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

Abstract

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.

Keywords: AFM; Alzheimer’s disease; Huntington’s disease; Parkinson’s disease; amyloids; atomic force microscopy; force spectroscopy; nanoimaging; nanomedicine.

Figure 1. Schematic of AFM force spectroscopy experiment used to measure the interactions of molecules immobilized on the AFM tip and surface via flexible tethers

The experimental force-distance curve is shown with blue. Red line is the fit of the experimental data with Worm-Like Chain (WLC) model. Cartoon (A) shows the approach stage at which tethered molecules are far from each other. Cartoon (B) illustrates the complex formation, which occurs at small tip-sample distance. The retraction of the tip as shown in cartoon (C) is accompanied by stretching of the tether and this entropy driven process is approximated with the WLC fit (red curve). After full stretching of the tether, the rupture occurs at the position indicated with a red arrow. After the complex rupture, there is no interaction between the molecules as evidenced by no change of the force on the tip-sample distance (horizontal red line).

Figure 2. Schematic for single-molecule probing of interactions between amyloid β peptides Aβ 40

The protein is immobilized onto an APS functionalized mica surface via a flexible PEG tether. The same protein is also immobilized to the AFM tip using the MAS tethering method [38,42].

Figure 3. Contour length analysis of rupture events for Aβ 40 dimers

(A) The individual force curve (blue) approximated with the worm-like chain model (WLC, red line). The values for the rupture force (F_R = 111 pN) and contour length (L_c = 45 nm) are indicated. (B) A set of four force curves with different rupture lengths. The rupture positions are indicated with arrows. (C) The model for the formation of misfolded Aβ40 dimers with different conformations corresponding to different contour lengths, L_c1, L_c2 and L_c3, as measured in the force probing experiments. See paper [42] for details. Copyright © 2011, American Chemical Society.

Figure 4. The contour length distributions for Aβ 40 and Aβ 42 dimers are shown in A and B, respectively

The positions of interacting segments are aligned according to the contour length distribution. The dotted lines denote the overall distribution profiles and the peaks are approximated according to the profiles. The arrows indicate major interacting segments.

Figure 5. Contour length distribution for α-Syn variants—group B force-distance curves with a single rupture

A) wild type [with maxima at 28 ± 2 nm (peak 0), 34 ± 2 nm (peak 1), 44 ± 4 nm (peak 2), 54 ± 3 nm (peak 3) and 68 ± 4 nm (peak 4)], B) A30P mutant (with maxima at 30 ± 1, 36 ± 2, 41 ± 2 and 49 ± 2 nm), C) A53T mutant (with maxima at 26 ± 3, 33 ± 4 and 44 ± 3 nm), D) E46K mutant (with maxima at 17 ± 3, 31 ± 4 and 40 ± 2 nm). The figure was reproduced from [54]. Copyright © 2013, American Chemical Society.

Figure 6. AFM spectroscopy in the presence and absence of Cu²⁺ cations at pH 7.4

The columns include, from left to right: the overlap of all raw force curves, the distribution of contour length (middle), and the distribution of rupture forces (right). The L_c and F_r denote the most probable contour length and the most probable rupture force, respectively. Figure was reproduced from [44]. Copyright © 2012, Springer Science+Business Media New York.

Figure 7. DFS analysis of α-Syn interactions

(A) DFS plot for AFM probing of α-Syn interactions measured at pH 5.1. The linear relationship indicates that the dissociation of an α-Syn dimer follows a one-barrier path shown in (B). The energy of the barrier is 28.1 k_BT; the complex lifetime is 0.27 ± 0.13 s. The figure was reproduced from [47]. Copyright © 2008, Elsevier Ltd..

Figure 8. Computational studies of Aβ (14–23) interactions

(A) The time-dependent of the N-N distance for the monomers, obtained from the all-atom MD simulation of two closely positioned monomers. Snapshots of the monomers conformations for selected points on the time trajectory are indicated above the graph. (B) Time-dependent secondary structure of the monomers (DSSP diagram) for the next 400 ns simulations. The snapshot for the representative conformation of the dimer is indicated above the graph. Amber-ff99sb*-ILDN force field was used in the simulations. The figure was reproduced from [67]. Copyright © 2013, American Chemical Society.

Figure 9. A schematic representation of protein dynamics

The transitions of the folded (native) state of the protein to unfolded and misfolded states are shown for simplicity only. Characteristic times for the transition between these states and the dimer lifetime are shown above the arrows.