Direct Detection of α-Synuclein Dimerization Dynamics: Single-Molecule Fluorescence Analysis

Lv, Zhengjian; Krasnoslobodtsev, Alexey V.; Zhang, Yuliang; Ysselstein, Daniel; Rochet, Jean‐Christophe; Blanchard, Scott C.; Lyubchenko, Yuri L.

doi:10.1016/j.bpj.2015.03.010

Cited by 51 publications

(79 citation statements)

References 64 publications

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“…This suggests that dimers of α-Syn protein are stable and dissociate in the range of seconds. These measurements are in line with our recent results obtained by entirely different experimental approach—tethered single molecule fluorescence [65]. The dimer lifetime in that experiments was obtained directly from measurements of the fluorescence duration time produced by fluorescently labeled α-Syn monomer bound to the surface tethered α-Syn.…”

Section: Molecular Mechanisms Of Protein Assembly Into Disease-prosupporting

confidence: 87%

Amyloid misfolding, aggregation, and the early onset of protein deposition diseases: insights from AFM experiments and computational analyses

Lyubchenko

2015

AIMS Molecular Science

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The development of Alzheimer’s disease is believed to be caused by the assembly of amyloid β proteins into aggregates and the formation of extracellular senile plaques. Similar models suggest that structural misfolding and aggregation of proteins are associated with the early onset of diseases such as Parkinson’s, Huntington’s, and other protein deposition diseases. Initially, the aggregates were structurally characterized by traditional techniques such as x-ray crystallography, NMR, electron microscopy, and AFM. However, data regarding the structures formed during the early stages of the aggregation process were unknown. Experimental models of protein deposition diseases have demonstrated that the small oligomeric species have significant neurotoxicity. This highlights the urgent need to discover the properties of these species, to enable the development of efficient diagnostic and therapeutic strategies. The oligomers exist transiently, making it impossible to use traditional structural techniques to study their characteristics. The recent implementation of single-molecule imaging and probing techniques that are capable of probing transient states have enabled the properties of these oligomers to be characterized. Additionally, powerful computational techniques capable of structurally analyzing oligomers at the atomic level advanced our understanding of the amyloid aggregation problem. This review outlines the progress in AFM experimental studies and computational analyses with a primary focus on understanding the very first stage of the aggregation process. Experimental approaches can aid in the development of novel sensitive diagnostic and preventive strategies for protein deposition diseases, and several examples of these approaches will be discussed.

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Section: Molecular Mechanisms Of Protein Assembly Into Disease-prosupporting

confidence: 87%

Amyloid misfolding, aggregation, and the early onset of protein deposition diseases: insights from AFM experiments and computational analyses

Lyubchenko

2015

AIMS Molecular Science

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“…The volume measurements for the 48 h sample reveal a major peak at 39 nm 3 which is very close to the monomer volume (Fig. 3D)20. There is a secondary minor peak with a volume value of 66 nm 3 that can be assigned to dimers-trimers.…”

Section: Resultsmentioning

confidence: 59%

“…Given that α-Syn is more than three times larger than Aβ-42 and the fact that we were previously able to image α-Syn monomers20, we anticipated being able to visualize the formation of oligomers starting with dimers. The surface-mediated aggregation experiment was carried out similarly to the analyses described above.…”

Section: Resultsmentioning

confidence: 99%

“…To characterize their sizes, volume measurements as described in ref. 20 were performed. These measurements are shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Intracellular crowding effects18 can facilitate the aggregation process, but these do not solve the problem. Recently, we have found that dimers of α-synuclein (α-Syn) could be assembled at nanomolar concentrations, if the target monomer is tethered to a surface20. These data led us to hypothesize that binding to the surface can be a factor dramatically facilitating the aggregation process.…”

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confidence: 99%

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A novel pathway for amyloids self-assembly in aggregates at nanomolar concentration mediated by the interaction with surfaces

Banerjee

Hashemi

et al. 2017

Sci Rep

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A limitation of the amyloid hypothesis in explaining the development of neurodegenerative diseases is that the level of amyloidogenic polypeptide in vivo is below the critical concentration required to form the aggregates observed in post-mortem brains. We discovered a novel, on-surface aggregation pathway of amyloidogenic polypeptide that eliminates this long-standing controversy. We applied atomic force microscope (AFM) to demonstrate directly that on-surface aggregation takes place at a concentration at which no aggregation in solution is observed. The experiments were performed with the full-size Aβ protein (Aβ42), a decapeptide Aβ(14-23) and α-synuclein; all three systems demonstrate a dramatic preference of the on-surface aggregation pathway compared to the aggregation in the bulk solution. Time-lapse AFM imaging, in solution, show that over time, oligomers increase in size and number and release in solution, suggesting that assembled aggregates can serve as nuclei for aggregation in bulk solution. Computational modeling performed with the all-atom MD simulations for Aβ(14-23) peptide shows that surface interactions induce conformational transitions of the monomer, which facilitate interactions with another monomer that undergoes conformational changes stabilizing the dimer assembly. Our findings suggest that interactions of amyloidogenic polypeptides with cellular surfaces play a major role in determining disease onset.

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Effect of acidic pH on the stability of α‐synuclein dimers

Krasnoslobodtsev

Zhang

et al. 2016

Biopolymers

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Environmental factors, such as acidic pH, facilitate the assembly of α-synuclein (α-Syn) in aggregates, but the impact of pH on the very first step of α-Syn aggregation remains elusive. Recently, we developed a single-molecule approach enabling us to measure directly the stability of α-Syn dimers. Unlabeled α-Syn monomers were immobilized on a substrate, and fluorophore-labeled monomers were added to the solution to allow them to form dimers with immobilized α-Syn monomers. The dimer lifetimes were measured directly from the fluorescence bursts on the time trajectories. Herein, we applied the single-molecule tethered approach for probing of intermolecular interaction to characterize the effect of acidic pH on the lifetimes of α-Syn dimers. The experiments were performed at pH 5 and pH 7 for the wild-type α–Syn and two mutants containing familial type mutations E46K and A53T. We demonstrate that decrease of pH resulted in more than 3-fold increase in the α-Syn dimers lifetimes with some variability between the α-Syn species. We hypothesize that the stabilization effect is explained by neutralization of residues 96–140 of α-Syn and this electrostatic effect facilitates the association of the two monomers. Given the fact that dimerization is the first step of α-Syn aggregation, we posit that the electrostatic effect contributes to accelerating α-Syn aggregation at acidic pH.

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Direct Detection of α-Synuclein Dimerization Dynamics: Single-Molecule Fluorescence Analysis

Cited by 51 publications

References 64 publications

Amyloid misfolding, aggregation, and the early onset of protein deposition diseases: insights from AFM experiments and computational analyses

Amyloid misfolding, aggregation, and the early onset of protein deposition diseases: insights from AFM experiments and computational analyses

A novel pathway for amyloids self-assembly in aggregates at nanomolar concentration mediated by the interaction with surfaces

Effect of acidic pH on the stability of α‐synuclein dimers

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