Highly Sensitive FRET-FCS Detects Amyloid β-Peptide Oligomers in Solution at Physiological Concentrations

Wennmalm, Stefan; Chmyrov, Volodymyr; Widengren, Jerker; Tjernberg, Lars O.

doi:10.1021/acs.analchem.5b02630

Cited by 51 publications

(50 citation statements)

References 40 publications

(92 reference statements)

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“…The distributions of R h for the Aβ40 and Aβ42 also overlap to a large extent (Figure 1(b)), and yield similar ensemble-averages (Table 1). For both the sequences, the R h values are in excellent agreement with (1.5-1.7 nm) NMR (53) and FCS experiments (54). The ratio R h /Rg for both Aβ40 and Aβ42 is ≈ 0.9, which implies that neither peptide behaves as a Gaussian chain, for which the ratios should be 0.640 and 0.665, respectively (55).…”

Section: Resultssupporting

confidence: 54%

“…The departure from standard homo-polymer theories is the first hint that sequence-specific effects as well as the inherent polyampholyte-like characteristic of the Aβ peptides, that are masked in moments of the distribution functions, need to be accounted for when describing their conformational ensembles. other measure of a polymer size, which is routinely estimated using dynamic light scattering (DLS), NMR, and FCS experiments (53,54). The distributions of R h for the Aβ40 and Aβ42 also overlap to a large extent (Figure 1(b)), and yield similar ensemble-averages (Table 1).…”

Section: Resultsmentioning

confidence: 90%

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Differences in the free energies between the excited states of Aβ40 and Aβ42 monomers encode their distinct aggregation propensities

Chakraborty¹,

Straub

Thirumalai³

2020

Preprint

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of 17Cluster 5Cluster 6 Cluster 8 Cluster 12 Cluster 1+2+3+7 Fig. S8. Clusters 4,5,6,8: Conformational clusters in Aβ40 with conformations with different extents of residual structuring near the N-terminus. Residues 1-10, which are disordered in the fibril structure, are shown in magenta, and residues 11-40, which are ordered in the fibril structure, are shown in light green. These clusters also consist of RC-like structures (see Table S3). The supercluster formed by clusters 1,2,3 and 7 consists of exclusively RC-like structures, and is characterized by a featureless contact map. Clusters in which the conformations with structural signatures of the fibril state are shown in the main text. 13 of 17Cluster 1Cluster 10Cluster 11 Cluster 2+3+8+9 Fig. S9. Different cluster families constituting the Aβ42 monomer ensemble. In cluster 1, the conformations have some structure in the C-terminus. Cluster 10 consists of structures with an ordered N-terminus. In cluster 11, both the termini are disordered. However, some residual structure is present near the middle of the peptide. In these clusters, residues 1-16, which form the disordered region in the fibril structure are shown in magenta, and residues 17-42, which form the ordered region of the fibril, are in green. These clusters also consist of RC-like conformations in addition to those exhibiting different extents of residual structuring. The supercluster formed by clusters 2,3,8 and 9 has a featureless contact map, and consists of exclusively RC-like conformations. The clusters which contain structures that have some resemblance to the monomer units of different Aβ42 polymorphs are shown in the main text.

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Section: Resultssupporting

confidence: 54%

Section: Resultsmentioning

confidence: 90%

Differences in the free energies between the excited states of Aβ40 and Aβ42 monomers encode their distinct aggregation propensities

Chakraborty¹,

Straub

Thirumalai³

2020

Preprint

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“…0.9-1.8 nm. [13][14][15][16][17] The average end to end distance follows a similar trend as the radius of gyration, with a value of 3.2 nm for Amber99SB*-ILDN and 2.7 and 2.8 nm for Charmm36mW without and with NaCl, respectively. The wide distributions of the end to end distance, shown in Figure S3, and the standard deviations of ∼1 nm indicate similar types of structural fluctuations in all three simulations.…”

Section: Resultsmentioning

confidence: 99%

“…10,11 In terms of overall dimensions, the hydrodynamic radius of Aβ40 was measured to be ∼1.6 nm at room temperature 12 while for Aβ42 it adopts a range of values between 0.9-1.8 nm. [13][14][15][16][17] Computationally, the Aβ42 monomer structure has been studied with various simulating techniques and force fields. 18 The general picture that emerges from the numerous molecular dynamics (MD) simulation studies on Aβ monomer is that the observed structural characteristics are very diverse and highly dependent on the simulation conditions.…”

Section: Introductionmentioning

confidence: 99%

Monomeric amyloid β-peptide (1-42) significantly populates compact fibril-like conformations

