scholarly journals Thioflavin T as an amyloid dye: fibril quantification, optimal concentration and effect on aggregation

2017 ◽  
Vol 4 (1) ◽  
pp. 160696 ◽  
Author(s):  
Christine Xue ◽  
Tiffany Yuwen Lin ◽  
Dennis Chang ◽  
Zhefeng Guo
Keyword(s):  
Amyloid Fibrils ◽  
Prion Diseases ◽  
Thioflavin T ◽  
Aggregation Kinetics ◽  
Fluorescence Signal ◽  
Amyloid Formation ◽  
Yeast Prion ◽  
Tht Fluorescence ◽  
Wide Range ◽  
Human Disorders

Formation of amyloid fibrils underlies a wide range of human disorders, including Alzheimer's and prion diseases. The amyloid fibrils can be readily detected thanks to thioflavin T (ThT), a small molecule that gives strong fluorescence upon binding to amyloids. Using the amyloid fibrils of Aβ40 and Aβ42 involved in Alzheimer's disease, and of yeast prion protein Ure2, here we study three aspects of ThT binding to amyloids: quantification of amyloid fibrils using ThT, the optimal ThT concentration for monitoring amyloid formation and the effect of ThT on aggregation kinetics. We show that ThT fluorescence correlates linearly with amyloid concentration over ThT concentrations ranging from 0.2 to 500 µM. At a given amyloid concentration, the plot of ThT fluorescence versus ThT concentration exhibits a bell-shaped curve. The maximal fluorescence signal depends mostly on the total ThT concentration, rather than amyloid to ThT ratio. For the three proteins investigated, the maximal fluorescence is observed at ThT concentrations of 20–50 µM. Aggregation kinetics experiments in the presence of different ThT concentrations show that ThT has little effect on aggregation at concentrations of 20 µM or lower. ThT at concentrations of 50 µM or more could affect the shape of the aggregation curves, but this effect is protein-dependent and not universal.

scholarly journals Effect of spin labelling on the aggregation kinetics of yeast prion protein Ure2

2021 ◽  
Vol 8 (3) ◽  
Author(s):  
Emilie N. Liu ◽  
Giovanna Park ◽  
Junsuke Nohara ◽  
Zhefeng Guo
Keyword(s):  
Protein Aggregation ◽  
Amyloid Fibrils ◽  
Prion Diseases ◽  
Aggregation Kinetics ◽  
Amyloid Formation ◽  
Yeast Prion ◽  
Aggregation Rate ◽  
Rate Limiting Step ◽  
Wide Range ◽  
Spin Labelling

Amyloid formation is involved in a wide range of neurodegenerative diseases including Alzheimer's and prion diseases. Structural understanding of the amyloid is critical to delineate the mechanism of aggregation and its pathological spreading. Site-directed spin labelling has emerged as a powerful structural tool in the studies of amyloid structures and provided structural evidence for the parallel in-register β-sheet structure for a wide range of amyloid proteins. It is generally accepted that spin labelling does not disrupt the structure of the amyloid fibrils, the end product of protein aggregation. The effect on the rate of protein aggregation, however, has not been well characterized. Here, we employed a scanning mutagenesis approach to study the effect of spin labelling on the aggregation rate of 79 spin-labelled variants of the Ure2 prion domain. The aggregation of Ure2 protein is the basis of yeast prion [URE3]. We found that all spin-labelled Ure2 mutants aggregated within the experimental timeframe of 15 to 40 h. Among the 79 spin-labelled positions, only five residue sites (N23, N27, S33, I35 and G42) showed a dramatic delay in the aggregation rate as a result of spin labelling. These positions may be important for fibril nucleation, a rate-limiting step in aggregation. Importantly, spin labelling at most of the sites had a muted effect on Ure2 aggregation kinetics, showing a general tolerance of spin labelling in protein aggregation studies.

