Thioflavin T Fluorescence Intensity Research Articles

An Aggregate Weight-Normalized Thioflavin-T Measurement Scale for Characterizing Polymorphic Amyloids and Assembly Intermediates.

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 qualitative detection of amyloid in tissue samples and for quantitation of particular fibril preparations with gravimetric linearity. The quantitative 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 weight concentration of aggregates. Furthermore, the aggregated intermediates commonly observed in amyloid assembly reactions can exhibit aggregate weight-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 quantitatively interpret, without additional 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 determinations with an analytical 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 addition, the combination of these data with ThT measurements of the same reaction time points reveals important aspects of average 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.

Fluorometric determination of lead(II) and mercury(II) based on their interaction with a complex formed between graphene oxide and a DNAzyme.

The authors have designed a DNAzyme where graphene oxide (GO) interacts with the ssDNA stem loop region. The DNAzyme strand and substrate strand are hybridized and bind to the surface of GO which act as a signal reporter, while GO act as a strong quencher. The presence of Pb(II) ion disturbs the GO-DNAzyme complex and causes internal cleavage of the DNAzyme complex. On addition of Thioflavin T (ThT) as a quadruplex inducer, fluorescence intensity (best measured at excitation/emission peaks of 425/490nm) is strongly enhanced. Subsequent addition of Hg(II) to ThT/G-quadruplex complex decreases fluorescence because the G-quadruplex is unwinding to form a T-Hg(II)-T dsDNA system. Therefore, the change in fluorescence intensity of ThT is directly correlated to the concentration of Pb(II) and Hg(II). As a result, the assay is highly selective and sensitive. The limits of detection are 96 pM for Pb(II) and 356 pM for Hg(II). Moreover, the method was applied to the detection of the two ions in spiked real samples and gave satisfactory results. Graphical abstract A label free sensitive and selective "on-off" fluorescent assay for detection of Pb(II) and Hg(II) based on graphene oxide -DNAzyme complex with fluorogenic dye thioflavin T. The limits of detection are 96 pM (Pb2+) and 356 pM (Hg2+).

Tuning supramolecular architectures of KI4K amphiphiles via varying terminal variations

We report here how to manipulate the self-assembly behavior of KI4K by the terminal variations. The results demonstrated that both the supramolecular architectures and their corresponding secondary structure can be tuned just by terminal variations. Twisted or helical ribbons with width over the range of 40–50nm and a bilayed height were dominant for the peptide CH3COKI4KCOOH due to the hydrogen bonding interactions resulted from the uncapping COOH. As a comparison, nanotubes with much larger width but only monolayer wall thickness were dominant for CH3COKI4KCONH2. As for FmocKI4KCOOH, the steric hindrance effect can limit the width growth but the π−π stacking interactions can promote further association of the primary aggregate along the height direction to form nanofiber bundles. Both circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) results indicated that the CH3COKI4KCOOH and FmocKI4KCOOH adopted a mixture of β-sheet and random coil structure while random coil structure was dominant for both CH3COKI4KCONH2 and NH2KI4KCOOH. The small angle neutron scattering (SANS) results indicated the basic lamellar structure for both CH3COKI4KCOOH and CH3COKI4KCONH2 but they go through different pathways to form ribbons and nanotubes with varied shape and size. The Thioflavin-T (ThT) results indicated their abilities to enhance ThT fluorescence intensity was possibly correlated with the amount of aggregates in solutions except for the aggregate morphologies. The peptides involved in the present study provide simple molecular models for the investigation of both COOH and Fmoc in contribution to varied non-covalent interactions and the final aggregate morphologies.

Correlation of cholinergic drug induced quenching of acetylcholinesterase bound thioflavin-T fluorescence with their inhibition activity

The development of new acetylcholinesterase inhibitors (AChEIs) and subsequent assay of their inhibition efficiency is considered to be a key step for AD treatment. The fluorescence intensity of thioflavin-T (ThT) bound in the active site of acetylcholinesterase (AChE) quenches substantially in presence of standard AChEI drugs due to the dynamic replacement of the fluorophore from the AChE active site as confirmed from steady state emission as well as time-resolved fluorescence anisotropy measurement and molecular dynamics simulation in conjunction with docking calculation. The parametrized % quenching data for individual system shows excellent correlation with enzyme inhibition activity measured independently by standard Ellman AChE assay method in a high throughput plate reader system. The results are encouraging towards design of a fluorescence intensity based AChE inhibition assay method and may provide a better toolset to rapidly evaluate as well as develop newer AChE-inhibitors for AD treatment.

