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

Abstract

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.

Trypsin Induced Degradation of Amyloid Fibrils

“…According to the literature, ThT incorporates into the grooves formed by the side chains of amino acids of the amyloid fibril backbone along the fiber axis perpendicular to the beta-sheets [ 59 ], and ANS is a hydrophobic probe interacting with protein associates [ 45 , 46 ]. Thus, the dyes are likely to bind to different sites of amyloids, which is in line with the results of the work [ 48 ]. We believe that it explains the difference of kinetic dependences of fibril degradation monitored by ANS and ThT fluorescence.…”

“…In order to reveal the structural changes of the studied lysozyme amyloid fibrils induced by trypsin, we analyzed the results obtained using various spectroscopic methods, such as intrinsic UV fluorescence of proteins and CD spectroscopy of fibrils in the far UV region, and also an approach based on the analysis of the interaction of amyloid fibrils with fluorescent probes. The amyloid-specific dye thioflavin T (ThT) [ 42 , 43 , 44 ] and the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) [ 45 , 46 , 47 ] were used, which are widely known probes to detect the formation of amyloid fibrils and study their structure and stability [ 48 , 49 , 50 , 51 ].…”

Amyloidogenic proteins in the SARS-CoV and SARS-CoV-2 proteomes

“…After the computational prediction of APRs in the SARS-CoV and SARS-CoV-2 proteomes, we investigated the in vitro aggregation behaviour of various proteins and peptides. For this purpose, we selected pH 7.4 and temperature 37 °C, and traced the aggregation process using the fluorescent dye thioflavin T (ThT), which interacts with amyloid fibrils and gives a maximum emission peak at ~490 nm upon binding to β-sheets in amyloid fibrils [27][28]. As protein aggregation has been shown to occur via a nucleation–polymerization mechanism [29,30], we studied this reaction using ThT fluorescence ( λ max at 490 nm) in presence of a fixed volume of incubated samples (25 μM).…”

Alpha-B-Crystallin Effect on Mature Amyloid Fibrils: Different Degradation Mechanisms and Changes in Cytotoxicity

“…The degradation process of amyloid fibrils was also confirmed by a decrease in the fluorescence intensity of the amyloid-specific dye thioflavin T (ThT) ( Figure 2 G,O), which is widely used to diagnose the occurrence and study the structure of amyloid fibrils [ 56 , 57 , 58 , 59 ]. Conversely, the fluorescence intensity of the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid, ANS, which integrates into hydrophobic cavities between protein associates forming large aggregates [ 60 , 61 , 62 ], as well as interacting with amyloid fibrils (probably due to electrostatic interactions) [ 63 ], and can be used to analyze of their degradation [ 64 ]), increased, which may indicate an increase in the number/size of protein aggregates in the sample despite the degradation of amyloid fibrils ( Figure 2 H,P). In can be noted that the sample contains alpha-B-crystallin in both bound to amyloids and free states.…”