation can give misleading results.     

Metal Cations Defibrillize the Amyloid Beta-Protein Fibrils

Amyloid beta-protein (A) is the major constituent of amyloid fibrils composing -amyloid plaques and cerebrovascular amyloid in Alzheimer's disease (AD). We studied the effect of metal cations on preformed fibrils of synthetic A by Thioflavin T (ThT) fluorescence spectroscopy and electronmicroscopy (EM) in negative staining. The amount of cross beta-pleated sheet structure of A 1–40 fibrils was found to decrease by metal cations in a concentration-dependent manner as measured by ThT fluorescence spectroscopy. The order of defibrillization of A 1–40 fibrils by metal cations was: Ca²⁺ and Zn²⁺ (IC₅₀ = 100 M) > Mg²⁺ (IC₅₀ = 300 M) > Al³⁺ (IC₅₀ =1.1 mM). EM analysis in negative staining showed that A 1–40 fibrils in the absence of cations were organized in a fine network with a little or no amorphous material. The addition of Ca²⁺, Mg²⁺, and Zn²⁺ to preformed A 1–40 fibrils defibrillized the fibrils or converted them into short rods or to amorphous material. Al³⁺ was less effective, and reduced the fibril network by about 80 % of that in the absence of any metal cation. Studies with A 1–42 showed that this peptide forms more dense network of fibrils as compared to A 1–40. Both ThT fluorescence spectroscopy and EM showed that similar to A 1–40, A 1–42 fibrils are also defibrillized in the presence of millimolar concentrations of Ca²⁺. These studies suggest that metal cations can defibrillize the fibrils of synthetic A.     

K114 (trans,trans)‐bromo‐2,5‐bis(4‐hydroxystyryl)benzene is an efficient detector of cationic amyloid fibrils

Cationic amyloid fibrils found in human semen enhance the transmission of the human immunodeficiency virus (HIV) and thus, are named semen‐derived enhancer of virus infection (SEVI). The mechanism for the enhancement of transmission is not completely understood but it has been proposed that SEVI neutralizes the repulsion that exists between the negatively charged viral envelope and host cell membrane. Consistent with this view, here we show that the fluorescence of cationic thioflavin T (ThT) in the presence of SEVI is weak, and thus ThT is not an efficient detector of SEVI. On the other hand, K114 ((trans, trans)‐bromo‐2,5‐bis(4‐hydroxystyryl)benzene) forms a highly fluorescent, phenolate‐like species on the cationic surface of SEVI. This species does not form in the presence of amyloid fibrils from insulin and amyloid‐β protein, both of which are efficiently detected by ThT fluorescence. Together, our results show that K114 is an efficient detector of SEVI.     

Surface-modified magnetite nanoparticles affect lysozyme amyloid fibrillization

BackgroundThe surface of nanoparticles (NPs) is an important factor affecting the process of poly/peptides' amyloid aggregation. We have investigated the in vitro effect of trisodium citrate (TC), gum arabic (GA) and citric acid (CA) surface-modified magnetite nanoparticles (COAT-MNPs) on hen egg-white lysozyme (HEWL) amyloid fibrillization and mature HEWL fibrils.MethodsDynamic light scattering (DLS) was used to characterize the physico-chemical properties of studied COAT-MNPs and determine the adsorption potential of their surface towards HEWL. The anti-amyloid properties were studied using thioflavin T (ThT) and tryptophan (Trp) intrinsic fluorescence assays, and atomic force microscopy (AFM). The morphology of amyloid aggregates was analyzed using Gwyddion software. The cytotoxicity of COAT-MNPs was determined utilizing Trypan blue (TB) assay.ResultsAgents used for surface modification affect the COAT-MNPs physico-chemical properties and modulate their anti-amyloid potential. The results from ThT and intrinsic fluorescence showed that the inhibitory activities result from the more favorable interactions of COAT-MNPs with early pre-amyloid species, presumably reducing nuclei and oligomers formation necessary for amyloid fibrillization. COAT-MNPs also possess destroying potential, which is presumably caused by the interaction with hydrophobic residues of the fibrils, resulting in the interruption of an interface between β-sheets stabilizing the amyloid fibrils.ConclusionCOAT-MNPs were able to inhibit HEWL fibrillization and destroy mature fibrils with different efficacy depending on their properties, TC-MNPs being the most potent nanoparticles.General significanceThe study reports findings regarding the general impact of nanoparticles' surface modifications on the amyloid aggregation of proteins.     

Synthesis and in vitro evaluation of fluorinated styryl benzazoles as amyloid-probes

The formation of proteinaceous aggregates is a pathognomonic hallmark of several neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. To date, the final diagnostic for these diseases can only be achieved by immunostaining of post-mortem brain tissues with the commonly used congo red and Thioflavin T/S amyloid-dyes. The interest in developing amyloid-avid radioprobes to be used for protein aggregates imaging by positron emission tomography has grown substantialy, due to the promise in assisting diagnosis of these disorders. To this purpose, the present work describes the synthesis and characterization of four novel fluorinated styryl benzazole derivatives 1–4 by means of the Wittig reaction, as well as their in vitro evaluation as amyloid-probing agents. All compounds were obtained as mixtures of geometric E and Z isomers, with the preferable formation of the E isomer. Photoisomerization reactions allowed for the maximization of the minor Z isomers. The authentic 1–4E/Z isomers were isolated after purification by column chromatography under dark conditions. Profiting from the fluorescence properties of the different geometric isomers of 1–4, their binding affinities towards amyloid fibrils of insulin, α-synuclein and β-amyloid peptide were also measured. These compounds share similarities with Thioflavin T, interacting specifically with fibrillary species with a red-shift in the excitation wavelengths along with an increase in the fluorescence emission intensity. Apparent binding constants were determined and ranged between 1.22 and 23.96 μM⁻¹. The present data suggest that the novel fluorinated styryl benzazole derivatives may prove useful for the design of ¹⁸F-labeled amyloid radioprobes.     

Michler's hydrol blue: a sensitive probe for amyloid fibril detection

Michler's hydrol blue (MHB) is investigated with respect to photophysical properties in varied solvent environment and when bound to insulin and lysozyme fibrils. The MHB chromophore is shown to act like a molecular rotor and bind well to amyloid fibrils, where it exhibits a characteristic red-shift in its excitation spectrum and an increase in the emission quantum yield upon binding. MHB is more sensitive to environmental changes than Thioflavin T (ThT) and furthermore, in contrast to the latter amyloid probe, can differentiate between insulin and lysozyme fibrils by a more red-shifted excitation spectrum for insulin fibrils. To support the experimental observations, time-dependent density functional theory (TDDFT) calculations were performed on MHB at several levels of theory. The predicted changes of spectral properties as a function of the environment are in good agreement with the experimental results. Linear dichroism (LD) is used to determine the orientation of the MHB within the fibrils. It was shown through LD and molecular modeling that MHB aligns itself preferentially parallel with the amyloid fiber at an angle of 14°-22° to the fibril axis and along the grooves of the β-sheet.