Polyols (Glycerol and Ethylene glycol) mediated amorphous aggregate inhibition and secondary structure restoration of metalloproteinase-conalbumin (ovotransferrin)

Under physical or chemical stress, proteins tend to form aggregates either highly ordered (amyloid) or unordered (amorphous) causing many pathological disorders in human and loss of proteins functionality in both laboratory conditions and industries during production and storage at commercial level. We investigated the effect of increasing temperature on Conalbumin (CA) and induced aggregation at 65°C. The enhanced Thioflavin T (ThT) and ANS (1-anilinonaphtalene 8-sulfonic acid) fluorescence intensity, show no shift on Congo red binding, additionally, transmission and scanning electron microscopy (TEM) (SEM) reveal amorphous morphology of the aggregate. Our investigation clearly demonstrated that polyols namely Glycerol (GL) and Ethylene glycol (EG) are so staunch to inhibit amorphous aggregates via restoring secondary conformation. Addition of polyols (15% GL and 35% EG) significantly decrease the turbidity, Rayleigh scattering ThT and ANS fluorescence intensity. The dynamic light scattering (DLS) data show that hydrodynamic radii (Rh) of the aggregates is ∼20 times higher than native CA while nearly similar for GL and EG protected CA due to condensation of core size with little difference.

Thioflavin T behaves as an efficient fluorescent ligand for label-free ATP aptasensor

Here, we for the first time demonstrated thioflavin T (ThT) as an efficient fluorescent ligand for 27-mer ATP-binding aptamer (ABA27), providing a novel signal readout mode for label-free selective ATP detection. ABA27 can promote the fluorescence emission of ThT with an unprecedentedly high efficiency, attributed to the specific structure of ABA27 rather than the G-tracts. Polyacrylamide gel electrophoresis, fluorescence spectroscopy, and fluorometric titration reveal that ThT interacts with ABA27 with a lower binding affinity (Kd ~89μM) than ATP, which allows ATP to easily compete with ThT for the DNA binder. In the presence of ThT, adding ATP induces ABA27 to undergo a structural change, thereby not favoring the binding to ThT, verified by circular dichroism and UV-Vis absorption spectroscopy. As a result, the fluorescence intensity of ThT decreases dramatically, enabling the sensitive detection of ATP with high selectivity over other analogs. Such a sensing strategy may make ThT able to serve as a facile signal reporter for DNA nanomechanical devices fueled with ATP. Graphical Abstract The principle of the displacement of ThT by ATP.

Combined thioflavin T–Congo red fluorescence assay for amyloid fibril detection

Fluorescence represents one of the most powerful tools for the detection and structural characterization of the pathogenic protein aggregates, amyloid fibrils. The traditional approaches to the identification and quantification of amyloid fibrils are based on monitoring the fluorescence changes of the benzothiazole dye thioflavin T (ThT) and absorbance changes of the azo dye Congo red (CR). In routine screening it is usually sufficient to perform only the ThT and CR assays, but both of them, when used separately, could give false results. Moreover, fibrillization kinetics can be measured only by ThT fluorescence, while the characteristic absorption spectra and birefringence of CR represent more rigid criteria for the presence of amyloid fibrils. Therefore, it seemed reasonable to use both these dyes simultaneously, combining the advantages of each technique. To this end, we undertook a detailed analysis of the fluorescence spectral behavior of these unique amyloid tracers upon their binding to amyloid fibrils from lysozyme, insulin and an N-terminal fragment of apolipoprotein A-I with Iowa mutation. The fluorescence measurements revealed several criteria for distinguishing between fibrillar and monomeric protein states: (i) a common drastic increase in ThT fluorescence intensity; (ii) a sharp decrease in ThT fluorescence upon addition of CR; (iii) an appearance of the maximum at 535–540 nm in the CR excitation spectra; (iv) increase in CR fluorescence intensity at 610 nm. Based on these findings we designed a novel combined ThT–CR fluorescence assay for amyloid identification. Such an approach not only strengthens the reliability of the ThT assay, but also provides new opportunities for structural characterization of amyloid fibrils.

