Thioflavin T Fluorescence Intensity Research Articles

Linalool acts as a chemical chaperone by inhibiting amyloid-β aggregation

Linalool is a neuroprotective monoterpene found in essential oils from aromatic plants. Linalool's effectiveness in AD animal models has been established previously, but its mechanisms of action remain unclear. Therefore, this study aims to investigate whether linalool binds directly to the amyloid beta (Aβ) fibrils to understand it's role in preventing neurodegeneration. The anti-aggregation ability of Linalool was determined using Dithiothreitol (DTT), and thermal aggregation assays followed by Thioflavin T (ThT) binding assay. AD animals were treated with Linalool, and Thioflavin T staining was used to check the binding of linalool to Aβ fibrils in rat brain tissue sections. Preliminary studies revealed the anti-aggregation potential of linalool under the thermal and chemical stimulus. Further, in ThT binding assay Linalool inhibited Aβ aggregation, binding directly to Aβ fibrils. The reduced fluorescence intensity of ThT in AD brain tissues following linalool administration, highlights its neuroprotective potential as a therapeutic agent for AD.

PCR-Free, Label-Free, and Centrifugation-Free Diagnosis of Multiplex Antibiotic Resistance Genes by Combining mDNA-Au@Fe3O4 from Heating Dry and DNA Concatamers with G-Triplex.

Accurate identification of antibiotic resistance genes (ARGs) is crucial for improving treatment and controlling the spread of antibiotic-resistant bacteria (ARB). Herein, a novel PCR-free, centrifugation-free, and label-free magnetic fluorescent biosensor (MFB) was developed by combining polyA-medium DNA-polyT (mDNA, which contained a partial sequence of a target DNA), gold nanoparticle (AuNP)-anchored magnetic nanoparticle (Au@Fe3O4), complementary strand DNA (CS) of the target DNA, DNA concatamer with G-triplex (G3), and thioflavin T (ThT). Thereinto, Au@Fe3O4 nanoparticles were first capped by mDNA strands within 20 min using a simple hot drying method, and then CS was added and hybridized with mDNA on Au@Fe3O4. Second, a DNA concatamer was used to bind with CS on Au@Fe3O4. When an ARG was present in the sample, the CS would recognize it and release the DNA concatamer into solution by a toehold-mediated strand displacement reaction. Finally, under magnetic separation, the free DNA concatamers with G3 were taken out easily and bound with ThT, resulting in strong fluorescence signals. The fluorescence intensity of ThT was positively correlated with the concentration of the ARG. The whole analysis was accomplished within 1.5 h using 96-well plates. Remarkably, our MFB was universal; eight ARGs were detected by replacing the corresponding mDNA and CS in this study. To verify the practicability of our method, 12 clinically isolated strains were analyzed. The results of the MFB method were in good agreement with those of the quantitative real-time PCR method with an area under the curve of 0.92 (95% confidence interval: 0.8479 to 0.9932), sensitivity of 92.00%, and specificity of 91.55%. Above all, the MFB assay established here is simple, low-cost, and universal and has great potential for applications in the identification of ARGs.

Structure-switching aptasensors for sensitive detection of ochratoxin A.

Ochratoxin A (OTA) is a toxic metabolite commonly found in various foods and feedstuffs. Accurate and sensitive detection of OTA is needed for food safety and human health. Based on a common OTA-binding aptamer (OTABA), two structure-switching OTABAs, namely OTABA4 and OTABA3, were designed by configuring a split G-quadruplex and a split G-triplex, respectively, at the two ends of OTABA to construct aptasensors for the detection of OTA. The OTABA, G-quadruplex, and G-triplex all can capture the thioflavin T (ThT) probe, thereby enhancing the fluorescence intensity of ThT. Bonding with OTA could change the conformations of OTABA and G-quadruplex or G-triplex regions, resulting in the release of the captured ThT and diminution of its fluorescence intensity. Dual conformation changes in structure-switching OTABA synergistically amplified the fluorescence signal and improved the sensitivity of the aptasensor, especially for that with OTABA3. The detection limits of the OTABA4-ThT and OTABA3-ThT systems for OTA were 0.28 and 0.059 ng ml-1 , with a 1.4-fold and 6.7-fold higher sensitivity than that of the original OTABA-ThT system, respectively. They performed well in corn and peanut samples and met the requirements of the food safety inspections.

