title: Steady-state and time-resolved Thioflavin-T fluorescence can report on morphological differences in amyloid fibrils formed by Aβ(1-40) and Aβ(1-42).
abstract: Thioflavin-T (ThT) is one of the most commonly used dyes for amyloid detection, but the origin of its fluorescence enhancement is not fully understood. Herein we have characterised the ThT fluorescence response upon binding to the Aβ(1-40) and Aβ(1-42) variants of the Alzheimer's-related peptide amyloid-β, in order to explore how the photophysical properties of this dye relates to structural and morphological properties of two amyloid fibril types formed by peptides with a high degree of sequence homology. We show that the steady-state ThT fluorescence is 1.7 times more intense with Aβ(1-40) compared to Aβ(1-42) fibrils in concentration matched samples prepared under quiescent conditions. By measuring the excited state lifetime of bound ThT, we also demonstrate a distinct difference between the two fibril isoforms, with Aβ(1-42) fibrils producing a longer ThT fluorescence lifetime compared to Aβ(1-40). The substantial steady-state intensity difference is therefore not explained by differences in fluorescence quantum yield. Further, we find that the ThT fluorescence intensity, but not the fluorescence lifetime, is dependent on the fibril preparation method (quiescent versus agitated conditions). We therefore propose that the fluorescence lifetime is inherent to each isoform and sensitively reports on fibril microstructure in the protofilament whereas the total fluorescence intensity relates to the amount of exposed β-sheet in the mature Aβ fibrils and hence to differences in their morphology. Our results highlight the complexity of ThT fluorescence, and demonstrate its extended use in amyloid fibril characterisation.
authors: Department of Biology and Biological Engineering, Division of Chemical Biology, Chalmers University of Technology, Kemivägen 10, SE-41296 Gothenburg, Sweden.
authors: David J
authors: DJ
authors: Lindberg
authors: Department of Chemistry and Chemical Engineering, Division of Physical Chemistry, Chalmers University of Technology, Kemivägen 10, SE-41296 Gothenburg, Sweden.
authors: Moa S
authors: MS
authors: Wranne
authors: Department of Chemistry and Chemical Engineering, Division of Physical Chemistry, Chalmers University of Technology, Kemivägen 10, SE-41296 Gothenburg, Sweden.
authors: Mélina
authors: M
authors: Gilbert Gatty
authors: Department of Biology and Biological Engineering, Division of Chemical Biology, Chalmers University of Technology, Kemivägen 10, SE-41296 Gothenburg, Sweden.
authors: Fredrik
authors: F
authors: Westerlund
authors: Department of Biology and Biological Engineering, Division of Chemical Biology, Chalmers University of Technology, Kemivägen 10, SE-41296 Gothenburg, Sweden. Electronic address: eline@chalmers.se.
authors: Elin K
authors: EK
authors: Esbjörner
mesh_terms: D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry* / Q000648:ultrastructure*; D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000648:ultrastructure*; D052160:Benzothiazoles; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000648:ultrastructure*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013844:Thiazoles / Q000737:chemistry*
chemical_list: D000682:Amyloid; D016229:Amyloid beta-Peptides; D052160:Benzothiazoles; D010446:Peptide Fragments; D013844:Thiazoles; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42); C009462:thioflavin T
keywords: Alzheimer's disease; Amyloid dye; Amyloid fibrils; Amyloid-β; Fluorescence lifetime; Thioflavin-T