State-of-the-Art Fluorescence Fluctuation-Based Spectroscopic Techniques for the Study of Protein Aggregation

Kitamura, Akira; Kinjo, Masataka

doi:10.3390/ijms19040964

Cited by 44 publications

(25 citation statements)

References 123 publications

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“…1 b ). The detected fluorescence real-time fluctuations and autocorrelation function provide the thermodynamic or kinetic parameters such as diffusion coefficient (DC) or time, nucleation boundary radius and the free energy barrier for nucleation (Medina and Schwille, 2002; Garai et al ., 2008; Kitamura and Kinjo, 2018). Knowing these parameters can assist in developing an exact thermodynamic model of A β aggregation.…”

Section: Smts For Biophysical Studies Of Amyloid Proteinsmentioning

confidence: 99%

Single-molecule studies of amyloid proteins: from biophysical properties to diagnostic perspectives

Cao

Loch

et al. 2020

Quart. Rev. Biophys.

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In neurodegenerative diseases, a wide range of amyloid proteins or peptides such as amyloid-beta and α-synuclein fail to keep native functional conformations, followed by misfolding and self-assembling into a diverse array of aggregates. The aggregates further exert toxicity leading to the dysfunction, degeneration and loss of cells in the affected organs. Due to the disordered structure of the amyloid proteins, endogenous molecules, such as lipids, are prone to interact with amyloid proteins at a low concentration and influence amyloid cytotoxicity. The heterogeneity of amyloid proteinscomplicates the understanding of the amyloid cytotoxicity when relying only on conventional bulk and ensemble techniques. As complementary tools, single-molecule techniques (SMTs) provide novel insights into the different subpopulations of a heterogeneous amyloid mixture as well as the cytotoxicity, in particular as involved in lipid membranes. This review focuses on the recent advances of a series of SMTs, including single-molecule fluorescence imaging, single-molecule force spectroscopy and single-nanopore electrical recording, for the understanding of the amyloid molecular mechanism. The working principles, benefits and limitations of each technique are discussed and compared in amyloid protein related studies.. We also discuss why SMTs show great potential and are worthy of further investigation with feasibility studies as diagnostic tools of neurodegenerative diseases and which limitations are to be addressed.

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Section: Smts For Biophysical Studies Of Amyloid Proteinsmentioning

confidence: 99%

Single-molecule studies of amyloid proteins: from biophysical properties to diagnostic perspectives

Cao

Loch

et al. 2020

Quart. Rev. Biophys.

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“…FtsZ polymerization has also been studied by fluorescence correlation spectroscopy (FCS), a single molecule technique that obtains autocorrelation curves from fluctuations in fluorescence intensities, which can then be used to derive translational diffusion parameters of the fluorescent molecules [ 63 , 86 ]. FCS methods are suitable for high throughput screening [ 87 ].…”

Section: Methods To Identify Drugs Targeting Ftsz Polymerization Amentioning

confidence: 99%

FtsZ Interactions and Biomolecular Condensates as Potential Targets for New Antibiotics

et al. 2021

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FtsZ is an essential and central protein for cell division in most bacteria. Because of its ability to organize into dynamic polymers at the cell membrane and recruit other protein partners to form a “divisome”, FtsZ is a leading target in the quest for new antibacterial compounds. Strategies to potentially arrest the essential and tightly regulated cell division process include perturbing FtsZ’s ability to interact with itself and other divisome proteins. Here, we discuss the available methodologies to screen for and characterize those interactions. In addition to assays that measure protein-ligand interactions in solution, we also discuss the use of minimal membrane systems and cell-like compartments to better approximate the native bacterial cell environment and hence provide a more accurate assessment of a candidate compound’s potential in vivo effect. We particularly focus on ways to measure and inhibit under-explored interactions between FtsZ and partner proteins. Finally, we discuss recent evidence that FtsZ forms biomolecular condensates in vitro, and the potential implications of these assemblies in bacterial resistance to antibiotic treatment.

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“…Figure adapted from ref. [ 36 ] with permission. ( C ) Single molecule total internal reflection fluorescence (TIRF) imaging of surface-immobilized amyloid oligomers.…”

Section: Figurementioning

confidence: 99%

“…The confocal setup can also be employed for fluorescence correlation spectroscopy (FCS) which is based on the fluorescence fluctuations originating from the fluorescent molecules diffusing across the focal volume [ 34 , 35 ]. By analyzing the auto-correlation curves of fluorescence fluctuation, quantitative information such as the concentration, diffusional coefficient and dynamic properties of molecules can be obtained [ 36 ]. SmFRET and FCS have been applied to study the conformations and dynamics of amyloidogenic proteins including the Alzheimer’s-related amyloid β-peptide (Aβ) [ 37 ] and Tau [ 38 , 39 , 40 ], the Parkinson’s-related protein α-synuclein [ 41 , 42 , 43 , 44 , 45 , 46 ], the amyotrophic lateral sclerosis-related protein TAR DNA-binding protein 43 (TDP-43) [ 47 ], Huntington’s disease-related huntingtin [ 48 ], and the yeast prion proteins Ure2 [ 49 ] and Sup35 [ 50 ].…”

Section: Introductionmentioning

confidence: 99%

Single Molecule Characterization of Amyloid Oligomers

Yang

Perrett

2021

Molecules

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The misfolding and aggregation of polypeptide chains into β-sheet-rich amyloid fibrils is associated with a wide range of neurodegenerative diseases. Growing evidence indicates that the oligomeric intermediates populated in the early stages of amyloid formation rather than the mature fibrils are responsible for the cytotoxicity and pathology and are potentially therapeutic targets. However, due to the low-populated, transient, and heterogeneous nature of amyloid oligomers, they are hard to characterize by conventional bulk methods. The development of single molecule approaches provides a powerful toolkit for investigating these oligomeric intermediates as well as the complex process of amyloid aggregation at molecular resolution. In this review, we present an overview of recent progress in characterizing the oligomerization of amyloid proteins by single molecule fluorescence techniques, including single-molecule Förster resonance energy transfer (smFRET), fluorescence correlation spectroscopy (FCS), single-molecule photobleaching and super-resolution optical imaging. We discuss how these techniques have been applied to investigate the different aspects of amyloid oligomers and facilitate understanding of the mechanism of amyloid aggregation.

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State-of-the-Art Fluorescence Fluctuation-Based Spectroscopic Techniques for the Study of Protein Aggregation

Cited by 44 publications

References 123 publications

Single-molecule studies of amyloid proteins: from biophysical properties to diagnostic perspectives

Single-molecule studies of amyloid proteins: from biophysical properties to diagnostic perspectives

FtsZ Interactions and Biomolecular Condensates as Potential Targets for New Antibiotics

Single Molecule Characterization of Amyloid Oligomers

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