A G-triplex and G-quadruplex concatemer-enhanced fluorescence probe coupled with hybridization chain reaction for ultrasensitive aptasensing of ochratoxin A

Ochratoxin A (OTA), a typical mycotoxin contaminant found in various agricultural products and foods, poses a serious threat to human health. In this study, an aptasensor based on a novel fluorescence probe comprising a G-rich DNA sequence (G43) and thioflavin T (ThT) was designed via hybridization chain reaction (HCR) for the ultrasensitive detection of OTA. G43 is a concatemer of G-quadruplex and G-triplex (a G-quadruplex-like structure with one G-quartet removed), which can drastically enhance the fluorescence intensity of ThT. For this strategy to work, the OTA aptamer is pro-locked in a hairpin structure, denoted “hairpin-locked aptamer” (HL-Apt). OTA binds to HL-Apt, opens the hairpin structure, releases the trigger sequence, and initiates the HCR reaction to form a long DNA duplex and numerous side chains. The side chains combine entirely with the complementary DNA and liberate the pro-locked G43 DNA, dramatically enhancing the intensity of the ThT fluorescence signal. The fluorescence intensity correlates linearly with the OTA concentration between 0.02 and 2.00 ng mL−1, and the method has a detection limit of 0.008 ng mL−1. The developed aptasensor was used to detect OTA in foodstuffs with satisfactory results.

Suppression of amyloid fibril formation by UV irradiation

Ultraviolet (UV) irradiation originating in the space environment has been increasing because of the destruction of the ozone layer. Although UV irradiation damages DNA, peptides, and cell structures, it also has the beneficial effects of suppressing excessive immune responses and cell proliferation. However, the effects of UV irradiation on amyloid fibril formation are unknown. Here, we analyzed its effects on amyloid fibril formation of two types of amyloidogenic proteins, human amyloid β42 (Aβ42) and human transthyretin (TTR). To study these effects, we incubated Aβ42 samples in neutral pH buffer and wild-type TTR (TTRwt) samples in acidic buffer, with and without UV irradiation. We analyzed amyloidogenicity via a thioflavin T (ThT) method and morphological changes by using electron microscopy. UV irradiation reduced the ThT fluorescence intensity of the human Aβ42 samples. For the electron microscopic analysis of Aβ42 samples, the UV irradiation induced thinning of the amyloid fiber tips. Although UV irradiation reduced the ThT fluorescence intensity of human TTRwt samples, the amounts of the aggregates did not differ with or without UV irradiation. Our experiments suggest that UV irradiation may become a useful tool for reducing the amyloid fibrils and amyloid like substances.

Epigallocatechin-3-gallate mediated self-assemble behavior and gelling properties of the ovalbumin with heating treatment

