Binding of thioflavin T by albumins: An underestimated role of protein oligomeric heterogeneity

Rovnyagina, Nataliya R.; Sluchanko, Nikolai N.; Tikhonova, T.; Фадеев, В. В.; Litskevich, Artur Yu.; Маскевич, А. А.; Shirshin, Evgeny A.

doi:10.1016/j.ijbiomac.2017.12.002

Cited by 31 publications

(32 citation statements)

References 35 publications

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“…Figure 2 demonstrates the dependence of the ThT photophysical parameters on the concentration of model globular proteins (β-lactoglobulin (β-LG) and soybean trypsin inhibitor (STI)) in the solution. In agreement with the results for other systems, namely, albumins [ 32 ], and α-lactalbumin (α-LA) [ 26 ], we observed that the ThT fluorescence lifetime saturates at lower protein concentration compared to the ThT fluorescence intensity. This asynchrony was attributed to the presence of a bound ThT subpopulation with ultrafast relaxation [ 26 ].…”

Section: Resultssupporting

confidence: 91%

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

Rovnyagina

Budylin

Vainer

et al. 2020

IJMS

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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.

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Section: Resultssupporting

confidence: 91%

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

Rovnyagina

Budylin

Vainer

et al. 2020

IJMS

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“…Benzothiazole dye-thioflavin T (ThT) was used to quantify amyloid fibrils in vitro. The interaction of ThT with globular proteins, and even with their amorphous aggregates is insignificant when compared to the interaction with protein fibrils [43]. ThT becomes incorporated with the amyloid fibrils, has a maximum at 450 nm and is dependent on solvent polarity [44].…”

Section: In-vitro Quantitative Assesment Of Amyloid Fibrilsmentioning

confidence: 99%

“…Although the mechanism of ThT interaction with amyloid fibrils is still poorly understood, it is widely accepted that ThT molecules intercalate inside the furrows (channels) between exposed to solvent side chains of amyloid fibrils located parallel to the long axis of fibrils. However, the interaction between ThT and amyloid fibrils is stoichiometric, saturated, and the fluorescence of the ThT-amyloid fibril system provides an accurate quantitative assessment of amyloid fibril formation [43,51]. Taboada et al used Congo red as a dye that is able to bind with fibrils.…”

Section: In-vitro Quantitative Assesment Of Amyloid Fibrilsmentioning

confidence: 99%

Human Serum Albumin Aggregation/Fibrillation and its Abilities to Drugs Binding

et al. 2020

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Human serum albumin (HSA) is a protein that transports neutral and acid ligands in the organism. Depending on the environment’s pH conditions, HSA can take one of the five isomeric forms that change its conformation. HSA can form aggregates resembling those in vitro formed from amyloid at physiological pH (neutral and acidic). Not surprisingly, the main goal of the research was aggregation/fibrillation of HSA, the study of the physicochemical properties of formed amyloid fibrils using thioflavin T (ThT) and the analysis of ligand binding to aggregated/fibrillated albumin in the presence of dansyl-l-glutamine (dGlu), dansyl-l-proline (dPro), phenylbutazone (Phb) and ketoprofen (Ket). Solutions of human serum albumin, both non-modified and modified, were examined with the use of fluorescence, absorption and circular dichroism (CD) spectroscopy. The experiments conducted allowed observation of changes in the structure of incubated HSA (HSAINC) in relation to nonmodified HSA (HSAFR). The formed aggregates/fibrillation differed in structure from HSA monomers and dimers. Based on CD spectroscopy, previously absent β-structural constructs have been registered. Whereas, using fluorescence spectroscopy, the association constants differing for fresh and incubated HSA solutions in the presence of dansyl-amino acids and markers for binding sites were calculated and allowed observation of the conformational changes in HSA molecule.

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“…In addition, the interaction of the dye with fibrils has a high specificity. ThT does not interact with globular proteins in a native state (other than with acetylcholinesterase [24] and serum albumins [25,26]), with molten globule and unfolded states or amorphous aggregates of proteins. Due to its unique properties, ThT is a sensitive tool for diagnostics of amyloid fibrils formation, studying the kinetics of fibrillogenesis, and more recently for the study their structure [27][28][29][30][31].…”

Section: Introductionmentioning

confidence: 99%

Effect of the fluorescent probes ThT and ANS on the mature amyloid fibrils

Sulatsky

Sulatskaya

Povarova

et al. 2020

Prion

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Fluorescent probes thioflavin T (ThT) and 1-anilino-8-naphthalene sulfonate (ANS) are widely used to study amyloid fibrils that accumulate in the body of patients with serious diseases, such as Alzheimer's, Parkinson's, prion diseases, etc. However, the possible effect of these probes on amyloid fibrils is not well understood. In this work, we investigated the photophysical characteristics, structure, and morphology of mature amyloid fibrils formed from two model proteins, insulin and lysozyme, in the presence of ThT and ANS. It turned out that ANS affects the secondary structure of amyloids (shown for fibrils formed from insulin and lysozyme) and their fibers clusterization (valid for lysozyme fibrils), while ThT has no such effects. These results confirm the differences in the mechanisms of these dyes interaction with amyloid fibrils. Observed effect of ANS was explained by the electrostatic interactions between the dye molecule and cationic groups of amyloid-forming proteins (unlike hydrophobic binding of ThT) that induce amyloids conformational changes. This interaction leads to weakening repulsion between positive charges of amyloid fibrils and can promote their clusterization. It was shown that when fibrillogenesis conditions and, consequently, fibrils structure is changing, as well as during defragmentation of amyloids by ultrasonication, the influence of ANS to amyloids does not change, which indicates the universality of the detected effects. Based on the obtained results, it was concluded that ANS should be used cautiously for the study of amyloid fibrils, since this fluorescence probe have a direct effect on the object of study.

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Binding of thioflavin T by albumins: An underestimated role of protein oligomeric heterogeneity

Cited by 31 publications

References 35 publications

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

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

Human Serum Albumin Aggregation/Fibrillation and its Abilities to Drugs Binding

Effect of the fluorescent probes ThT and ANS on the mature amyloid fibrils

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