Stoichiometry and Affinity of Thioflavin T Binding to Sup35p Amyloid Fibrils

• Published in: PloS one 2016-05, Vol.11 (5), p.e0156314-e0156314
• Main Authors: Sulatskaya, Anna I, Kuznetsova, Irina M, Belousov, Mikhail V, Bondarev, Stanislav A, Zhouravleva, Galina A, Turoverov, Konstantin K
• Format: Article
• Language: English
• Subjects: Absorption; Absorption spectra; Affinity; Affinity labeling; Amyloid - chemistry; Amyloid - metabolism; Amyloid beta-protein; Baking yeast; Binding proteins; Binding sites; Biology and Life Sciences; Biotechnology; Cellular biology; Dielectric properties; Dyes; Extinction coefficient; Fibrils; Fluorescence; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; Fluorescent indicators; Genetics; Grooves; Kinetics; Laboratories; Medicine and Health Sciences; Microdialysis; Molecular structure; Nervous system; Peptide Termination Factors - chemistry; Peptide Termination Factors - metabolism; Physical Sciences; Prion protein; Prions; Properties; Protein Binding; Proteins; Research and Analysis Methods; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - metabolism; Science; Spectrometry; Spectrum analysis; Stoichiometry; Thiazoles - chemistry; Thiazoles - metabolism
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0156314

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.