Ovalbumin (OVA), is the most abundant protein in the egg white. In the present study, the effects of Epigallocatechin-3-gallate (EGCG) on the self-assembly behavior and gelling properties of OVA with different heating time were investigated. Electrophoresis, surface hydrophobicity, Fourier transform infrared (FTIR) spectroscopy and molecular docking analysis indicated that hydrophobic force and hydrogen bond were the predominant interactions between EGCG and OVA. The binding of EGCG to OVA suppressed the formation of fibrils as evidenced by the results of Thioflavin T (ThT) intensity and atomic force microscopy (AFM) analysis. When heated for 15–30 min, EGCG addition, especially at 2.5 μM/g, resulted in a decrease of ThT fluorescence intensity and hydrophobicity of OVA samples, and a formation of dense linear aggregates. However, the OVA solution did not form a gel. In a longer heating time of 45 min, 5 μM/g EGCG induced a significant decrease in ThT fluorescence intensity and hydrophobicity of OVA samples, and a transition from the dense linear aggregates to the dense spherical clusters. Therefore, the OVA samples realized the transition from the solution state to the gel state with a higher storage modulus (G′). When the heating time was extended to 60 min, all the OVA samples formed gel and reached the highest G′ at 5 μM/g EGCG. These results suggested that the suitable hydrophobic interactions and hydrogen bonds between EGCG and the OVA improved gel quality. Polyphenol binding provided a new way to regulate the assembly behavior and gelling properties of OVA. • EGCG bound with OVA mainly through hydrophobic interactions and hydrogen bonds. • EGCG mediated self-assemble behavior and gelling properties of the OVA. • The binding of EGCG suppressed the heat-induced fibrils of the OVA. • EGCG (5 μM/g) changed the topology of the OVA aggregates heated for 45–60 min. • EGCG (5 μM/g) drove the formation of the OVA gel heated for 45–60 min.

Microscopic Insights into the Mechanism of White Light Generation by Disruptive Interaction between Human Serum Albumin Amyloid Fibrils and Surfactant-AIEgen Nanorods.

Dimethyl-2,5-bis[4-(methoxyphenyl)amino] terephthalate (DBMPT) exhibits aggregation-induced enhancement of emission with Tween 40 and formation of nanorods with strong orange fluorescence. These nanorods disrupt fibrils of human serum albumin and lead to partial refolding of the protein, as monitored by circular dichroism and thioflavin T (ThT) fluorescence. The resultant milieu emits white light, the mechanism of which is explored in this study. It is established that direct excitation of the acceptor plays a significant role, even though Förster resonance energy transfer (FRET) is found to be operative to some extent. A decrease in the fluorescence intensity and lifetime of ThT with progressive addition of DBMPT, which is often used as the sole indicator of FRET, is ascribed to the disruption of the fibrils by the nanorods.

NMR and vibrational spectroscopic studies on the structure and self-assembly of Two de novo dipeptides in methanol

Short peptides are known to form self-organized nano-architectures useful for advanced biomedical applications. Both hydrogen-bonding and hydrophobic interactions play a pivotal role in molecular assembly, including self-assembly of proteins and peptides. In the current investigation, we reported the synthesis, self-assembly pattern and physicochemical properties of two de novo dipeptides: N-Boc-(O-benzyl)-tyro-(dimethyl)-glutamate and N-Boc-(O-benzyl)-glu-(O-benzyl)-tyrosine-methyl ester, abbreviated as P1 and P2, respectively. These two peptides produced different morphological features under similar conditions due to the presence of a small twist in their molecular structures. In methanol, P1 gave rise to a unique fibrillar network whereas P2 self-assembled into oligomers of 20-30 nm diameters. The UV-Vis, FT-IR and Raman spectra were recorded using experimental methods and compared with the computational spectra obtained by density functional method (B3LYP/6-31G(d)). The electronic absorption spectra of both P1 and P2 measured in methanol showed broad absorption peaks in the UV region with an absorption maximum at ∼ 273 nm. FT-IR and Raman spectra of both the dipeptides in solid state showed amide I vibration bands at ∼1650 cm−1 indicating a similar conformation state of the peptide bonds. However, H/D exchange 1H NMR measurements confirmed that the amide NH of P1 was strongly involved in H-bond formation compared to P2 amide NH and rapid H/D exchange occurred upon addition of D2O in the solution. Thioflavin T (ThT) fluorescence assay measurement suggested that incubation of P1 in methanol at room temperature produced fibrillar aggregates enriched with compact ß-sheet conformation. Formation of such ordered structure also caused a blue shift in the amide-carbonyl Raman frequency of the peptide to ∼1657 cm−1. P1 fibrils further showed gel formation behaviour in 1:1 methanol/water mixture. The minimum gelation concentration (MGC) of the dipeptide was found to be 0.5% w/v and the calculated gelation temperature (Tgel) of the corresponding MGC was 67°C. On the other hand, the amide carbonyl stretching vibration of P2 appeared at ∼1650 cm−1 in the Raman spectra and it was not significantly altered in its oligomeric state (produced in methanol).Moreover, no significant enhancement of ThT fluorescence intensity of P2 indicating absence of compact ß-sheet conformation of the peptide. The P2 oligomers also showed no gel like phase transformation in methanol/water mixtures.

