ThT 101: a primer on the use of thioflavin T to investigate amyloid formation

Malmos, Kirsten Gade; Blancas‐Mejia, Luis M.; Weber, Benedikt; Büchner, Johannes; Ramı́rez-Alvarado, Marina; Naiki, Hironobu; Otzen, Daniel E.

doi:10.1080/13506129.2017.1304905

Cited by 287 publications

(256 citation statements)

References 125 publications

(165 reference statements)

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“…Briefly, each protein was desalted from a stock solution containing 8 M urea (which keeps the protein monomeric under storage conditions) into buffer and diluted to a final concentration of 0.2 mg/mL. Samples were incubated with shaking in a plate reader at 37 °C, and thioflavin T (ThT), a compound known to fluoresce in the presence of β-sheet-rich amyloid fibrils [28], enabled monitoring of protein fibril formation in real time. Conversion from random coil to β-sheet secondary structure was verified by circular dichroism spectroscopy (CD) (SI Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Note that it was not uncommon to see a decay in ThT signal intensity from the plateau reached after the transition region; this is frequently observed for fibrillating proteins, and while no definitive explanation for its occurrence has been provided, it may be caused by slow isomerization of ThT or bundling of the initially formed fibrils rather than reflecting conformational transitions at the level of the individual molecule. Therefore, we like others chose to disregard it in our analysis of the data [28]. Overall, the most significant changes resulted from GVNIAA→KFDDTK mutation in R3.…”

Section: Resultsmentioning

confidence: 99%

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Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms

Bleem

Christiansen

Madsen

et al. 2018

Journal of Molecular Biology

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Amyloids are typically associated with neurodegenerative diseases, but recent research demonstrates that several bacteria utilize functional amyloid fibrils to fortify the biofilm extracellular matrix and thereby resist antibiotic treatments. In Pseudomonas aeruginosa, these fibrils are composed predominantly of FapC, a protein with high-sequence conservation among the genera. Previous studies established FapC as the major amyloid subunit, but its mechanism of fibril formation in P. aeruginosa remained largely unexplored. Here, we examine the FapC sequence in greater detail through a combination of bioinformatics and protein engineering, and we identify specific motifs that are implicated in amyloid formation. Sequence regions of high evolutionary conservation tend to coincide with regions of high amyloid propensity, and mutation of amyloidogenic motifs to a designed, non-amyloidogenic motif suppresses fibril formation in a pH-dependent manner. We establish the particular significance of the third repeat motif in promoting fibril formation and also demonstrate emergence of soluble oligomer species early in the aggregation pathway. The insights reported here expand our understanding of the mechanism of amyloid polymerization in P. aeruginosa, laying the foundation for development of new amyloid inhibitors to combat recalcitrant biofilm infections.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms

Bleem

Christiansen

Madsen

et al. 2018

Journal of Molecular Biology

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“…The most commonly used approach to investigate chaperone activity are assays that monitor the aggregation of proteins in vitro, either via light scatter or, in the case of amyloid fibril formation, fluorescent dyes such as Thioflavin T 41 . Indeed, we demonstrate via light scattering assay that Bc is able to effectively inhibit the heat-induced aggregation of CLIC1.…”

Section: Discussionmentioning

confidence: 99%

Single-molecule fluorescence-based approach reveals novel mechanistic insights into small heat shock protein chaperone function

Johnston

Marzano

Paudel

et al. 2020

Preprint

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Small heat shock proteins (sHsps) are a family of ubiquitous intracellular molecular chaperones that are up-regulated under stress conditions and play a vital role in protein homeostasis (proteostasis). It is commonly accepted that these chaperones work by trapping misfolded proteins to prevent their aggregation, however fundamental questions regarding the molecular mechanism by which sHsps interact with misfolded proteins remain unanswered. Traditionally, it has been difficult to study sHsp function due to the dynamic and heterogenous nature of the species formed between sHsps and aggregation-prone proteins. Single-molecule techniques have emerged as a powerful tool to study dynamic protein complexes and we have therefore developed a novel single-molecule fluorescence-based approach to observe the chaperone action of human B-crystallin (Bc, HSPB5). Using this approach we have, for the first time, determined the stoichiometries of complexes formed between Bc and a model client protein, chloride intracellular channel 1 (CLIC1). By examining the polydispersity and stoichiometries of these complexes over time, and in response to different concentrations of Bc, we have uncovered unique and important insights into a two-step mechanism by which Bc interacts with misfolded client proteins to prevent their aggregation. Understanding this fundamental mechanism of sHsp action is crucial to understanding how these molecular chaperone function to protect the cell from protein misfolding and their overall role in the cellular proteostasis network.2 Introduction:

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“…ThT binds equally well to amyloid fibrils and cross β-sheet-rich structures of biological (proteins) and synthetic origin (peptides) [17, 18], displaying a characteristic red shift in the emission spectrum [15, 17, 19], and an enhanced fluorescence [20] when bonded, which makes it a cheap, fast, and reliable amyloid probe [15, 21, 22]. …”

Section: Methodsmentioning

confidence: 99%

Assays for Light Chain Amyloidosis Formation and Cytotoxicity

Blancas‐Mejia

Misra

Dick

et al. 2018

Methods in Molecular Biology

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Common biophysical techniques like absorption and fluorescence spectroscopy, microscopy, and light scattering studies have been in use to investigate fibril assembly for a long time. However, there is sometimes a lack of consensus from the findings of an individual technique when compared in parallel with the other techniques. In this chapter, we aim to provide a concise compilation of techniques that can effectively be used to obtain a comprehensive representation of the structural, aggregation, and toxicity determinants in immunoglobulin light chain amyloidosis. We start by giving a brief introduction on amyloid assembly and the advantages of using simple and readily available techniques to study aggregation. After an overview on preparation of protein to set up parallel experiments, we provide a systematic description of the in vitro techniques used to study aggregation in AL protein. Additionally, we thoroughly discuss the steps needed in our experience during the individual experiments for better reproducibility and data analysis.

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ThT 101: a primer on the use of thioflavin T to investigate amyloid formation

Cited by 287 publications

References 125 publications

Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms

Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms

Single-molecule fluorescence-based approach reveals novel mechanistic insights into small heat shock protein chaperone function

Assays for Light Chain Amyloidosis Formation and Cytotoxicity

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