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

Iu A Antifeeva,K K Turoverov,A I Sulatskaya,I M Kuznetsova,O I Povarova,M I Sulatsky

doi:10.1080/19336896.2020.1720487

Iu A Antifeeva, K K Turoverov + Show 4 more

Open Access

https://doi.org/10.1080/19336896.2020.1720487

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Journal: Prion	Publication Date: Jan 1, 2020
Citations: 47	License type: open-access

Affiliation: Peter the Great St. Petersburg Polytechnic University

Abstract

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.

Highlights

The formation of ordered protein aggregates, amyloid fibrils, is associated with serious human diseases, such as Alzheimer’s, Parkinson’s, prion diseases, etc [1,2,3,4,5,6,7]
The benzothiazole dye thioflavin T (ThT) is the ‘gold standard’ for amyloid investigation [16,17,18,19,20,21]. This is due to a significant increase in fluorescence intensity and fluorescence lifetime of the dye accompanying its binding to amyloid fibrils in comparison to that of free ThT in aqueous solution which is caused by the molecular rotor nature of this dye [22,23]
Amyloid fibrils formed from insulin and lysozyme under the same conditions have differences in the secondary structure and tendency to clasterization

Summary

Introduction

The formation of ordered protein aggregates, amyloid fibrils, is associated with serious human diseases, such as Alzheimer’s, Parkinson’s, prion diseases, etc [1,2,3,4,5,6,7]. A wide range of physicochemical methods is used to study amyloid fibrils [13,14,15] One of these approaches is the use of fluorescent probes that change their photophysical characteristics during its interaction with amyloid fibrils. Such probes, in particular, are thioflavin T (ThT) and 1-anilino-8-naphthalene sulfonate (ANS). 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]

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Conclusion