Inflammasome assembly is required for intracellular formation of β2-microglobulin amyloid fibrils, leading to IL-1β secretion.

IntroductionDialysis-related amyloidosis (DRA) caused by β2-microgloblin (B2M) fibrils is a serious complication for patients with kidney failure on long-term dialysis. Deposition of B2M amyloid fibrils is thought to be due not only to serum extracellular B2M but also to infiltrating inflammatory cells, which may have an important role in B2M amyloid deposition in osteoarticular tissues in patients with DRA. Here, we asked whether B2M amyloid fibrils activate the inflammasome and contribute to formation and deposition of amyloid fibrils in cells.MethodsAmyloid formation was confirmed by a thioflavin T (ThT) spectroscopic assay and scanning electron microscopy (SEM). Activation of inflammasomes was assessed by detecting interleukin (IL)-1β in culture supernatants from human embryonic kidney (HEK) 293T cells ectopically expressing inflammasome components. IL-1β secretion was measured by enzyme-linked immunosorbent assay. Expression and co-localization were analyzed by immunohistochemistry and dual immunofluorescence microscopy.ResultsB2M amyloid fibrils interacted directly with NLRP3/Pyrin and to activate the NLRP3/Pyrin inflammasomes, resulting in IL-1β secretion. When HEK293T cells were transfected with inflammasome components NLRP3 or Pyrin, along with ASC, pro-caspase-1, pro-IL-1β, and B2M, ThT fluorescence intensity increased. This was accompanied by IL-1β secretion, which increased in line with the amount of transfected B2M. In this case, morphological glowing of amyloid fibrils was observed by SEM. In the absence of ASC, there was no increase in ThT fluorescence intensity or IL-1β secretion, or any morphological glowing of amyloid fibrils. NLRP3 or Pyrin and B2M were co-localized in a “speck” in HEK293T cells, and co-expressed in infiltrated monocytes/macrophages in the osteoarticular synovial tissues in a patient with DRA.ConclusionTaken together, these data suggest that inflammasome assembly is required for the subsequent triggering of intracellular formation of B2M amyloid fibrils, which may contribute to osteoarticular deposition of B2M amyloid fibrils and inflammation in patients with DRA.

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.

Effect of Ionic Strength on Thioflavin-T Affinity to Amyloid Fibrils and Its Fluorescence Intensity.

The formation of amyloid fibrils is linked to multiple neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease. Despite years of research and countless studies on the topic of such aggregate formation, as well as their resulting structure, the current knowledge is still fairly limited. One of the main aspects prohibiting effective aggregation tracking is the environment’s effect on amyloid-specific dyes, namely thioflavin-T (ThT). Currently, there are only a few studies hinting at ionic strength being one of the factors that modulate the dye’s binding affinity and fluorescence intensity. In this work we explore this effect under a range of ionic strength conditions, using insulin, lysozyme, mouse prion protein, and α-synuclein fibrils. We show that ionic strength is an extremely important factor affecting both the binding affinity, as well as the fluorescence intensity of ThT.

