scholarly journals Single Molecule Characterization of Amyloid Oligomers

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 948
Author(s):  
Jie Yang ◽  
Sarah Perrett ◽  
Si Wu
Keyword(s):  
Single Molecule ◽  
Amyloid Fibrils ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Correlation Spectroscopy ◽  
Amyloid Formation ◽  
Amyloid Aggregation ◽  
Wide Range ◽  
Amyloid Oligomers

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.

scholarly journals Allosteric modulation of the SARS-CoV-2 spike conformation

2021 ◽  
Author(s):  
Marco A Diaz-Salinas ◽  
Qi Li ◽  
Monir Ejemel ◽  
Yang Wang ◽  
James B Munro
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Data ◽  
Correlation Spectroscopy ◽  
Host Cells ◽  
Parallel Solution ◽  
Real Time Analysis ◽  
Multiple Conformations

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells through binding to angiotensin-converting enzyme 2 (ACE2), which is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural data and real-time analysis of conformational dynamics have shown that S can adopt multiple conformations, which mediate the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Förster resonance energy transfer (smFRET) imaging we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and the B.1 variant (D614G). We found that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicated antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.

Quantitative single molecule FRET efficiencies using TIRF microscopy

2015 ◽  
Vol 184 ◽  
pp. 131-142 ◽  
Author(s):  
Lasse L. Hildebrandt ◽  
Søren Preus ◽  
Victoria Birkedal
Keyword(s):  
Single Molecule ◽  
Structural Information ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Complexes ◽  
Distance Information ◽  
Single Molecule Level ◽  
Single Molecule Fret ◽  
Wide Range ◽  
Intermolecular Distances

Förster resonance energy transfer (FRET) microscopy at the single molecule level has the potential to yield information on intra and intermolecular distances within the 2–10 nm range of molecules or molecular complexes that undergo frequent conformation changes. A pre-requirement for obtaining accurate distance information is to determine quantitative instrument independent FRET efficiency values. Here, we applied and evaluated a procedure to determine quantitative FRET efficiencies directly from individual fluorescence time traces of surface immobilized DNA molecules without the need for external calibrants. To probe the robustness of the approach over a wide range of FRET efficiencies we used a set of doubly labelled double stranded DNA samples, where the acceptor position was varied systematically. Interestingly, we found that fluorescence contributions arising from direct acceptor excitation following donor excitation are intrinsically taken into account in these conditions as other correction factors can compensate for inaccurate values of these parameters. We give here guidelines, that can be used through tools within the iSMS software (http://www.isms.au.dk), for determining quantitative FRET and assess uncertainties linked with the procedure. Our results provide insights into the experimental parameters governing quantitative FRET determination, which is essential for obtaining accurate structural information from a wide range of biomolecules.

scholarly journals Affinity of Skp to OmpC revealed by single-molecule detection

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sichen Pan ◽  
Chen Yang ◽  
Xin Sheng Zhao
Keyword(s):  
Single Molecule ◽  
Molecular Chaperones ◽  
Outer Membrane Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Hill Coefficient ◽  
Stoichiometric Ratio ◽  
Single Molecule Level

Abstract Outer membrane proteins (OMPs) are essential to gram-negative bacteria, and molecular chaperones prevent the OMPs from aggregation in the periplasm during the OMPs biogenesis. Skp is one of the molecular chaperones for this purpose. Here, we combined single-molecule fluorescence resonance energy transfer and fluorescence correlation spectroscopy to study the affinity and stoichiometric ratio of Skp in its binding with OmpC at the single-molecule level. The half concentration of the Skp self-trimerization (C1/2) was measured to be (2.5 ± 0.7) × 102 nM. Under an Skp concentration far below the C1/2, OmpC could recruit Skp monomers to form OmpC·Skp3. The affinity to form the OmpC·Skp3 complex was determined to be (5.5 ± 0.4) × 102 pM with a Hill coefficient of 1.6 ± 0.2. Under the micromolar concentrations of Skp, the formation of OmpC·(Skp3)2 was confirmed, and the dissociation constant of OmpC·(Skp3)2 was determined to be 1.2 ± 0.4 μM. The precise information will help us to quantitatively depict the role of Skp in the biogenesis of OMPs.

scholarly journals Nanometer-accuracy distance measurements between fluorophores at the single-molecule level

