Polymorph-specific distribution of binding sites determines thioflavin-T fluorescence intensity in α-synuclein fibrils

Arshdeep Sidhu* (Corresponding Author), Jonathan Vaneyck, Christian Blum, Ine Segers-Nolten (Corresponding Author), Vinod Subramaniam

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

5 Citations (Scopus)
46 Downloads (Pure)

Abstract

Thioflavin-T (ThT) is the most commonly used fluorescent dye for following amyloid formation semi-quantitatively in vitro, specifically probing the fibrillar cross-β-sheet content. In recent years, structural polymorphism of amyloid fibrils has been shown to be an important aspect of amyloid formation, both in vitro and in neurodegenerative diseases. Therefore, understanding ThT–amyloid interactions in the context of structural polymorphism of amyloids is necessary for correct interpretation of ThT fluorescence data. Here we study the influence of fibril morphology on ThT fluorescence and ThT binding sites, with two morphologically distinct but chemically identical α-synuclein polymorphs. In ThT fluorescence assays the two polymorphs show type-specific fluorescence intensity behaviour although their β-sheet content has been shown to be similar. Further, fluorescence lifetime measurements of fibril-bound ThT reveal the presence of at least two qualitatively different ThT binding sites on the polymorphs. The relative distributions of the binding sites on the fibril surfaces appear to be morphology dependent, thus determining the observed polymorph-specific ThT fluorescence intensities. These results, highlighting the role of fibril morphology in ThT-based amyloid studies, underline the relevance of polymorphs in ThT–amyloid interaction and can explain the variability often observed in ThT amyloid binding assays.

Original languageEnglish
Pages (from-to)189-196
Number of pages8
JournalAmyloid
Volume25
Issue number3
DOIs
Publication statusPublished - 3 Jul 2018

Fingerprint

Synucleins
Fluorescence
Binding Sites
Amyloid
thioflavin T
Fluorescent Dyes
Neurodegenerative Diseases

Keywords

  • UT-Hybrid-D
  • atomic force microscopy
  • polymorphism
  • thioflavin-T
  • α-synuclein
  • Amyloid

Cite this

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title = "Polymorph-specific distribution of binding sites determines thioflavin-T fluorescence intensity in α-synuclein fibrils",
abstract = "Thioflavin-T (ThT) is the most commonly used fluorescent dye for following amyloid formation semi-quantitatively in vitro, specifically probing the fibrillar cross-β-sheet content. In recent years, structural polymorphism of amyloid fibrils has been shown to be an important aspect of amyloid formation, both in vitro and in neurodegenerative diseases. Therefore, understanding ThT–amyloid interactions in the context of structural polymorphism of amyloids is necessary for correct interpretation of ThT fluorescence data. Here we study the influence of fibril morphology on ThT fluorescence and ThT binding sites, with two morphologically distinct but chemically identical α-synuclein polymorphs. In ThT fluorescence assays the two polymorphs show type-specific fluorescence intensity behaviour although their β-sheet content has been shown to be similar. Further, fluorescence lifetime measurements of fibril-bound ThT reveal the presence of at least two qualitatively different ThT binding sites on the polymorphs. The relative distributions of the binding sites on the fibril surfaces appear to be morphology dependent, thus determining the observed polymorph-specific ThT fluorescence intensities. These results, highlighting the role of fibril morphology in ThT-based amyloid studies, underline the relevance of polymorphs in ThT–amyloid interaction and can explain the variability often observed in ThT amyloid binding assays.",
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author = "Arshdeep Sidhu and Jonathan Vaneyck and Christian Blum and Ine Segers-Nolten and Vinod Subramaniam",
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Polymorph-specific distribution of binding sites determines thioflavin-T fluorescence intensity in α-synuclein fibrils. / Sidhu, Arshdeep (Corresponding Author); Vaneyck, Jonathan; Blum, Christian; Segers-Nolten, Ine (Corresponding Author); Subramaniam, Vinod.

In: Amyloid, Vol. 25, No. 3, 03.07.2018, p. 189-196.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Polymorph-specific distribution of binding sites determines thioflavin-T fluorescence intensity in α-synuclein fibrils

AU - Sidhu, Arshdeep

AU - Vaneyck, Jonathan

AU - Blum, Christian

AU - Segers-Nolten, Ine

AU - Subramaniam, Vinod

N1 - Taylor & Francis deal

PY - 2018/7/3

Y1 - 2018/7/3

N2 - Thioflavin-T (ThT) is the most commonly used fluorescent dye for following amyloid formation semi-quantitatively in vitro, specifically probing the fibrillar cross-β-sheet content. In recent years, structural polymorphism of amyloid fibrils has been shown to be an important aspect of amyloid formation, both in vitro and in neurodegenerative diseases. Therefore, understanding ThT–amyloid interactions in the context of structural polymorphism of amyloids is necessary for correct interpretation of ThT fluorescence data. Here we study the influence of fibril morphology on ThT fluorescence and ThT binding sites, with two morphologically distinct but chemically identical α-synuclein polymorphs. In ThT fluorescence assays the two polymorphs show type-specific fluorescence intensity behaviour although their β-sheet content has been shown to be similar. Further, fluorescence lifetime measurements of fibril-bound ThT reveal the presence of at least two qualitatively different ThT binding sites on the polymorphs. The relative distributions of the binding sites on the fibril surfaces appear to be morphology dependent, thus determining the observed polymorph-specific ThT fluorescence intensities. These results, highlighting the role of fibril morphology in ThT-based amyloid studies, underline the relevance of polymorphs in ThT–amyloid interaction and can explain the variability often observed in ThT amyloid binding assays.

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KW - UT-Hybrid-D

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