Assembly behavior, structural characterization and rheological properties of legume proteins based amyloid fibrils

The assembly behavior of three selected legume proteins was monitored using thioflavin T (ThT) fluorescence intensity, atomic force microscopy (AFM) and dynamic light scattering (DLS) in this test. It was found that the cowpea protein (CoP) had the highest ability to form amyloid fibrils due to the higher ThT fluorescence intensity of CoP fibrils than chickpea protein (ChP) fibrils and lentil protein (LP) fibrils. These legume proteins were partially denatured during initial heating period, and then the elongation of fibrils was occurred with prolonged heating time. Correspondingly, the particle size of legume proteins decreased firstly, probably resulting from the degraded polypeptides and disrupted structure under the heat treatment and acid hydrolysis. Then the particle size of proteins increased with increasing heating time due to the reorganization of ordered structure. From the results of SDS-PAGE and AFM, vicilin fractions were more beneficial to the formation of fibrils, and the longer and flexible fibrils were observed in the vicilin enriched CoP fibrils, whereas the semiflexible fibrils and rigid fibrils were occurred in LP and ChP fibrils, respectively. In addition, the rheological properties of CoP fibrils were superior to ChP and LP fibrils, owing to the tight entanglement of the longer and flexible CoP fibrils. This work will strengthen the understanding of the formation mechanism of different legume proteins based fibrils and explore their potential application.

A Label-free DNA-based Fluorescent Sensor for Cisplatin Detection

Cisplatin, an anti-cancer drug, has been used extensively and has become a “gold standard” treatment for various types of cancer. However, overdosage has serious side effects. Thus, this drug should not be over-administered. The current study presents a label-free but sensitive fluorescent sensor for cisplatin detection based on G-quadruplex (GQ) and fluorescent dye Thioflavin T (ThT). In the absence of cisplatin, ThT binds to GQ, resulting in high fluorescence emission. In the presence of cisplatin, the GQ structure is destroyed after cisplatin–DNA adduct formation, leaving no binding scaffold for ThT, thereby causing a decrease in ThT fluorescence. The amount of cisplatin can be quantified from the changes in ThT fluorescence intensity. Parameters that affect the sensor performance were tested, including pH and concentration of potassium phosphate buffer, type of GQ, number of quartets, loop length, incubation time, and temperature. The sensor provided a linear dynamic range of 10–500 nM with limit of quantification at 10 nM. The sensor was successfully applied to detect cisplatin in urine samples from patients undergoing treatment with high accuracy (recoveries between 74 ± 14 and 112.8 ± 5.0%) and precision (relative standard deviations between 1.15% and 2.12%).

Fluorescence Lifetime and Intensity of Thioflavin T as Reporters of Different Fibrillation Stages: Insights Obtained from Fluorescence Up-Conversion and Particle Size Distribution Measurements.

Thioflavin T (ThT) assay is extensively used for studying fibrillation kinetics in vitro. However, the differences in the time course of ThT fluorescence intensity and lifetime and other physical parameters of the system, such as particle size distribution, raise questions about the correct interpretation of the aggregation kinetics. In this work, we focused on the investigation of the mechanisms, which underlay the difference in sensitivity of ThT fluorescence intensity and lifetime to the formation of protein aggregates during fibrillation by the example of insulin and during binding to globular proteins. The assessment of aggregate sizes and heterogeneity was performed using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Using the sub-nanosecond resolution measurements, it was shown that the ThT lifetime is sensitive to the appearance of as much as a few percent of ThT bound to the high-affinity sites that occur simultaneously with an abrupt increase of the average particle size, particles concentration, and size heterogeneity. The discrepancy between ThT fluorescence intensity and a lifetime can be explained as the consequence of a ThT molecule fraction with ultrafast decay and weak fluorescence. These ThT molecules can only be detected using time-resolved fluorescence measurements in the sub-picosecond time domain. The presence of a bound ThT subpopulation with similar photophysical properties was also demonstrated for globular proteins that were attributed to non-specifically bound ThT molecules with a non-rigid microenvironment.