Detection of type I collagen fibrils formation and dissociation by a fluorescence method based on thioflavin T

In this study, fibrillogenesis and thermal dissociation of pepsin-soluble collagen (PSC), extracted from snakehead (Channa argus) skin, were monitored by fluorescence method based on thioflavin T (Th-T), where the accuracy and sensitivity were evaluated and compared with those of turbidity assay. The fluorescence method revealed the fibrillogenesis dynamics of collagen with better sensitivity, especially at nucleation and plateau stages. The melting temperature (Tm) of PSC was estimated to be 47°C by circular dichroism spectroscopy; below this temperature, the triple-helical structure should be intact. After that, the dynamic process of collagen dissociation was explored by the fluorescence method, and verified by morphological analysis of the fibrils and the proportion of retained fibrils. The thermal dissociation critical temperature (TDCT) of PSC fibrils was confirmed to be 39°C. The fluorescence intensity of fibril-incorporated Th-T gradually decreases in the dissociation process, and the decrease rate can be accelerated by increasing temperature. Finally, the thermal stability of triple-helical structures of free-, assembled- and dissociated-PSC was compared. Thus, we demonstrated the formation and thermal dissociation of collagen fibrils in vitro by a fluorescence method based on Th-T. This approach may advance the understanding of fibril formation and inverse dissociation of fish-sourced collagen in vitro.

Dual relaxation channel in thioflavin-T: An ultrafast spectroscopic study

In the present contribution, the ultrafast excited state dynamics of thioflavin T (ThT) has been investigated in methanol and chloroform. The first hand data of 30 times higher fluorescence quantum yield and observation of slow rise time in fluorescence intensity of ThT in chloroform compared to methanol indicate the complicated photophysics of the molecule. Time resolved fluorescence data along with temperature dependence on fluorescence quantum yield, TD-DFT calculations and femtosecond transient absorption study vividly suggest the involvement of one more twisted intramolecular charge transfer state (TICT-2) along with the TICT-1 state, in the excited state manifold of ThT, which was not identified earlier. It was also established that the depletion of the molecule from the locally excited state to the TICT-1 state probably barrierless in nature, however, an inherent activation energy barrier is present between LE and TICT-2 states. This activation energy barrier in methanol (0.59 kcal mol−1) was found to be exactly same as that available at room temperature, whereas the same in chloroform is found to be quite high (1.58 kcal mol−1). This barrier is proposed to be responsible for the high quantum yield of ThT in chlorinated solvents and we demand a revisit to the mechanism of fibril sensing by ThT.

Photophysical Properties of Thioflavin T. Does it form Excimers When Integrated into Amyloid Fibrils?

Thioflavin T (ThT) is a proven tool to detect and study of the amyloid fibrils that frequently accompany the development of serious human diseases, including Alzheimer's, Parkinson's, prion, and more than 25 other diseases. Obviously, the success of ThT use for investigation of fibrils largely depends on the correct understanding of its photophysical properties and mechanism of interaction with amyloid fibrils. Using the specially elaborated approach for recoded fluorescence intensity correction on the primary and secondary inner filter effects and spectrofluorimeter with the horizontal slits we for the first time observed the fluorescence band with a maximum at 570 nm for ThT aqueous solutions with high concentrations and proved that this band can be attributed to dye excimers. We showed that ThT excimer fluorescence spectrum has nothing in common with the fluorescence of ThT incorporated into amyloid fibrils, which has maximum at about 490 nm. Furthermore we proved that the increase in the ThT fluorescence intensity when the dye binds to amyloid fibrils can be explained only by the molecular rotor nature of ThT. Thus, we concluded that there is no basis to suggest that the fluorescence of ThT bound to fibrils is caused by its excimers.This work was supported in part by grant 14-08-90024_Bel (K.K.T.), 13-04-02068 (A.I.S.) from the Russian Foundation of Basic Research, the Program “Molecular and Cell Biology” of the Russian Academy of Sciences, and RF President fellowship SP-1982.2015.4 (A.I.S.).

