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...

Full description

Bibliographic Details
Main Authors: Anna I. Sulatskaya, Natalia P. Rodina, Maksim I. Sulatsky, Olga I. Povarova, Iuliia A. Antifeeva, Irina M. Kuznetsova, Konstantin K. Turoverov
Format: Article
Language:English
Published: MDPI AG 2018-08-01
Series:International Journal of Molecular Sciences
Subjects:
Online Access:http://www.mdpi.com/1422-0067/19/9/2486
id doaj-a2a5e2dee2824fa0910812b47d411fa3
record_format Article
spelling 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
work_keys_str_mv AT annaisulatskaya investigationofasynucleinamyloidfibrilsusingthefluorescentprobethioflavint
AT nataliaprodina investigationofasynucleinamyloidfibrilsusingthefluorescentprobethioflavint
AT maksimisulatsky investigationofasynucleinamyloidfibrilsusingthefluorescentprobethioflavint
AT olgaipovarova investigationofasynucleinamyloidfibrilsusingthefluorescentprobethioflavint
AT iuliiaaantifeeva investigationofasynucleinamyloidfibrilsusingthefluorescentprobethioflavint
AT irinamkuznetsova investigationofasynucleinamyloidfibrilsusingthefluorescentprobethioflavint
AT konstantinkturoverov investigationofasynucleinamyloidfibrilsusingthefluorescentprobethioflavint
_version_ 1716744893536141312