Enzyme-Mediated Self-Assembly of Highly Ordered Structures From Disordered Proteins

2008 ◽  
Author(s):  
Ahmad Athamneh ◽  
Justin Barone
Keyword(s):  
Self Assembly ◽  
Amyloid Fibrils ◽  
Prion Diseases ◽  
Wheat Gluten ◽  
Disordered Proteins ◽  
X Ray Diffraction ◽  
Ordered Structures ◽  
Wide Range ◽  
Trypsin Hydrolysis ◽  
Hydrolysis Of

Trypsin hydrolysis of wheat gluten produced glutamine-rich short peptides with a tendency to self-assemble into supermolecular structures extrinsic to native wheat gluten. Fourier transform infrared and X-ray diffraction data suggested that the new structures formed resembled that of cross-β amyloid fibrils found in some insect silk and implicated in prion diseases. The superstructures were about 10 μm in diameter with clear right-handed helical configuration and appeared to be bundles of smaller fibrils of about 63 nm in diameter. Results demonstrate the potential for utilizing cheap protein sources and natural mechanisms of protein self-assembly to design advanced nanomaterials that can provide a wide range of structural and chemical functionality.

scholarly journals The many faces of amyloid: Protein misfolding: failure or function?

2011 ◽  
Vol 33 (5) ◽  
pp. 6-9
Author(s):  
Elizabeth B. Sawyer ◽  
Sarah Perrett
Keyword(s):  
Protein Misfolding ◽  
Amyloid Fibrils ◽  
Prion Diseases ◽  
Three Dimensional ◽  
Epigenetic Inheritance ◽  
Dimensional Structure ◽  
Spongiform Encephalopathy ◽  
Wide Range ◽  
The Many ◽  
And Function

The ability of proteins to recognize, bind and manipulate a wide range of other molecules lies at the heart of virtually every cellular process. In order to achieve this, proteins must fold into a precise three-dimensional structure. A failure to achieve this structure, and the associated loss of protein stability and function, results in diseases such as muscular dystrophy and cystic fibrosis. In addition, the misfolding and aggregation of proteins to form fibrillar species is associated with the progression of amyloid diseases such as Alzheimer's and Huntington's and prion diseases including Creutzfeldt– Jakob disease and bovine spongiform encephalopathy (BSE, or ‘mad cow disease’). In this article, we consider advances in the study of protein folding and misfolding and their relevance to biological function. We also explore the issue of protein ‘misfolding’ to form functional aggregated structures, such as the mode of epigenetic inheritance mediated by fungal prions and the formation of amyloid fibrils with positive biological functions in bacteria.

scholarly journals Codon 129 polymorphism of the human prion protein influences the kinetics of amyloid formation

2006 ◽  
Vol 87 (8) ◽  
pp. 2443-2449 ◽  
Author(s):  
Patrick A. Lewis ◽  
M. Howard Tattum ◽  
Samantha Jones ◽  
Daljit Bhelt ◽  
Mark Batchelor ◽  
...  
Keyword(s):  
Prion Protein ◽  
Amyloid Fibrils ◽  
Prion Diseases ◽  
Amyloid Formation ◽  
Native Structure ◽  
Prion Strain ◽  
Profound Influence ◽  
Human Prion Protein ◽  
Kinetics Of ◽  
Codon 129

The human prion protein (PrP) has a common polymorphism at residue 129, which can be valine or methionine. This polymorphism has a strong influence on susceptibility to prion diseases and on prion-strain properties. Previous work has shown that this amino acid variation has no measurable effect on the native structure of cellular PrP (PrPC). Here, it is shown that the polymorphism does not change the efficiency of conversion to the β-PrP conformation or affect the binding of copper(II) ions. However, in a partially denatured conformation, the polymorphic variation has a profound influence on the ability of the protein to form amyloid fibrils spontaneously.

scholarly journals Kinetic diversity of amyloid oligomers

2020 ◽  
Vol 117 (22) ◽  
pp. 12087-12094 ◽  
Author(s):  
Alexander J. Dear ◽  
Thomas C. T. Michaels ◽  
Georg Meisl ◽  
David Klenerman ◽  
Si Wu ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Mechanistic Models ◽  
Cellular Toxicity ◽  
Parkinson’S Diseases ◽  
Wide Range ◽  
Amyloid Oligomers ◽  
Human Disorders ◽  
Physicochemical Features ◽  
Pathogenic Agents ◽  
The Relationship

