title: Stoichiometry and Affinity of Thioflavin T Binding to Sup35p Amyloid Fibrils.
abstract: In this work two modes of binding of the fluorescent probe thioflavin T to yeast prion protein Sup35p amyloid fibrils were revealed by absorption spectrometry of solutions prepared by equilibrium microdialysis. These binding modes exhibited significant differences in binding affinity and stoichiometry. Moreover, the absorption spectrum and the molar extinction coefficient of the dye bound in each mode were determined. The fluorescence quantum yield of the dye bound in each mode was determined via a spectrofluorimetric study of the same solutions in which the recorded fluorescence intensity was corrected for the primary inner filter effect. As previously predicted, the existence of one of the detected binding modes may be due to the incorporation of the dye into the grooves along the fiber axis perpendicular to the β-sheets of the fibrils. It was assumed that the second type of binding with higher affinity may be due to the existence of ThT binding sites that are localized to areas where amyloid fibrils are clustered.
authors.affiliation: Laboratory for Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Science, St. Petersburg, Tikhoretsky Ave. 4, 194064, Russia.
authors.forename: Anna I
authors.initials: AI
authors.lastname: Sulatskaya
authors.affiliation: Laboratory for Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Science, St. Petersburg, Tikhoretsky Ave. 4, 194064, Russia.
authors.forename: Irina M
authors.initials: IM
authors.lastname: Kuznetsova
authors.affiliation: Department of Genetics and Biotechnology, Saint Petersburg State University, Saint Petersburg, Universitetskaya Emb. 7-9, 199034, Russia.
authors.forename: Mikhail V
authors.initials: MV
authors.lastname: Belousov
authors.affiliation: Department of Genetics and Biotechnology, Saint Petersburg State University, Saint Petersburg, Universitetskaya Emb. 7-9, 199034, Russia.|Laboratory of Amyloid Biology, Saint Petersburg State University, Saint Petersburg, 199034, Russia.
authors.forename: Stanislav A
authors.initials: SA
authors.lastname: Bondarev
authors.affiliation: Department of Genetics and Biotechnology, Saint Petersburg State University, Saint Petersburg, Universitetskaya Emb. 7-9, 199034, Russia.|Laboratory of Amyloid Biology, Saint Petersburg State University, Saint Petersburg, 199034, Russia.
authors.forename: Galina A
authors.initials: GA
authors.lastname: Zhouravleva
authors.affiliation: Laboratory for Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Science, St. Petersburg, Tikhoretsky Ave. 4, 194064, Russia.|Institute of Physics, Nanotechnology and Telecommunications, Peter the Great St.-Petersburg Polytechnic University, St. Petersburg, Polytechnicheskaya 29, 195251, Russia.
authors.forename: Konstantin K
authors.initials: KK
authors.lastname: Turoverov
mesh_terms: D000682:Amyloid / Q000737:chemistry / Q000378:metabolism*; D052160:Benzothiazoles; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D010454:Peptide Termination Factors / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000682:Amyloid; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D010454:Peptide Termination Factors; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013844:Thiazoles; C009462:thioflavin T