Sulatskaya, A.I.; Rodina, N.P.; Sulatsky, M.I.; Povarova, O.I.; Antifeeva, I.A.; Kuznetsova, I.M.; Turoverov, K.K. Investigation of α-Synuclein Amyloid Fibrils Using the Fluorescent Probe Thioflavin T. Int. J. Mol. Sci.2018, 19, 2486.
Sulatskaya, A.I.; Rodina, N.P.; Sulatsky, M.I.; Povarova, O.I.; Antifeeva, I.A.; Kuznetsova, I.M.; Turoverov, K.K. Investigation of α-Synuclein Amyloid Fibrils Using the Fluorescent Probe Thioflavin T. Int. J. Mol. Sci. 2018, 19, 2486.
In this work α-synuclein amyloid fibrils, formation of which is a biomarker of the Parkinson’s disease, were investigated with the use of fluorescent probe thioflavin T (ThT). Experimental conditions of the protein fibrillogenesis were chosen so that a sufficient number of continuous measurements can be performed to characterize and analyze all stages of this process. The reproducibility of fibrillogenesis and the structure of the obtained aggregates (that is a critical point for their further investigation) were proved using a wide range of physical-chemical methods. For determination of ThT—α-synuclein amyloid fibrils binding parameters sample and reference solutions were prepared with the use of equilibrium microdialysis. By absorption spectroscopy of these solutions ThT—fibrils binding mode with the binding constant about 104 M−1 and stoichiometry of ThT per protein molecule 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 to determine another mode of ThT binding to fibrils with the binding constant about 106 M−1 and stoichiometry about 1:2500. Analysis of photophysical characteristics of the dye molecules bound to the sites of different binding modes allowed to assume the possible localization of these sites. Obtained differences in the ThT binding parameters to 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 fibrils polymorphism.
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.