The spontaneous assembly of proteins into amyloid fibrils is a phenomenon central to many increasingly common and currently incurable human disorders, including Alzheimer’s and Parkinson’s diseases. Oligomeric species form transiently during this process and not only act as essential intermediates in the assembly of new filaments but also represent major pathogenic agents in these diseases. While amyloid fibrils possess a common, defining set of physicochemical features, oligomers, by contrast, appear much more diverse, and their commonalities and differences have hitherto remained largely unexplored. Here, we use the framework of chemical kinetics to investigate their dynamical properties. By fitting experimental data for several unrelated amyloidogenic systems to newly derived mechanistic models, we find that oligomers present with a remarkably wide range of kinetic and thermodynamic stabilities but that they possess two properties that are generic: they are overwhelmingly nonfibrillar, and they predominantly dissociate back to monomers rather than maturing into fibrillar species. These discoveries change our understanding of the relationship between amyloid oligomers and amyloid fibrils and have important implications for the nature of their cellular toxicity.

scholarly journals The Fluorescent Dye 1,6-Diphenyl-1,3,5-Hexatriene Binds to Amyloid Fibrils Formed by Human Amylin and Provides a New Probe of Amylin Amyloid Kinetics

2021 ◽  
Author(s):  
Ming-Hao Li ◽  
Lakshan Manathunga ◽  
Erwin London ◽  
Daniel Raleigh
Keyword(s):  
Time Course ◽  
Amyloid Fibrils ◽  
Fold Increase ◽  
Fluorescent Dye ◽  
Thioflavin T ◽  
Lag Phase ◽  
Amyloid Formation ◽  
Islet Amyloid ◽  
Human Amylin ◽  
Mutant Forms

The fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) is widely used as a probe of membrane order. We show that DPH also interacts with amyloid fibrils formed by human amylin (also known as islet amyloid polypeptide) in solution and this results in a 100-fold increase in DPH fluorescence for a sample of microM human amylin and 0.25 microM DPH. No increase in DPH fluorescence is observed with the non-amyloidogenic rat amylin or with freshly dissolved, non-fibrillar human amylin. The time course of amyloid formation by amylin was followed by monitoring the fluorescence of added DPH as a function of time and was similar to that monitored by the standard fluorescent probe thioflavin-T. The inclusion of DPH in the buffer did not perturb the time course of amyloid formation under the conditions examined and the time course was independent of the range of DPH concentrations tested (0.25 to 5 microM). Maximum final fluorescence intensity is observed at substoichiometric ratios of DPH to amylin. No significant increase in fluorescence was observed during the lag phase of amyloid formation, and the implications for the structure of amylin pre-fibril oligomers are discussed. Human amylin contains three aromatic residues. A triple aromatic to leucine mutant forms amyloid and DPH binds to the resulting fibrils, indicating that interactions with aromatic side chains are not required for DPH amylin amyloid interactions. DPH may be especially useful for studies on mutant amylins and other polypeptides in which changes in charged residues might complicate interpretation of thioflavin-T fluorescence.

scholarly journals Familial Alzheimer's Disease Mutations within the Amyloid Precursor Protein Alter the Aggregation and Conformation of the Amyloid-β Peptide

2017 ◽  
Vol 292 (8) ◽  
pp. 3172-3185 ◽  
Author(s):  
Asa Hatami ◽  
Sanaz Monjazeb ◽  
Saskia Milton ◽  
Charles G. Glabe
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Monoclonal Antibodies ◽  
Amyloid Precursor Protein ◽  
Amyloid Β ◽  
Thioflavin T ◽  
Precursor Protein ◽  
Aggregation Kinetics ◽  
Wide Range ◽  
Amyloid Structures