The effect of three polyphenols and some other antioxidant substances on amyloid fibril formation by Human cystatin C

Human cystatin C (CysC) is an amyloid forming protein involved in the hereditary cerebral amyloid angiopathy (HCCAA) that affects arteries in the brain and the peripheral nervous system. In this study we measured the influence of several substances on human CysC aggregation and amyloid fibril formation, induced at pH 4 in vitro. The effect of three polyphenols: resveratrol, quercetin and curcumin and of two antioxidants: vitamin C (VitC) and N-acetyl-L-cysteine (NAC) was explored as well as the effect of sulphoraphane (SF) and α-lipoic acid (AL). The formation of amyloid fibrils was followed by Thioflavin T (ThT) fluorescence and by transmission electron microscopy (TEM). Effects on the length of the lag phase were revealed by following the increase of ThT fluorescence intensity with time. The amount and morphology of fibrils in comparison to prefibrillar aggregates and globular oligomers were evaluated by TEM at the plateau stage of the reaction. Thermal stabilization of the CysC monomer by the small compounds was measured by differential scanning fluorimetry (DSF). NAC, VitC and SF exhibited the largest inhibitory effect on amyloid fibril growth. The effects of polyphenols were not significant, apart from resveratrol, which partly inhibited the amyloid fibril growth.

A dual-function oligonucleotide-based ratiometric fluorescence sensor for ATP detection

Adenosine triphosphate (ATP) is the main energy currency of life that plays a vital role in supporting physiological activities in living organisms, including humans. Therefore, accurate and sensitive detection of ATP concentration is necessary in biochemical research and clinical diagnosis. Herein, a ratiometric fluorescence aptasensor was developed for ATP detection. A dual-function DNA strand comprising an ATP-binding aptamer (ABA) and berberine-binding aptamer (BBA) was designed and optimized, in which ABA can capture ATP and thioflavin T (ThT), whereas BBA can capture berberine. Interestingly, the fluorescence intensity of both berberine and ThT were enhanced as they were captured by this dual-function DNA strand. In the presence of ATP, the ABA on the 3′-end of the DNA bound specifically to its target, causing ThT release and a significant drop in ThT fluorescence. However, ATP had no significant effect on the interaction between berberine and DNA, remaining the enhanced fluorescence intensity of berberine stable. Based on this interesting phenomenon, a ratiometric fluorescence sensor was constructed that used the enhanced fluorescence intensity of berberine as reference to measure the fluorescence intensity of ThT for ATP detection. This ratiometric fluorescence strategy had excellent selectivity and high sensitivity towards ATP with a detection limit (3σ) as low as 24.8 nM. The feasibility of application of this method in biological samples was evaluated in human serum and urine samples, where it exhibited a good detection performance.

Preferential Binding of Thioflavin T to AT-Rich DNA: White Light Emission through Intramolecular Förster Resonance Energy Transfer.

Herein we report the effect of different nucleobase pair compositions on the association-induced fluorescence enhancement property of Thioflavin T (ThT), upon binding with 20 base pair long double-stranded DNA (dsDNA). Analysis of binding and decay constants along with the association (Kass) and dissociation (Kdiss) rate constants obtained from the fluctuation in the fluorescence intensity of ThT after binding with different DNA revealed selective affinity of ThT toward AT-rich dsDNA. Molecular docking also substantiates the experimental results. We also observed that addition of orange-emitting ethidium bromide (EtBr) to cyan-emitting ThT-DNA complexes leads to bright white light emission (WLE) through Förster resonance energy transfer. Additionally, the emission of white light is far greater in the case of intra-DNA strands. Besides endorsing the binding insights of ThT to AT-rich dsDNA, the present investigations open a new perspective for realizing promising WLE from two biomarkers without labeling the DNA.

