Investigation of α-Synuclein Amyloid Fibrils Using the Fluorescent Probe Thioflavin T

International Journal of Molecular Sciences
Anna I SulatskayaKonstantin K Turoverov

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 c...Continue Reading

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Citations

Feb 28, 2020·Chemical Communications : Chem Comm·Zsofia Lengyel-ZhandRobert H Mach
Mar 18, 2020·Microscopy Research and Technique·Giuseppe SancataldoValeria Vetri
May 30, 2020·The Journal of Biological Chemistry·Eliana NachmanCarmen Nussbaum-Krammer
Aug 5, 2020·Journal of Neurochemistry·Narayanaperumal PravinMaheswaran Shanmugam
Oct 22, 2020·International Journal of Molecular Sciences·Olga V StepanenkoA I Sulatskaya
Oct 25, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·María Florencia PignataroVerónica Isabel Dodero
Oct 4, 2019·The Journal of Physical Chemistry. B·Mantas Ziaunys, Vytautas Smirnovas
Nov 30, 2019·Journal of the American Chemical Society·Patricia BondiaCristina Flors
Mar 4, 2019··Elena N. VelichkoElina K. Nepomnyashchaya

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Methods Mentioned

BETA
electron microscopy
fluorescence spectroscopy
fluorescence
equilibrium microdialysis
light scattering
confocal microscopy

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