Most cases of Alzheimer's disease (AD) are sporadic, but a small percentage of AD cases, called familial AD (FAD), are associated with mutations in presenilin 1, presenilin 2, or the amyloid precursor protein. Amyloid precursor protein mutations falling within the amyloid-β (Aβ) sequence lead to a wide range of disease phenotypes. There is increasing evidence that distinct amyloid structures distinguished by amyloid conformation-dependent monoclonal antibodies have similarly distinct roles in pathology. It is possible that this phenotypic diversity of FAD associated with mutations within the Aβ sequence is due to differences in the conformations adopted by mutant Aβ peptides, but the effects of FAD mutations on aggregation kinetics and conformational and morphological changes of the Aβ peptide are poorly defined. To gain more insight into this possibility, we therefore investigated the effects of 11 FAD mutations on the aggregation kinetics of Aβ, as well as its ability to form distinct conformations recognized by a panel of amyloid conformation-specific monoclonal antibodies. We found that most FAD mutations increased the rate of aggregation of Aβ. The FAD mutations also led to the adoption of alternative amyloid conformations distinguished by monoclonal antibodies and resulted in the formation of distinct aggregate morphologies as determined by transmission electron microscopy. In addition, several of the mutant peptides displayed a large reduction in thioflavin T fluorescence, despite forming abundant fibrils indicating that thioflavin T is a probe of conformational polymorphisms rather than a reliable indicator of fibrillization. Taken together, these results indicate that FAD mutations falling within the Aβ sequence lead to dramatic changes in aggregation kinetics and influence the ability of Aβ to form immunologically and morphologically distinct amyloid structures.

scholarly journals A Biocompatible Fluorescent Probe for the Selective Detection of Amyloid Fibrils

2020 ◽  
Author(s):  
Anirban Das ◽  
Tanoy Dutta ◽  
Laxmikant Gadhe ◽  
Apurba Koner ◽  
Ishu Saraogi
Keyword(s):  
Amyloid Fibrils ◽  
Intramolecular Charge Transfer ◽  
Aggregation Kinetics ◽  
Amyloid Formation ◽  
Cellular Internalization ◽  
Protein Fibrils ◽  
Model Protein ◽  
Low Toxicity ◽  
Kinetics Of ◽  
Biological Milieu

The misfolding and aggregation of proteins leading to amyloid formation has been linked to numerous diseases, necessitating the development of tools to monitor the fibrillation process. Here we report an intramolecular charge transfer (ICT) dye, DMNDC, as an alternative to Thioflavin-T (ThT), most commonly used for monitoring amyloid fibrils. Using insulin as a model protein, we show that DMNDC efficiently detects all stages of fibril formation, namely, nucleation, elongation, and saturation. An approximately 70 nm hypsochromic shift along with a large increase in emission intensity was observed upon binding of DMNDC to protein fibrils. The aggregation kinetics of insulin remained unaffected at excess DMNDC concentration, suggesting that DMNDC does not inhibit insulin aggregation. Additionally, the efficient cellular internalization and low toxicity of DMNDC make it highly suited for sensing and imaging of amyloid fibrils in the complex biological milieu.<br>

Abstract 312: Amyloids of Oxidized Apolipoprotein A-I Induce Amyloid Fibril Formation in Intact Apolipoprotein A-I

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Gary K Chan ◽  
Andrzej Witkowski ◽  
Giorgio Cavigiolio
Keyword(s):  
Amyloid Fibrils ◽  
Amyloid Fibril ◽  
Fibril Formation ◽  
Atherosclerotic Plaques ◽  
Amyloid Formation ◽  
Atherosclerotic Lesions ◽  
Apolipoprotein A ◽  
Amyloid Deposits ◽  
Amyloid Fibril Formation ◽  
Tht Fluorescence