Steady-state and time-resolved Thioflavin-T fluorescence can report on morphological differences in amyloid fibrils formed by Aβ(1-40) and Aβ(1-42)

Thioflavin-T (ThT) is one of the most commonly used dyes for amyloid detection, but the origin of its fluorescence enhancement is not fully understood. Herein we have characterised the ThT fluorescence response upon binding to the Aβ(1-40) and Aβ(1-42) variants of the Alzheimer's-related peptide amyloid-β, in order to explore how the photophysical properties of this dye relates to structural and morphological properties of two amyloid fibril types formed by peptides with a high degree of sequence homology. We show that the steady-state ThT fluorescence is 1.7 times more intense with Aβ(1-40) compared to Aβ(1-42) fibrils in concentration matched samples prepared under quiescent conditions. By measuring the excited state lifetime of bound ThT, we also demonstrate a distinct difference between the two fibril isoforms, with Aβ(1-42) fibrils producing a longer ThT fluorescence lifetime compared to Aβ(1-40). The substantial steady-state intensity difference is therefore not explained by differences in fluorescence quantum yield. Further, we find that the ThT fluorescence intensity, but not the fluorescence lifetime, is dependent on the fibril preparation method (quiescent versus agitated conditions). We therefore propose that the fluorescence lifetime is inherent to each isoform and sensitively reports on fibril microstructure in the protofilament whereas the total fluorescence intensity relates to the amount of exposed β-sheet in the mature Aβ fibrils and hence to differences in their morphology. Our results highlight the complexity of ThT fluorescence, and demonstrate its extended use in amyloid fibril characterisation.

Investigation of the kinetics of insulin amyloid fibrils formation

Investigation of the structure of ordered protein aggregates--amyloid fibrils, the influence of the native structure of the protein and the environment on the process of fibrillation is currently the subject of intensive research. The present work is devoted to the study of the kinetics of insulin amyloid fibrils formation at low pH values (which are produced at many stages of the isolation and purification of the protein) using a fluorescent probe thioflavin T (ThT). It has been shown that the increase of fluorescence intensity of ThT during the formation of amyloid fibrils is described by a sigmoidal curve, in which 3 areas can be distinguished: the lag phase, the growth and the plateau, which characterize the various stages of fibril formation. Despite the variation in the length of the lag phase at the same experimental conditions (pH and temperature), we have found its reduction with stirring the solution and seeding. Data obtained using electron microscopy showed that the formed fibrils are long, linear filament having a diameter of -20 nm. With increasing incubation time fibril diameter did not change while their length increases to 2-3 μm, which was accompanied by a significant increase in the number of aggregates of fibrils. All the experimental data shows that, regardless of the kinetics of the formation of amyloid fibrils, their properties after the fibrillation process are identical. The results of this work together with the previously studies of insulin amyloid fibrils might be important for clarification the mechanism of their formation, as well as for the treatment of amyloidosis associated with the aggregation of insulin.

Selective recognition of ds-DNA cavities by a molecular rotor: switched fluorescence of thioflavin T

Thioflavin T (ThT) has been widely utilized as a fluorescent marker for amyloid fibrils. However, the use of ThT as an efficient reporter for a specific DNA structure still remains in question. Here, we report that the fluorescence intensity of ThT is obviously enhancement in when it binds to ds-DNAs which contain cavity structures such as an abasic site, gap site or mismatch site. Such enhancement in fluorescence cannot be achieved for DNA without these cavity structures. The DNA cavities provide appropriate spaces to accommodate ThT and allow the occurrence of some specific interactions. The stacking interaction of the bound ThT with the cavity context bases is the main driving force for ThT binding to the cavities. This interaction restricts the excited state's rapid torsional rotation around the single C-C bond between the benzothiazole and dimethylaminobenzene moieties and thus results in a decreased population of the nonradiative twisted internal charge-transfer (TICT) state. It is impossible for this stacking interaction to occur in DNA without these cavities. This property can be used to recognize DNA cavities with high selectivity and sensitivity. We expect that the ability of ThT to target these DNA structures has the potential to be developed into practical and functional biomaterials for DNA sensors or devices.

Macromolecular crowding modulates the kinetics and morphology of amyloid self-assembly by β-lactoglobulin

The formation of amyloid fibrils by β-lactoglobulin in the presence of GndHCl has been monitored by using thioflavin T (ThT) fluorescence, Congo Red and transmission electron microscopy (TEM). Large quantities of aggregated protein are formed by incubating β-lactoglobulin in 2M GndHCl at room temperature and pH 7.0 for about 20 days. The kinetics of fibrillation process can be described by the lag time for formation of stable nuclei (nucleation) and the apparent rate constant for the growth of fibrils (elongation). Moreover, the effects of macromolecular crowding agents, Dextran 70 and polyethylene glycols (PEG), on the amyloid formation of β-lactoglobulin at pH 7.0 are studied. The results show that the increase in macromolecular crowding agent concentrations results in shorter lag time and faster growth of fibrils. It proves that macromolecular crowding can effectively accelerate the fibril formation of β-lactoglobulin at neutral pH. At the same time, it can be observed that the amplitude of the ThT fluorescence intensity decreases as the Dextran 70 concentration is increased. The observation suggests that the yield of amyloid fibrils is significantly reduced by the addition of macromolecular crowding agents. The conclusion is further confirmed by the transmission electron microscopy. In addition, the results of transmission electron microscopy also indicate that macromolecular crowding can alter the fibril morphology of β-lactoglobulin. In brief, our findings demonstrate that the effects of macromolecular crowding are essential to the understanding of protein amyloid self-assembly occurring in vivo.

