Does Thioflavin-T Detect Oligomers Formed During Amyloid Fibril Assembly

Persichilli, Christopher; Hill, Shannon E.; Mast, Jason; Muschol, Martin

doi:10.1016/j.bpj.2010.12.3140

Cited by 7 publications

(4 citation statements)

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“…88,89 ThT binds to β sheet stacks common in amyloid structures and this binding enhances its fluorescence emission; 90 importantly, ThT is insensitive to lysozyme amyloid oligomers and only interacts with protofibrils and their more complex structures. 80,81,91 The fluorescent intensities in Figure 3d demonstrate that ANS binding to lysozyme heated to 65 °C is indistinguishable from that of native lysozyme; heating to 80 °C induces binding comparable to that of native lysozyme, indicating that few new hydrophobic sites were exposed to the solution owing to these treatments. ANS strongly binds to lysozyme heated to 90 °C, suggesting numerous exposed hydrophobic sites.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Polymorphism of Lysozyme Condensates

et al. 2017

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Protein condensates play essential roles in physiological processes and pathological conditions. Recently discovered mesoscopic protein-rich clusters may act as crucial precursors for the nucleation of ordered protein solids, such as crystals, sickle hemoglobin polymers, and amyloid fibrils. These clusters challenge settled paradigms of protein condensation as the constituent protein molecules present features characteristic of both partially misfolded and native proteins. Here we employ the antimicrobial enzyme lysozyme and examine the similarities between mesoscopic clusters, amyloid structures, and disordered aggregates consisting of chemically modified protein. We show that the mesoscopic clusters are distinct from the other two classes of aggregates. Whereas cluster formation and amyloid oligomerization are both reversible, aggregation triggered by reduction of the intramolecular S-S bonds is permanent. In contrast to the amyloid structures, protein molecules in the clusters retain their enzymatic activity. Furthermore, an essential feature of the mesoscopic clusters is their constant radius of less than 50 nm. The amyloid and disordered aggregates are significantly larger and rapidly grow. These findings demonstrate that the clusters are a product of limited protein structural flexibility. In view of the role of the clusters in the nucleation of ordered protein solids, our results suggest that fine-tuning the degree of protein conformational stability is a powerful tool to control and direct the pathways of protein condensation.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Polymorphism of Lysozyme Condensates

et al. 2017

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“…The kinetics of amyloid assembly have typically been investigated in bulk assays, − in which the recorded signal reflects a convolution of molecularly distinct events: nucleation, growth, fragmentation, and fibril surface-catalyzed nucleation. It is hard to disentangle these processes .…”

Section: Introductionmentioning

confidence: 99%

Steady, Symmetric, and Reversible Growth and Dissolution of Individual Amyloid-β Fibrils

Safari

et al. 2019

ACS Chem. Neurosci.

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Oligomers and fibrils of the amyloid-β (Aβ) peptide are implicated in the pathology of Alzheimer’s disease. Here, we monitor the growth of individual Aβ40 fibrils by time-resolved in situ atomic force microscopy and thereby directly measure fibril growth rates. The measured growth rates in a population of fibrils that includes both single protofilaments and bundles of filaments are independent of the fibril thickness, indicating that cooperation between adjacent protofilaments does not affect incorporation of monomers. The opposite ends of individual fibrils grow at similar rates. In contrast to the “stop-and-go” kinetics that has previously been observed for amyloid-forming peptides, growth and dissolution of the Aβ40 fibrils are relatively steady for peptide concentration of 0–10 μM. The fibrils readily dissolve in quiescent peptide-free solutions at a rate that is consistent with the microscopic reversibility of growth and dissolution. Importantly, the bimolecular rate coefficient for the association of a monomer to the fibril end is significantly smaller than the diffusion limit, implying that the transition state for incorporation of a monomer into a fibril is associated with a relatively high free energy.

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“…At low concentrations (∼0.5–0.9 μM) the ThT fluorescence emission decreases due to the low fibril concentration. Moreover, oligomeric intermediates interact poorly with ThT, resulting in low fluorescence emission as well . For ∼0.7 μM Aβ concentration, which was the lowest concentration that could be still monitored with ThT fluorescence, the half-time was determined to be ∼40 h.…”

Section: Resultsmentioning

confidence: 99%

Interaction of Amyloid-β-(1–42) Peptide and Its Aggregates with Lipid/Water Interfaces Probed by Vibrational Sum-Frequency Generation Spectroscopy

et al. 2021

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In this study, we use surface-sensitive vibrational sum-frequency generation (VSFG) spectroscopy to investigate the interaction between model lipid monolayers and Aβ(1–42) in its monomeric and aggregated states. Combining VSFG with atomic force microscopy (AFM) and thioflavin T (ThT) fluorescence measurements, we found that only small aggregates with probably a β-hairpin-like structure adsorbed to the zwitterionic lipid monolayer (DOPC). In contrast, larger aggregates with an extended β-sheet structure adsorbed to a negatively charged lipid monolayer (DOPG). The adsorption of small, initially formed aggregates strongly destabilized both monolayers, but only the DOPC monolayer was completely disrupted. We showed that the intensity of the amide-II′ band in achiral (SSP) and chiral (SPP) polarization combinations increased in time when Aβ(1–42) aggregates accumulated at the DOPG monolayer. Nevertheless, almost no adsorption of preformed mature fibrils to DOPG monolayers was detected. By performing spectral VSFG calculations, we revealed a clear correlation between the amide-II′ signal and the degree of amyloid aggregates (e.g., oligomers or (proto)fibrils) of various Aβ(1–42) structures. The calculations showed that only structures with a significant amyloid β-sheet content have a strong amide-II′ intensity, in line with previous Raman studies. The combination of the presented results substantiates the amide-II(′) band as a legitimate amyloid marker.

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Does Thioflavin-T Detect Oligomers Formed During Amyloid Fibril Assembly

Cited by 7 publications

References 0 publications

Polymorphism of Lysozyme Condensates

Polymorphism of Lysozyme Condensates

Steady, Symmetric, and Reversible Growth and Dissolution of Individual Amyloid-β Fibrils

Interaction of Amyloid-β-(1–42) Peptide and Its Aggregates with Lipid/Water Interfaces Probed by Vibrational Sum-Frequency Generation Spectroscopy

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