2019 ◽  
Vol 116 (10) ◽  
pp. 4275-4284 ◽  
Author(s):  
Stefan Niekamp ◽  
Jongmin Sung ◽  
Walter Huynh ◽  
Gira Bhabha ◽  
Ronald D. Vale ◽  
...  
Keyword(s):  
Single Molecule ◽  
Open Source Software ◽  
Single Molecules ◽  
Coiled Coil ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Distance Measurements ◽  
Single Molecule Level ◽  
Wide Range ◽  
Different Color

Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. Single-molecule Förster resonance energy transfer can measure intramolecular distance changes of single molecules in the range of 2 to 8 nm. However, current superresolution measurements become error-prone below 25 nm. Thus, new single-molecule methods are needed for measuring distances in the 8- to 25-nm range. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ∼1-nm accuracy at distances >2 nm. These techniques can be implemented in high throughput using a standard total internal reflection fluorescence microscope and open-source software. We applied our two-color localization method to uncover an unexpected ∼4-nm nucleotide-dependent conformational change in the coiled-coil “stalk” of the motor protein dynein. We anticipate that these methods will be useful for high-accuracy distance measurements of single molecules over a wide range of length scales.

scholarly journals Real-Time Monitoring of Apoptosis by Caspase-3-Like Protease Induced FRET Reduction Triggered by Amyloid Aggregation

2008 ◽  
Vol 2008 ◽  
pp. 1-12 ◽  
Author(s):  
Johan F. Paulsson ◽  
Sebastian W. Schultz ◽  
Martin Köhler ◽  
Ingo Leibiger ◽  
Per-Olof Berggren ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Energy Transfer ◽  
Caspase 3 ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cell Number ◽  
Amyloid Formation ◽  
Amyloid Aggregation ◽  
Caspase Cascade

Amyloid formation is cytotoxic and can activate the caspase cascade. Here, we monitor caspase-3-like activity as reduction of fluorescence resonance energy transfer (FRET) using the contstruct pFRET2-DEVD containing enhanced cyan fluorescent protin (EYFP) linked by the caspase-3 specific cleavage site residues DEVD. Beta-TC-6 cells were transfected, and the fluoorescence was measured at 440 nm excitation and 535 nm (EYFP) and 480 nm (ECFP) emission wavelength. Cells were incubated with recombinant pro lset Amyloid Polypeptide (recprolAPP) or the processing metabolites of prolAPP; the N-terminal flanking peptide withIAPP (recN+IAPP); IAPP with the C-terminal flanking peptied (recIAPP+C) and lslet Amyloid Polypeptide (recIAPP) . Peptides were added in solubilized from (50 μM) or as performed amyloid-like fibrils, or as a combination of these. FRET was measured and incubation with a mixture of solubilized peptide and performed fibrils resulted in loss of FRET and apoptosis was determined to occure in cells incubated withrecproIAPP (49%),recN+IAPP (46%),recIAPP (72%) andrecIAPP+C (59%). These results show that proIAPP and the processing intermediates reside the same cell toxic capacity as IAPP, and they can all have a central role in the reduction of beta-cell number in type 2 diabetes.

scholarly journals Staphylococcus aureustoxin LukSF dissociates from its membrane receptor target to enable renewed ligand sequestration

2018 ◽  
Author(s):  
Karita Haapasalo ◽  
Adam J. M. Wollman ◽  
Carla de Haas ◽  
Kok van Kessel ◽  
Jos van Strijp ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Lysis ◽  
C5a Receptor ◽  
Inflammatory Reactions ◽  
Microbial Infections ◽  
New Antibiotics ◽  
Receptor Clusters

ABSTRACTbackgroundStaphylococcus aureusPanton Valentine Leukocidin (PVL) is a pore-forming toxin targeting the human C5a receptor (hC5aR), enabling this pathogen to battle the immune response by destroying phagocytes through targeted lysis. The mechanisms that contribute to rapid cell lysis are largely unexplored.ResultsHere we show that cell lysis may be enabled by a process of toxins targeting receptor clusters and receptor ‘recycling’ which allows multiple toxin pores to be formed close together. Using live cell single-molecule super-resolution imaging, Förster resonance energy transfer (FRET) and nanoscale total internal reflection fluorescence (TIRF) colocalization microscopy we visualized toxin pore formation in the presence of its natural docking ligand.ConclusionsWe demonstrate disassociation of hC5aR from toxin complexes and simultaneous binding of new ligands. This effect may free mobile receptors to amplify hyper inflammatory reactions in early stages of microbial infections and have implications for several other similar bi-component toxins and the design of new antibiotics.

scholarly journals High accuracy measurements of nanometer-scale distances between fluorophores at the single-molecule level