Free and bound Thioflavin T molecules with ultrafast relaxation: implications for assessment of protein binding and aggregation

Fluorescent dye Thioflavin T (ThT) is a widely used probe for the detection of amyloid fibrils, which are protein aggregates involved in the pathogenesis of neurodegenerative disorders. Upon the formation of a complex with amyloids, the fluorescence quantum yield of ThT increases 1000-fold due to a dramatic reduction of the nonradiative decay rate. This is accompanied by a remarkable change of ThT fluorescence lifetime τ from ~1 to ~1000 ps, thus making it possible to assess ThT binding to different systems using τ as an indicator. However, when measuring ThT interaction with proteins, one can observe that the binding affinity determined from the ThT fluorescence intensity’s dependence on protein concentration may be orders of magnitude lower than that determined using τ. Here we show that this discrepancy at least partly originates from a limited temporal resolution when determining the fluorescence lifetime of ThT in the ThT-protein system using the time-correlated single photon counting technique (TCSPC), which is usually characterized by a ~100 ps instrument response function. This results in the situation when a small fraction (~1%) of ThT molecules with a relatively slow decay (τ ~ 1000 ps) completely disguises the impact of ThT molecules with an ultrafast decay (τ ~ 1 ps) to the overall measured fluorescence decay curve. Moreover, using the femtosecond-resolved fluorescence up-conversion technique, we demonstrate that not only free ThT molecules but also a subpopulation of protein-bound ThT molecules exhibits fluorescence decay on a 1 ps timescale. The obtained results are of critical importance for a reliable interpretation of protein binding and aggregation experiments when using a ThT assay with a fluorescence lifetime determined by the TCSPC as an indicator.

Impact of N-Terminal Acetylation on α-Synuclein Amyloid Formation

The accumulation of α-synuclein (αSyn) as β-sheet-rich amyloid in Lewy bodies is one of the hallmarks of Parkinson's disease. Recent data have demonstrated that αSyn is constitutively N-terminally acetylated in mammalian cells, a co-translational modification that has been shown to increase α-helical propensity in the N-terminal region of the protein and modulate membrane-binding behavior. The results of in vitro experiments comparing the amyloid formation kinetics of acetylated (Ac-αSyn) and the more widely studied non-acetylated a-synuclein (NH3-αSyn) have been contradictory. However, these assays largely rely on the extrinsic dye thioflavin T (ThT), which displays a dramatic increase in quantum yield upon binding to the cross-β sheet structure in amyloids. We find that the ThT response is approximately tenfold lower in Ac-αSyn, and that Ac-αSyn aggregates more slowly than NH3-αSyn at physiological pH. Intrinsic fluorescence of Trp mutants confirms that ThT emission accurately reports on the kinetics of amyloid formation for both forms of the protein. Seeding experiments indicate that the ThT fluorescence intensity is determined by the final fibrillar structure templated from the seeds rather than the presence or absence of the N-terminal acetyl group. Structural differences of the de novo and seeded fibrils are corroborated by Raman spectroscopy, limited proteolysis experiments, and transmission electron microscopy. These results indicate that N-terminal acetylation has important consequences for the aggregation propensity and amyloid structure of αSyn, and that nonacetylated αSyn is not a substitute for the biological form of the protein.

Dissection of the deep-blue autofluorescence changes accompanying amyloid fibrillation

Pathogenesis of numerous diseases is associated with the formation of amyloid fibrils. Extrinsic fluorescent dyes, including Thioflavin T (ThT), are used to follow the fibrillation kinetics. It has recently been reported that the so-called deep-blue autofluorescence (dbAF) is changing during the aggregation process. However, the origin of dbAF and the reasons for its change remain debatable. Here, the kinetics of fibril formation in model proteins were comprehensively analyzed using fluorescence lifetime and intensity of ThT, intrinsic fluorescence of proteinaceous fluorophores, and dbAF. For all systems, intensity enhancement of the dbAF band with similar spectral parameters (∼350 nm excitation; ∼450 nm emission) was observed. Although the time course of ThT lifetime (indicative of protofibrils formation) coincided with that of tyrosine residues in insulin, and the kinetic changes in the ThT fluorescence intensity (reflecting formation of mature fibrils) coincided with changes in ThT absorption spectrum, the dbAF band started to increase from the beginning of the incubation process without a lag-phase. Our mass-spectrometry data and model experiments suggested that dbAF could be at least partially related to oxidation of amino acids. This study scrutinizes the dbAF features in the context of the existing hypotheses about the origin of this spectral band.