Background: Amyloid deposition in atherosclerotic plaques increases with aging. Although a correlation between arterial amyloid deposits and cardiovascular events is yet to be established, the high incidence of amyloids associated with aortic intima and with atherosclerotic lesions indicates that amyloid deposits may contribute to atherosclerosis progression. Remarkably, apolipoprotein A-I (apoA-I) is the main component of these amyloids. We previously demonstrated that oxidation of apoA-I methionines by myeloperoxidase, at a concentration similar to that produced by activated macrophages in atherosclerotic lesions, promotes apoA-I amyloid fibril formation. Furthermore, recent studies revealed a hundred-fold increase in the amount of lipid-free apoA-I in atherosclerotic arteries compared to normal arteries. Notably, this apoA-I is heavily oxidized. Thus in the atherosclerotic plaques, high concentration of lipid-free apoA-I and an oxidative milieu are favorable conditions for apoA-I amyloid formation. Hypothesis: We tested the hypothesis that amyloid fibrils constituted of oxidized apoA-I can transfer the dysfunctional phenotype to intact apoA-I. Methods: Pre-formed amyloid fibrils constituted of oxidized apoA-I were incubated with a 10-fold excess of intact apoA-I at 37 °C, pH 6.0, with continuous vortexing. Kinetics of amyloid fibril formation by the pool of intact apoA-I were derived by measuring Thioflavin-T (ThT) fluorescence over a 6-day period. Results: After a lag-phase of 24-48 h, fibril formation proceeded with typical sigmoidal kinetics and reached plateau levels after about 6 days. In control samples, in which intact apoA-I was incubated in the absence of pre-formed fibrils, no significant changes in ThT fluorescence were detected for the same time course. Conclusions: Oxidized apoA-I amyloid fibrils can catalyze the aggregation of a large excess of intact protein. This observation bears important pathophysiological implications. In vivo , a small amount of amyloid fibrils could be produced by oxidized apoA-I in specific microenvironments of the atheroma; when transferred to the surrounding tissues, these amyloid seeds could induce extended amyloid formation in the large available pool of lipid-free apoA-I.

scholarly journals α-Synuclein Amyloid Fibrils Investigation with the Use of Fluorescent Probe Thioflavin T

2018 ◽  
Author(s):  
Anna I. Sulatskaya ◽  
Natalia P. Rodina ◽  
Maksim I. Sulatsky ◽  
Olga I. Povarova ◽  
Iuliia A. Antifeeva ◽  
...  
Keyword(s):  
Fluorescent Probe ◽  
Binding Constant ◽  
Amyloid Fibrils ◽  
Inner Filter Effect ◽  
Alpha Synuclein ◽  
Thioflavin T ◽  
Binding Modes ◽  
Binding Parameters ◽  
Experimental Conditions ◽  
Wide Range

In this work &alpha;-synuclein amyloid fibrils, formation of which is a biomarker of the Parkinson&rsquo;s disease, were investigated with the use of fluorescent probe thioflavin T (ThT). Experimental conditions of the protein fibrillogenesis were chosen so that a sufficient number of continuous measurements can be performed to characterize and analyze all stages of this process. The reproducibility of fibrillogenesis and the structure of the obtained aggregates (that is a critical point for their further investigation) were proved using a wide range of physical-chemical methods. For determination of ThT&mdash;&alpha;-synuclein amyloid fibrils binding parameters sample and reference solutions were prepared with the use of equilibrium microdialysis. By absorption spectroscopy of these solutions ThT&mdash;fibrils binding mode with the binding constant about 104 M&minus;1 and stoichiometry of ThT per protein molecule about 1:8 was observed. Fluorescence spectroscopy of the same solutions with the subsequent correction of the recorded fluorescence intensity on the primary inner filter effect allowed to determine another mode of ThT binding to fibrils with the binding constant about 106 M&minus;1 and stoichiometry about 1:2500. Analysis of photophysical characteristics of the dye molecules bound to the sites of different binding modes allowed to assume the possible localization of these sites. Obtained differences in the ThT binding parameters to amyloid fibrils formed from &alpha;-synuclein and other amyloidogenic proteins, as well as in the photophysical characteristics of the bound dye, confirmed the hypothesis of amyloid fibrils polymorphism.

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