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.

Small Amphipathic Molecules Modulate Secondary Structure and Amyloid Fibril-forming Kinetics of Alzheimer Disease Peptide Aβ1–42

Amyloid fibril formation is associated with a number of debilitating systemic and neurodegenerative diseases. One of the most prominent is Alzheimer disease in which aggregation and deposition of the Aβ peptide occur. Aβ is widely considered to mediate the extensive neuronal loss observed in this disease through the formation of soluble oligomeric species, with the final fibrillar end product of the aggregation process being relatively inert. Factors that influence the aggregation of these amyloid-forming proteins are therefore very important. We have screened a library of 96 amphipathic molecules for effects on Aβ(1-42) aggregation and self-association. We find, using thioflavin T fluorescence and electron microscopy assays, that 30 of the molecules inhibit the aggregation process, whereas 36 activate fibril formation. Several activators and inhibitors were subjected to further analysis using analytical ultracentrifugation and circular dichroism. Activators typically display a 1:10 peptide:detergent stoichiometry for maximal activation, whereas the inhibitors are effective at a 1:1 stoichiometry. Analytical ultracentrifugation and circular dichroism experiments show that activators promote a mixture of unfolded and β-sheet structures and rapidly form large aggregates, whereas inhibitors induce α-helical structures that form stable dimeric/trimeric oligomers. The results suggest that Aβ(1-42) contains at least one small molecule binding site, which modulates the secondary structure and aggregation processes. Further studies of the binding of these compounds to Aβ may provide insight for developing therapeutic strategies aimed at stabilizing Aβ in a favorable conformation.

Self-assembly pathways of E22Δ-type amyloid β peptide mutants generated from non-aggregative O-acyl isopeptide precursors

The recently identified E22Δ-type amyloid β peptide (Aβ) mutants are reported to favor oligomerization over fibrillization and to exhibit more-potent synaptotoxicity than does wild-type (WT) Aβ. Aβ(E22Δ) mutants can thus be expected to serve as tools for clarifying the impact of Aβ oligomers in Alzheimer’s disease (or Alzheimer’s-type dementia). However, the biochemical and biophysical properties of Aβ(E22Δ) have not been conclusively determined. Here, we evaluated the self-assembly pathways of Aβ(E22Δ) mutants generated from water-soluble, non-aggregative O-acyl isopeptide precursors. Circular dichroism spectroscopy, Western blot analysis, and thioflavin-T fluorescence intensity and cellular toxicity assays suggest that the self-assembly pathways of Aβ(E22Δ) differed from those of Aβ(WT). Aβ1–40(E22Δ) underwent a rapid random coil→β-sheet conformational change in its monomeric or low-molecular-weight oligomeric states, whereas Aβ1–40(WT) self-assembled gradually without losing its propensity to form random coil structures. The Aβ1–42(E22Δ) monomer formed β-sheet-rich oligomers more rapidly than did Aβ1–42(WT). Additionally, the Aβ1–42(E22Δ) oligomers appear to differ from Aβ1–42(WT) oligomers in size, shape, or both. These results should provide new insights into the functions of Aβ(E22Δ) mutants.

Thioflavin T as a Molecular Rotor: Fluorescent Properties of Thioflavin T in Solvents with Different Viscosity

