Investigation of α-synuclein amyloid fibrils using the fluorescent probe thioflavin T

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Abstract

In this work, α-synuclein amyloid fibrils—the formation of which is a biomarker of Parkinson’s disease—were investigated using the fluorescent probe thioflavin T (ThT). The experimental conditions of protein fibrillogenesis were chosen so that a sufficient number of continuous measurements could be performed to characterize and analyze all stages of this process. The reproducibility of fibrillogenesis and the structure of the obtained aggregates (which is a critical point for further investigation) were proven using a wide range of physical-chemical methods. For the determination of ThT-α-synuclein amyloid fibril binding parameters, the sample and reference solutions were prepared using equilibrium microdialysis. By utilizing absorption spectroscopy of these solutions, the ThT-fibrils binding mode with a binding constant of about 104 M−1 and stoichiometry of ThT per protein molecule of about 1:8 was observed. Fluorescence spectroscopy of the same solutions with the subsequent correction of the recorded fluorescence intensity on the primary inner filter effect allowed us to determine another mode of ThT binding to fibrils, with a binding constant of about 106 M−1 and stoichiometry of about 1:2500. Analysis of the photophysical characteristics of the dye molecules bound to the sites of different binding modes allowed us to assume the possible localization of these sites. The obtained differences in the ThT binding parameters to the amyloid fibrils formed from α-synuclein and other amyloidogenic proteins, as well as in the photophysical characteristics of the bound dye, confirmed the hypothesis of amyloid fibril polymorphism.

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Sulatskaya, A. I., Rodina, N. P., Sulatsky, M. I., Povarova, O. I., Antifeeva, I. A., Kuznetsova, I. M., & Turoverov, K. K. (2018). Investigation of α-synuclein amyloid fibrils using the fluorescent probe thioflavin T. International Journal of Molecular Sciences, 19(9). https://doi.org/10.3390/ijms19092486

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