Barz

Buell

Nath

2020

Preprint

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The aggregation of the amyloid β (Aβ) peptide is a major hallmark of Alzheimer's disease. This peptide can aggregate into oligomers, proto-fibrils, and mature fibrils, which eventually assemble into amyloid plaques. The peptide monomers are the smallest assembly units, and play an important role in most of the individual processes involved in amyloid fibril formation, such as primary and secondary nucleation and elongation. The structure of the Aβ monomer has been shown to be very dynamic and mostly disordered, both in experimental and in computational studies, similar to a random coil. This structural state of the monomer contrasts with the very stable and well defined structural core of the amyloid fibrils. An important question is whether the monomer can adopt transient fibril-like conformations in solution and what role 1 such conformations might play in the aggregation process. Here we use enhanced and extensive molecular dynamics simulations to study the Aβ42 monomer structural flexibility with different force fields, water models and salt concentrations. We show that the monomer behaves as a random coil under different simulation conditions. Importantly, we find a conformation with the N-terminal region structured very similarly to that of recent experimentally determined fibril models. This is to the best of our knowledge the first monomeric structural ensemble to show such a similarity with the fibril structure.

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“…Thus, investigation methods which can work at much lower concentrations, as optical techniques based on absorption and/or fluorescence detection, have been most widely employed to monitor in real-time the kinetics of the early aggregation processes in solution (20). In the last two decades techniques as fluorescence absorbance (22), photobleaching (19,23), self-fluorescent-quenching (20), fluorescence correlation spectroscopy (FCS) (24,25), fluorescence cross-correlation spectroscopy (FCCS) (26), dual-color fluorescence cross-correlation spectroscopy (dc-FCCS) (27), Förster resonance energy transfer combined with fluorescence correlation spectroscopy (FRET-FCS) (26), confocal two-color coincidence detection (cTCCD) (28), but also fluorescence imaging of labeled peptides (19,(29)(30)(31) or binding of Thioflavine T (ThT) (32) or Congo Red (33), have been widely applied for the study of such systems at very low concentrations. Despite the great sensitiveness of such optical techniques, one draw-back about using fluorescence is to be found in the inherent, yet necessary, modification of the original peptide system due to the attached fluorophore.…”

Section: Introductionmentioning

confidence: 99%

How fluorescent tags modify oligomer size distributions of the Alzheimer-peptide Aβ(1-40)

Wägele

Dey

Bruno

et al. 2018

Preprint

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Within the complex aggregation process of Aβ-peptides into fibrils, oligomeric species, play a central role and reveal fundamental properties of the underlying mechanism of aggregation. In particular, low molecular weight aggregates have attracted increasing interest because of their role in cytotoxicity and neuronal apoptosis, typical of aggregation related diseases. One of the main techniques used to characterize such early stages of aggregation is fluorescence spectroscopy. To this end, Aβ-peptide chains are functionalized with fluorescent tags, often covalently bound to the disordered N-terminus region of the peptide, with the assumption that functionalization and presence of the fluorophore will not modify the process of self-assembly nor the final fibrillar structure. Up to date, experimental findings reveal size distributions of thermodynamically stable oligomers ranging from very narrow distributions of dimers to octamers, to very broad distributions up to 50-mers. In the present investigation we systematically study the effects of five of the most commonly used fluorophores on the aggregation of Aβ(1-40)-peptides. Time-resolved and single-molecule fluorescence spectroscopy have been chosen to monitor the oligomer populations at different fibrillation times, TEM, AFM and X-ray diffraction to investigate the structure of mature fibrils. While the structures of the mature fibrils were only slightly affected by the fluorescent tags, the sizes of the detected oligomeric species varied significantly depending on the chosen fluorophore. In particular, we relate the presence of high molecular weight oligomers (as found for the fluorophores HiLyte 647, Atto 647N and Atto 655) to net-attractive, hydrophobic fluorophore-peptide interactions, which are weak in the case of HiLyte 488, and Atto 488. The latter form low molecular weight oligomers only. Our findings reveal the potentially high impact of the properties of fluorophores on transient aggregates which needs to be included in the interpretation of experimental data of oligomers of fluorescently labeled peptides.

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Highly Sensitive FRET-FCS Detects Amyloid β-Peptide Oligomers in Solution at Physiological Concentrations

Cited by 51 publications

References 40 publications

Differences in the free energies between the excited states of Aβ40 and Aβ42 monomers encode their distinct aggregation propensities

Differences in the free energies between the excited states of Aβ40 and Aβ42 monomers encode their distinct aggregation propensities

Monomeric amyloid β-peptide (1-42) significantly populates compact fibril-like conformations

How fluorescent tags modify oligomer size distributions of the Alzheimer-peptide Aβ(1-40)

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