The effect of solvent viscosity on thioflavin T (ThT) fluorescent properties is analyzed to understand the molecular mechanisms of the characteristic increase in ThT fluorescence intensity accompanying its incorporation into the amyloid-like fibrils. To this end, the dependencies of the ThT quantum yield and fluorescence lifetime on temperature and glycerol content in the water-glycerol mixtures are studied. It has been found that fluorescent properties of ThT are typical for the specific class of fluorophores known as molecular rotors. It has been established that the low ThT fluorescence intensity in the solvents with low viscosity is caused by the nonradiative deactivation of the excited state associated with the torsional motion of the ThT benzthiazole and aminobenzene rings relative to each other, which results in the transition of ThT molecule to nonfluorescent twisted internal charge transfer (TICT) state. The rate of this process is determined by the solvent viscosity, whereas the emission does occur from the nonequilibrium locally excited (LE) state. High polarization degree of the ThT fluorescence (P = 0.45) observed for glycerol solutions of different viscosity confirms the nonequilibrium character of the emission from the LE state and testifies that rotational correlation time of the whole molecule is considerably greater than the time required to accomplish transition to the nonfluorescent TICT state. Torsional movements of the ThT fragments take place in the same temporal interval as solvent relaxation, which leads to nonexponential fluorescence decay of the dye in viscous solvents. This photophysical model successfully explains the fluorescent properties of ThT in solvents with different viscosities. The model is confirmed by the results of the quantum-chemical calculations, which showed that energy minimum for the ground state of ThT corresponds to conformation with torsional angle phi = 37 degrees between the benzthiazole and aminobenzene rings and in the excited-state twisted conformation of ThT with phi = 90 degrees has minimal energy. These data support the idea that the reason for the characteristic increase in the ThT fluorescence intensity accompanying its incorporation into the amyloid fibrils is determined by the rigidity of the dye environment, which prevents the rotation of the benzthiazole ring relative to the aminobenzene ring in the excited state.

Role of conformational change in the C-terminus of β2-microglobulin in dialysis-related amyloidosis

Beta(2)-microglobulin (beta(2)m) has been identified as the precursor protein of dialysis-related amyloidosis (DRA), which is a serious complication for haemodialysis (HD) patients. However, mechanisms underlying beta(2)m amyloid fibril formation remains to be elucidated. We previously demonstrated, in amyloid deposits from HD patients, a conformational isoform of beta(2)m with an unfolded C-terminus. However, no direct experiments have previously been performed to address whether unfolded beta(2)m in the C-terminus may be prone to form amyloid fibrils. To evaluate roles of C-terminal amino acids in beta(2)m-induced amyloid formation, we generated six types of recombinant beta(2)m with amino acid substitutions in the C-terminal region. To investigate their conformational change and amyloidogenicity, we measured circular dichroism spectra, the fluorescence intensity of tryptophan and thioflavin-T (ThT) of the recombinant beta(2)m. To analyse morphological change of beta(2)m, we performed electron microscopy (EM) on the samples with elevated ThT fluorescence intensity. We used ultrasonication to enhance beta(2)m destabilization of the protein. Beta(2)M Trp95Leu and Arg97Ala showed conformational changes and increased their amyloidgenicity compared with beta(2)m wild-type (WT). With ultrasonication, beta(2)m Trp95Leu and Arg97Ala generated more amyloid fibrils than did beta(2)m WT even in physiological solution. EM showed that beta(2)m formed amorphous debris containing typical amyloid fibrils at 24 hours, when ThT fluorescence intensity was three-fold lower than that at six hours. Conformational changes in the C-terminus of beta(2)m may play an important role in DRA and that ultrasonication is useful for analysis of beta(2)m amyloidogenesis.

Probing the Role of PrP Repeats in Conformational Conversion and Amyloid Assembly of Chimeric Yeast Prions

Oligopeptide repeats appear in many proteins that undergo conformational conversions to form amyloid, including the mammalian prion protein PrP and the yeast prion protein Sup35. Whereas the repeats in PrP have been studied more exhaustively, interpretation of these studies is confounded by the fact that many details of the PrP prion conformational conversion are not well understood. On the other hand, there is now a relatively good understanding of the factors that guide the conformational conversion of the Sup35 prion protein. To provide a general model for studying the role of oligopeptide repeats in prion conformational conversion and amyloid formation, we have substituted various numbers of the PrP octarepeats for the endogenous Sup35 repeats. The resulting chimeric proteins can adopt the [PSI+] prion state in yeast, and the stability of the prion state depends on the number of repeats. In vitro, these chimeric proteins form amyloid fibers, with more repeats leading to shorter lag phases and faster assembly rates. Both pH and the presence of metal ions modulate assembly kinetics of the chimeric proteins, and the extent of modulation is highly sensitive to the number of PrP repeats. This work offers new insight into the properties of the PrP octarepeats in amyloid assembly and prion formation. It also reveals new features of the yeast prion protein, and provides a level of control over yeast prion assembly that will be useful for future structural studies and for creating amyloid-based biomaterials.