Investigation of α-Synuclein Amyloid Fibrils Using the Fluorescent Probe Thioflavin T
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 num...
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doaj-a2a5e2dee2824fa0910812b47d411fa32020-11-24T21:15:33ZengMDPI AGInternational Journal of Molecular Sciences1422-00672018-08-01199248610.3390/ijms19092486ijms19092486Investigation of α-Synuclein Amyloid Fibrils Using the Fluorescent Probe Thioflavin TAnna I. Sulatskaya0Natalia P. Rodina1Maksim I. Sulatsky2Olga I. Povarova3Iuliia A. Antifeeva4Irina M. Kuznetsova5Konstantin K. Turoverov6Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology of the Russian Academy of Science, Tikhoretsky ave. 4, 194064 St. Petersburg, RussiaLaboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology of the Russian Academy of Science, Tikhoretsky ave. 4, 194064 St. Petersburg, RussiaLaboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology of the Russian Academy of Science, Tikhoretsky ave. 4, 194064 St. Petersburg, RussiaLaboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology of the Russian Academy of Science, Tikhoretsky ave. 4, 194064 St. Petersburg, RussiaLaboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology of the Russian Academy of Science, Tikhoretsky ave. 4, 194064 St. Petersburg, RussiaLaboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology of the Russian Academy of Science, Tikhoretsky ave. 4, 194064 St. Petersburg, RussiaLaboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology of the Russian Academy of Science, Tikhoretsky ave. 4, 194064 St. Petersburg, RussiaIn 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.http://www.mdpi.com/1422-0067/19/9/2486α-synucleinamyloid fibrilsfibrillogenesisthioflavin Tequilibrium microdialysisbinding parametersstructural polymorphism |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Anna I. Sulatskaya Natalia P. Rodina Maksim I. Sulatsky Olga I. Povarova Iuliia A. Antifeeva Irina M. Kuznetsova Konstantin K. Turoverov |
spellingShingle |
Anna I. Sulatskaya Natalia P. Rodina Maksim I. Sulatsky Olga I. Povarova Iuliia A. Antifeeva Irina M. Kuznetsova Konstantin K. Turoverov Investigation of α-Synuclein Amyloid Fibrils Using the Fluorescent Probe Thioflavin T International Journal of Molecular Sciences α-synuclein amyloid fibrils fibrillogenesis thioflavin T equilibrium microdialysis binding parameters structural polymorphism |
author_facet |
Anna I. Sulatskaya Natalia P. Rodina Maksim I. Sulatsky Olga I. Povarova Iuliia A. Antifeeva Irina M. Kuznetsova Konstantin K. Turoverov |
author_sort |
Anna I. Sulatskaya |
title |
Investigation of α-Synuclein Amyloid Fibrils Using the Fluorescent Probe Thioflavin T |
title_short |
Investigation of α-Synuclein Amyloid Fibrils Using the Fluorescent Probe Thioflavin T |
title_full |
Investigation of α-Synuclein Amyloid Fibrils Using the Fluorescent Probe Thioflavin T |
title_fullStr |
Investigation of α-Synuclein Amyloid Fibrils Using the Fluorescent Probe Thioflavin T |
title_full_unstemmed |
Investigation of α-Synuclein Amyloid Fibrils Using the Fluorescent Probe Thioflavin T |
title_sort |
investigation of α-synuclein amyloid fibrils using the fluorescent probe thioflavin t |
publisher |
MDPI AG |
series |
International Journal of Molecular Sciences |
issn |
1422-0067 |
publishDate |
2018-08-01 |
description |
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. |
topic |
α-synuclein amyloid fibrils fibrillogenesis thioflavin T equilibrium microdialysis binding parameters structural polymorphism |
url |
http://www.mdpi.com/1422-0067/19/9/2486 |
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