An Electrochemistry and Computational Study at an Electrified Liquid-Liquid Interface for Studying Beta-Amyloid Aggregation

Abstract

Amphiphilic peptides, such as Aß amyloids, can adsorb at an interface between two immiscible electrolyte solutions (ITIES). Based on previous work (vide infra), a hydrophilic/hydrophobic interface is used as a simple biomimetic system for studying drug interactions. The ITIES provides a 2D interface to study ion-transfer processes associated with aggregation, as a function of Galvani potential difference. Here, the aggregation/complexation behaviour of Aβ_(1-42) is studied in the presence of Cu (II) ions, together with the effect of a multifunctional peptidomimetic inhibitor (P6). Cyclic and differential pulse voltammetry proved to be particularly sensitive to the detection of the complexation and aggregation of Aβ_(1-42), enabling estimations of changes in lipophilicity upon binding to Cu (II) and P6. At a 1:1 ratio of Cu (II):Aβ_(1-42), fresh samples showed a single DPV (Differential Pulse Voltammetry) peak half wave transfer potential (E1/2) at 0.40 V. Upon increasing the ratio of Cu (II) two-fold, fluctuations were observed in the DPVs, indicating aggregation. The approximate stoichiometry and binding properties of Aβ_(1-42) during complexation with Cu (II) were determined by performing a differential pulse voltammetry (DPV) standard addition method, which showed two binding regimes. A pKa of 8.1 was estimated, with a Cu:Aβ_1-42 ratio~1:1.7. Studies using molecular dynamics simulations of peptides at the ITIES show that Aβ_(1-42) strands interact through the formation of β-sheet stabilised structures. In the absence of copper, binding/unbinding is dynamic, and interactions are relatively weak, leading to the observation of parallel and anti-parallel arrangements of β-sheet stabilised aggregates. In the presence of copper ions, strong binding occurs between a copper ion and histidine residues on two peptides. This provides a convenient geometry for inducing favourable interactions between folded β-sheet structures. Circular Dichroism spectroscopy (CD spectroscopy) was used to support the aggregation behaviour of the Aβ_(1-42) peptides following the addition of Cu (II) and P6 to the aqueous phase.

Keywords: aggregation; beta-amyloid; copper binding; drug–peptide interactions; electrified liquid–liquid interface; molecular dynamic simulations.

Figure 1

(a) Snapshot of the folded structure of Aβ_1-42 taken from the end of a 285 ns production run in water. (b) Protein secondary structure during the final 60 ns of the simulation, determined using the DSSP software [46,47].

Scheme 1

(A) Structure of amyloid-beta 1-42 and sequence showing electroactive amino acids and metal binding sites. (B) Possible Cu-Aβ complexes that are reported in literature. (C) Structure of peptidomimetic peptides (P6), P6 binds to Cu²⁺ through N-terminal glycine-histidine-lysine (GHK) groups and prevents its redox cycling in reducing conditions.

Scheme 1

(A) Structure of amyloid-beta 1-42 and sequence showing electroactive amino acids and metal binding sites. (B) Possible Cu-Aβ complexes that are reported in literature. (C) Structure of peptidomimetic peptides (P6), P6 binds to Cu²⁺ through N-terminal glycine-histidine-lysine (GHK) groups and prevents its redox cycling in reducing conditions.

Scheme 2

Configuration of the cells for the ITIES studies. x is the concentration ion in the aqueous phase. The double bar shows the polarised interface.

Figure 2

Snapshots from a 10 ns run showing the capture of the Aβ_1-42 peptide by a hydrophobic–hydrophilic interface, water molecules are shown in red (dots) and DCE molecules are shown in blue/gray (dots).

Figure 3

Snapshots showing aggregation of Aβ_1-42 peptides at a DCE–water interface. Four chains are shown in cyan, red, lime green and orange together with protein secondary structure. The dark blue box outline indicates the 2D profile of the periodic box, which shows an average dimension of ~6.25 nm × 6.15 nm.

Figure 4

Association of an Aβ_1-42 peptide dimer bound by copper at the dicholoroethane-water. The picture shows a snapshot form the end of a 1470.9 ns molecular dynamics run with a copper (II) ion linking two chains (as shown in Figures S3b and S4 (see ESI)). Individual chains are colour-coded in cyan and red, the copper ion is shown in yellow and the first residue of each chain is shown in white. The area of the interface is 9.95 nm × 9.94 nm.

Figure 5

(A): CVs of (a) background solutions (b) with 0.01 M Aβ_(1-42) added to the aqueous phase (c) with Cu(II)-Aβ_(1-42), 1:1 ratio in the aqueous phase, corresponding to the cells shown in Scheme 2, Cell 1, 2 and 3, using a scan rate 0.1 V/s. (B): CV of Cu-Aβ and P6, 1:4 ratio, using a scan rate 0.1 V/s at ITIES after 10 m and after 24 h.

Figure 6

Background, phosphate buffer, subtracted DPVs. (A): Solid line (Cell 2, Scheme 2): Aβ_1-42. Dotted line, CuAβ_1-42 1:1 (Cell 3, Scheme 2). Dashed line, CuAb_1-42; P6 1:4 (Cell 4, Scheme 2). (B): Effect of addition of 0.5 µM P6 to the 0.5 µM CuAβ_1-42 complex formed freshly. The signal starts decreasing when the ratio of P6 reaches 1:3. (C): Effect of excess Cu²⁺; (a): 0.02 μM Cu:0.01 μM Aβ_(1-42) in a ratio 2:1 showing increasing current with time. The fluctuations in current indicate aggregation phenomenon at the liquid–liquid interface. (b): DPV obtained upon addition of 0.03 μM P6, showing a decrease in current with time, (B).

Figure 7

Summary of the effect of time (0–60 min) on changes in current, at a potential of 0.46 V, observed at the electrified liquid–liquid interface for Aβ_(1-42), 1:1 Aβ_(1-42) Cu complex and upon addition of P₆ to the complex at an Aβ_(1-42) Cu:P6: 1:3 ratio.