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.

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 Temperature-Dependent Structural Variability of Prion Protein Amyloid Fibrils

2021 ◽  
Vol 22 (10) ◽  
pp. 5075
Author(s):  
Mantas Ziaunys ◽  
Andrius Sakalauskas ◽  
Kamile Mikalauskaite ◽  
Ruta Snieckute ◽  
Vytautas Smirnovas
Keyword(s):  
Protein Aggregation ◽  
Prion Protein ◽  
Amyloid Fibrils ◽  
Prion Diseases ◽  
Morphological Properties ◽  
Large Sample Size ◽  
Structural Variations ◽  
Temperature Dependent ◽  
Protein Fibrils ◽  
Propagation Properties

Prion protein aggregation into amyloid fibrils is associated with the onset and progression of prion diseases—a group of neurodegenerative amyloidoses. The process of such aggregate formation is still not fully understood, especially regarding their polymorphism, an event where the same type of protein forms multiple, conformationally and morphologically distinct structures. Considering that such structural variations can greatly complicate the search for potential antiamyloid compounds, either by having specific propagation properties or stability, it is important to better understand this aggregation event. We have recently reported the ability of prion protein fibrils to obtain at least two distinct conformations under identical conditions, which raised the question if this occurrence is tied to only certain environmental conditions. In this work, we examined a large sample size of prion protein aggregation reactions under a range of temperatures and analyzed the resulting fibril dye-binding, secondary structure and morphological properties. We show that all temperature conditions lead to the formation of more than one fibril type and that this variability may depend on the state of the initial prion protein molecules.

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 The Materials Science of Protein Aggregation

MRS Bulletin ◽  
2005 ◽  
Vol 30 (6) ◽  
pp. 452-457 ◽  
Author(s):  
D.L. Cox ◽  
H. Lashuel ◽  
K.Y.C. Lee ◽  
R.R.P. Singh
Keyword(s):  
Protein Aggregation ◽  
Conformational Change ◽  
Lipid Bilayers ◽  
Materials Science ◽  
Prion Diseases ◽  
Protein Structures ◽  
Biochemical Networks ◽  
Amyloid Formation ◽  
Biological Phenomena

AbstractNumerous human diseases are associated with conformational change and aggregation of proteins, including Alzheimer's, Parkinson's, prion diseases (such as mad cow disease), familial amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease), Huntington's, and type II (mature onset) diabetes. In many cases, it has been demonstrated that conformational change and aggregation can occur outside living cells and complex biochemical networks. Hence, approaches from materials and physical science have enhanced our understanding of the role of protein aggregation in these diseases at the molecular and nanoscale levels. In this article, we will review what is known about these protein structures from the perspective of materials science, focusing on the details of emergent oligomeric and nanotube-like structures, their interactions with model lipid bilayers, how the structures relate to observed biological phenomena, and how protein aggregation and amyloid formation can be employed for the good in biology and materials science.

scholarly journals Transthyretin microheterogeneity and molecular interactions: implications for amyloid formation

2014 ◽  
Vol 5 (3) ◽  
pp. 257-264
Author(s):  
Michael Landreh ◽  
Linus J. Östberg ◽  
Tom M. Pettersson ◽  
Hans Jörnvall
Keyword(s):  
Amyloid Fibrils ◽  
Binding Protein ◽  
Retinol Binding Protein ◽  
Amyloid Formation ◽  
Rate Limiting Step ◽  
Pharmacological Prevention ◽  
Dietary Components ◽  
Rate Limiting ◽  
Amyloid Diseases ◽  
Tetrameric Form

AbstractAggregation of transthyretin (TTR), a plasma-binding protein for thyroxine and retinol-binding protein, is the cause of several amyloid diseases. Disease-associated mutations are well known, but wild-type TTR is, to a lesser extent, also amyloidogenic. Monomerization, not oligomer formation as in several other depository diseases, is the rate-limiting step in TTR aggregation, and stabilization of the natively tetrameric form can inhibit amyloid formation. Modifications on Cys10, as well as interactions with native ligands in plasma, were early found to influence the equilibrium between tetrameric and monomeric TTR by dissociating or stabilizing the tetramer. Following these discoveries, synthetic ligands for pharmacological prevention of TTR aggregation could be developed. In this article, we outline how the different types of TTR interactions and its microheterogeneity in plasma are related to its propensity to form amyloid fibrils. We conclude that plasma constituents and dietary components may act as natural TTR stabilizers whose mechanisms of action provide cues for the amelioration of TTR amyloid disease.

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>

AggreRATE-Pred: a mathematical model for the prediction of change in aggregation rate upon point mutation

2019 ◽  
Author(s):  
Puneet Rawat ◽  
R Prabakaran ◽  
Sandeep Kumar ◽  
M Michael Gromiha
Keyword(s):  
Mathematical Model ◽  
Protein Aggregation ◽  
Correlation Coefficient ◽  
Single Point ◽  
Three Dimensional ◽  
Point Mutations ◽  
Supplementary Information ◽  
P Value ◽  
Aggregation Kinetics ◽  
Aggregation Rate

Abstract Motivation Protein aggregation is a major unsolved problem in biochemistry with implications for several human diseases, biotechnology and biomaterial sciences. A majority of sequence-structural properties known for their mechanistic roles in protein aggregation do not correlate well with the aggregation kinetics. This limits the practical utility of predictive algorithms. Results We analyzed experimental data on 183 unique single point mutations that lead to change in aggregation rates for 23 polypeptides and proteins. Our initial mathematical model obtained a correlation coefficient of 0.43 between predicted and experimental change in aggregation rate upon mutation (P-value <0.0001). However, when the dataset was classified based on protein length and conformation at the mutation sites, the average correlation coefficient almost doubled to 0.82 (range: 0.74–0.87; P-value <0.0001). We observed that distinct sequence and structure-based properties determine protein aggregation kinetics in each class. In conclusion, the protein aggregation kinetics are impacted by local factors and not by global ones, such as overall three-dimensional protein fold, or mechanistic factors such as the presence of aggregation-prone regions. Availability and implementation The web server is available at http://www.iitm.ac.in/bioinfo/aggrerate-pred/. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals A generic approach to decipher the mechanistic pathway of heterogeneous protein aggregation kinetics

2021 ◽  
Author(s):  
Baishakhi Tikader ◽  
Samir K. Maji ◽  
Sandip Kar
Keyword(s):  
Protein Aggregation ◽  
Generic Property ◽  
Aggregation Kinetics ◽  
Amyloid Formation ◽  
Mechanistic Pathway ◽  
Polypeptide Chains

Amyloid formation is a generic property of many protein/polypeptide chains.

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