Single-molecule probing of amyloid nano-ensembles using the polymer nanoarray approach

Maity, Sibaprasad; Viazovkina, Ekaterina; Gall, A. A.; Lyubchenko, Yuri L.

doi:10.1039/c7cp02691a

Cited by 12 publications

(21 citation statements)

References 40 publications

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“…AFM force spectroscopy studies revealed novel properties of transiently formed amyloid dimers, this method was limited to probing of dimers. To extend the force spectroscopy technique to oligomers longer than dimers, we developed an approach in which Aβ(14-23) trimers and tetramers were probed with the use of preformed Aβ(14-23) dimers (Maity et al, 2017b). The dimers were assembled using the recently developed flexible nano array (FNA)-oligomer approach in which monomers are tethered inside a flexible polymer, thereby keeping the monomers in close proximity due to the high flexibility of the FNA scaffold (Krasnoslobodtsev et al, 2015;Maity et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

AFM Probing of Amyloid-Beta 42 Dimers and Trimers

Maity

Lyubchenko

2020

Front. Mol. Biosci.

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Elucidating the molecular mechanisms in the development of such a devastating neurodegenerative disorder as Alzheimer's disease (AD) is currently one of the major challenges of molecular medicine. Evidence strongly suggests that the development of AD is due to the accumulation of amyloid β (Aβ) oligomers; therefore, understanding the molecular mechanisms defining the conversion of physiologically important monomers of Aβ proteins into neurotoxic oligomeric species is the key for the development of treatments and preventions of AD. However, these oligomers are unstable and unavailable for structural, physical, and chemical studies. We have recently developed a novel flexible nano array (FNA)-oligomer scaffold approach in which monomers tethered inside a flexible template can assemble spontaneously into oligomers with sizes defined by the number of tethered monomers. The FNA approach was tested on short decamer Aβ(14-23) peptides which were assembled into dimers and trimers. In this paper, we have extended our FNA technique for assembling full-length Aβ42 dimers. The FNA scaffold enabling the self-assembly of Aβ42 dimers from tethered monomeric species has been designed and the assembly of the dimers has been validated by AFM force spectroscopy experiments. Two major parameters of the force spectroscopy probing, the rupture forces and the rupture profiles, were obtained to prove the assembly of Aβ42 dimers. In addition, the FNA-Aβ42 dimers were used to probe Aβ42 trimers in the force spectroscopy experiments with the use of AFM tips functionalized with FNA-Aβ42 dimers and the surface with immobilized Aβ42 monomers. We found that the binding force for the Aβ42 trimer is higher than the dimer (75 ± 7 pN vs. 60 ± 3 pN) and the rupture pattern corresponds to a cooperative dissociation of the trimer. The rupture profiles for the dissociation of the Aβ42 dimers and trimers are proposed. Prospects for further extension of the FNA-based approach for probing of higher order oligomers of Aβ42 proteins are discussed.

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Section: Introductionmentioning

confidence: 99%

AFM Probing of Amyloid-Beta 42 Dimers and Trimers

Maity

Lyubchenko

2020

Front. Mol. Biosci.

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“…Additional analysis of the rupture process, as performed in [12] for Aβ(14-23) dimer, can reveal interesting properties of dimer assembly. The FNA approach was critical in probing interactions between Aβ(14-23) dimer and monomer and dimer-dimer interactions [11]. This suggests that FNA can be used to probe higher order oligomers with three or more conjugated monomer units.…”

Section: Methodsmentioning

confidence: 99%

“…We have recently introduced an approach that allowed us to characterize dimers formed by different amyloidogenic polypeptides [10]. The use of a flexible polymeric platform that contained the assembled oligomers allowed us to probe oligomers larger than dimers [11]. The flexible polymer is synthesized from the non-nucleoside PA spacers using a commercial DNA synthesizer.…”

Section: Introductionmentioning

confidence: 99%

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Polymer Nanoarray Approach for the Characterization of Biomolecular Interactions

Maity

Viazovkina²,

Gall³

et al. 2018

Methods in Molecular Biology

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Pair-wise interactions at the single-molecule level can be done with nanoprobing techniques, such as AFM force spectroscopy, optical tweezers, and magnetic tweezers. These techniques can be used to probe interactions between well-characterized assemblies of biomolecules, such as monomer-dimer, dimer-dimer, and trimer-monomer. An important step of these techniques is the proper assembly of dimers, trimers, and higher oligomers to enable the interactions to be probed. We have developed a novel approach in which a defined number of peptides are assembled along a flexible polymeric molecule that serves as a linear matrix, termed as flexible nanoarray (FNA). The construct is synthesized with the use of phosphoramidite chemistry (PA), in which non-nucleoside PA spacers and standard oligonucleotide synthesis are used to grow the polymeric chain with the desired length. The reactive sites are incorporated during FNA synthesis. As a result, the FNA polymer contains a set of predesigned reactive sites to which the peptides are covalently conjugated. We describe the protocol for the synthesis of FNA and the application of this methodology to measure the molecular interactions between amyloid peptides of monomer-monomer, monomer-dimer, and dimer-dimer.

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“…For experiments that extend a single protein (mechanical unfolding), some of these problems have been addressed by site-specific immobilization onto passivated surfaces 25 or by insertion of the protein of interest into a mechanically strong protein within a protein concatemer. 26 For protein-protein interaction studies, Lyubchenko and coworkers have developed a "flexible nanoarray" method 27,28 whereby pairs of aggregation-prone peptides are tethered onto an extensible linker at a defined separation, allowing dimerization events to be analyzed. 27 Extending the tether using the AFM allows dissociation to be measured away from the surface.…”

Section: Introductionmentioning

confidence: 99%

A peptide‐display protein scaffold to facilitate single molecule force studies of aggregation‐prone peptides

et al. 2018

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Protein aggregation is linked with the onset of several neurodegenerative disorders, including Parkinson's disease (PD), which is associated with the aggregation of a-synuclein (aSyn). The structural mechanistic details of protein aggregation, including the nature of the earliest protein-protein interactions, remain elusive. In this study, we have used single molecule force spectroscopy (SMFS) to probe the first dimerization events of the central aggregation-prone region of aSyn (residues 71-82) that may initiate aggregation. This region has been shown to be necessary for the aggregation of full length aSyn and is capable of forming amyloid fibrils in isolation. We demonstrate that the interaction of aSyn 71-82 peptides can be studied using SMFS when inserted into a loop of protein L, a mechanically strong and soluble scaffold protein that acts as a display system for SMFS studies. The corresponding fragment of the homolog protein c-synuclein (cSyn), which has a lower aggregation propensity, has also been studied here. The results from SMFS, together with native mass spectrometry and aggregation assays, demonstrate that the dimerization propensity of cSyn 71-82 is lower than that of aSyn [71][72][73][74][75][76][77][78][79][80][81][82] , but that a mixed aSyn 71-82 : cSyn 71-82 dimer forms with a similar propensity to the aSyn 71-82 homodimer, slowing amyloid formation. This work demonstrates the utility of a novel display method for SMFS studies of aggregation-prone peptides, which would otherwise be difficult to study.

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Single-molecule probing of amyloid nano-ensembles using the polymer nanoarray approach

Cited by 12 publications

References 40 publications

AFM Probing of Amyloid-Beta 42 Dimers and Trimers

AFM Probing of Amyloid-Beta 42 Dimers and Trimers

Polymer Nanoarray Approach for the Characterization of Biomolecular Interactions

A peptide‐display protein scaffold to facilitate single molecule force studies of aggregation‐prone peptides

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