Probing Intermolecular Interactions within the Amyloid β Trimer Using a Tethered Polymer Nanoarray

Abstract

Amyloid oligomers are considered the most neurotoxic species of amyloid aggregates. Spontaneous assembly of amyloids into aggregates is recognized as a major molecular mechanism behind Alzheimer's disease and other neurodegenerative disorders involving protein aggregation. Characterization of such oligomers is extremely challenging but complicated by their transient nature. Previously, we introduced a flexible nanoarray (FNA) method enabling us to probe dimers assembled by the amyloid β (14-23) [Aβ (14-23)] peptide. The study presented herein modifies and enhances this approach to assemble and probe trimers of Aβ (14-23). A metal-free click chemistry approach was used, in which dibenzocyclooctyne (DBCO) groups were incorporated at selected sites within the FNA template to click Aβ (14-23) monomers at their terminal azide groups. Atomic force microscopy (AFM) force spectroscopy was employed to characterize the assemblies. The force measurement data demonstrate that the dissociation of the trimer undergoes a stepwise pattern, in which the first monomer dissociates at the rupture force ∼48 ± 2.4 pN. The remaining dimer ruptures at the second step at a slightly larger rupture force (∼53 ± 3.2 pN). The assembled trimer was found to be quite dynamic, and transient species of this inherently dynamic process were identified.

AFM Probing of Amyloid-Beta 42 Dimers and Trimers

“…Analysis of such force curves was performed in the framework of the WLC model (see section "Materials and Methods"), which resulted in rupture forces of 55 and 109 pN for the first and second peaks, respectively. The first value corresponds well to the rupture force value for Aβ42 dimers obtained in our early force spectroscopy studies (Lv et al, 2013b;Kim and Lyubchenko, 2014), whereas the second peak value is in line with the rupture values of streptavidin-biotin bonds (Teulon et al, 2011;Maity et al, 2018a). This conclusion is supported by a statistical analysis of 202 rupture events out of several thousands of probing events.…”

“…The force distribution for the second peak is shown in Figure 3A, and the maxima of the Gaussian fitting occurs at 115 ± 7 pN. This value corresponds well to the biotinstreptavidin bonds dissociation strength (Guo et al, 2008;Teulon et al, 2011;Maity et al, 2018a). The contour length distribution for the final rupture event is shown in Figure 3B.…”

“…Mica surface was functionalized with streptavidin by using our previous protocol (Maity et al, 2018a). Briefly, a freshly cleaved mica surface was treated with 167 µM APS solution and incubated for 30 min in a humidified chamber, followed by multiple rinse steps with DI water.…”

“…The FNA tether contains a terminal thiol group separated from the Aβ anchoring point by one PA unit. The FNA was attached with the AFM probe at this terminal thiol by maleimidethiol reaction using our developed protocol (Maity et al, 2016(Maity et al, , 2018a. The other end of the FNA scaffold separated from another anchoring point for Aβ42 monomer is terminated with biotin and is used in the Aβ42 dimer stretching experiments, in which the surface is functionalized with streptavidin.…”

Trans-toxin ion-sensitivity of charybdotoxin-blocked potassium-channels reveals unbinding transitional states

“…This network of interacting partners, together with thermal agitation, could yield to several different energetically equivalent dissociation pathways. There are recent and old examples of rare and ephemeral intermediate states during protein folding (Raschke and Marqusee, 1997; Tapia-Rojo et al, 2019) or during protein oligomerization (Maity et al, 2018). Nevertheless, remain to be shown if states that arise during mechanically stretching the protein can be spontaneously visited.…”