2017 ◽  
Author(s):  
Stefan Niekamp ◽  
Jongmin Sung ◽  
Walter Huynh ◽  
Gira Bhabha ◽  
Ronald D. Vale ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Molecules ◽  
Coiled Coil ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Shape ◽  
High Accuracy ◽  
Distance Measurements ◽  
Distance Information ◽  
Wide Range

To uncover the mechanisms of molecular machines it is useful to probe their structural conformations. Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for measuring intra-molecular shape changes of single-molecules, but is confined to distances of 2-8 nm. Current super-resolution measurements are error prone at <25 nm. Thus, reliable high-throughput distance information between 8-25 nm is currently difficult to achieve. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ∼1 nm accuracy at any distance >2 nm, using a standard TIRF microscope and open-source software. We applied our two-color localization method to uncover a ∼4 nm conformational change in the “stalk” of the motor protein dynein, revealing unexpected flexibility in this antiparallel coiled-coil domain. These new methods enable high-accuracy distance measurements of single-molecules that can be used over a wide range of length scales.

scholarly journals Advanced Data Analysis for Fluorescence-Lifetime Single-Molecule Localization Microscopy

2021 ◽  
Vol 1 ◽  
Author(s):  
Jan Christoph Thiele ◽  
Oleksii Nevskyi ◽  
Dominic A. Helmerich ◽  
Markus Sauer ◽  
Jörg Enderlein
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Local Environment ◽  
Localization Microscopy ◽  
The Impact ◽  
Single Molecule Localization Microscopy

Fluorescence-lifetime single molecule localization microscopy (FL-SMLM) adds the lifetime dimension to the spatial super-resolution provided by SMLM. Independent of intensity and spectrum, this lifetime information can be used, for example, to quantify the energy transfer efficiency in Förster Resonance Energy Transfer (FRET) imaging, to probe the local environment with dyes that change their lifetime in an environment-sensitive manner, or to achieve image multiplexing by using dyes with different lifetimes. We present a thorough theoretical analysis of fluorescence-lifetime determination in the context of FL-SMLM and compare different lifetime-fitting approaches. In particular, we investigate the impact of background and noise, and give clear guidelines for procedures that are optimized for FL-SMLM. We do also present and discuss our public-domain software package “Fluorescence-Lifetime TrackNTrace,” which converts recorded fluorescence microscopy movies into super-resolved FL-SMLM images.

Evidence that GPVI is Expressed as a Mixture of Monomers and Dimers, and that the D2 Domain is not Essential for GPVI Activation

2021 ◽  
Author(s):  
Joanne C. Clark ◽  
Raluca A. I. Neagoe ◽  
Malou Zuidscherwoude ◽  
Deirdre M. Kavanagh ◽  
Alexandre Slater ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Glycoprotein Vi ◽  
D2 Domain ◽  
Microscopy Techniques

AbstractCollagen has been proposed to bind to a unique epitope in dimeric glycoprotein VI (GPVI) and the number of GPVI dimers has been reported to increase upon platelet activation. However, in contrast, the crystal structure of GPVI in complex with collagen-related peptide (CRP) showed binding distinct from the site of dimerization. Further fibrinogen has been reported to bind to monomeric but not dimeric GPVI. In the present study, we have used the advanced fluorescence microscopy techniques of single-molecule microscopy, fluorescence correlation spectroscopy (FCS) and bioluminescence resonance energy transfer (BRET), and mutagenesis studies in a transfected cell line model to show that GPVI is expressed as a mixture of monomers and dimers and that dimerization through the D2 domain is not critical for activation. As many of these techniques cannot be applied to platelets to resolve this issue, due to the high density of GPVI and its anucleate nature, we used Förster resonance energy transfer (FRET) to show that endogenous GPVI is at least partially expressed as a dimer on resting and activated platelet membranes. We propose that GPVI may be expressed as a monomer on the cell surface and it forms dimers in the membrane through diffusion, giving rise to a mixture of monomers and dimers. We speculate that the formation of dimers facilitates ligand binding through avidity.

Single-Molecule Resonance Energy Transfer and Fluorescence Correlation Spectroscopy of Calmodulin in Solution†

2004 ◽  
Vol 108 (29) ◽  
pp. 10388-10397 ◽  
Author(s):  
Brian D. Slaughter ◽  
Michael W. Allen ◽  
Jay R. Unruh ◽  
Ramona J. Bieber Urbauer ◽  
Carey K. Johnson
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation
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