Aggregation of the amyloid-beta (Aβ) peptide into insoluble plaques is a major factor in Alzheimer's disease (AD) pathology. Another major factor in AD is arguably metal ions, as metal dyshomeostasis is observed in AD patients, metal ions modulate Aβ aggregation, and AD plaques contain numerous metals including redox-active Cu and Fe ions. In vivo, Aβ is found in various cellular locations including membranes. So far, Cu(II)/Aβ interactions and ROS generation have not been investigated in a membrane environment. Here, we study Cu(II) and Zn(II) interactions with Aβ bound to SDS micelles or to engineered aggregation-inhibiting molecules (the cyclic peptide CP-2 and the ZAβ3(12-58)Y18L Affibody molecule). In all studied systems the Aβ N-terminal segment was found to be unbound, unstructured, and free to bind metal ions. In SDS micelles, Aβ was found to bind Cu(II) and Zn(II) with the same ligands and the same KD as in aqueous solution. ROS was generated in all Cu(II)/Aβ complexes. These results indicate that binding of Aβ to membranes, drugs, and other entities that do not interact with the Aβ N-terminal part, appears not to compromise the N-terminal segment's ability to bind metal ions, nor impede the capacity of N-terminally bound Cu(II) to generate ROS.

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The aggregation of amyloid-b (Ab) peptides is believed to be a major factor in the onset and progression of Alzheimer's disease. Molecules binding with high affinity and selectivity to Abpeptides are important tools for investigating the aggregation process. An Ab-binding Affibody molecule, Z Ab3 , has earlier been selected by phage display and shown to bind Ab(1-40) with nanomolar affinity and to inhibit Ab-peptide aggregation. In this study, we create truncated functional versions of the Z Ab3 Affibody molecule better suited for chemical synthesis production. Engineered Affibody molecules of different length were produced by solid phase peptide synthesis and allowed to form covalently linked homodimers by S-S-bridges. The N-terminally truncated Affibody molecules Z Ab3 (12-58), Z Ab3 (15-58), and Z Ab3 (18-58) were produced in considerably higher synthetic yield than the corresponding full-length molecule Z Ab3 (1-58). Circular dichroism spectroscopy and surface plasmon resonance-based biosensor analysis showed that the shortest Affibody molecule, Z Ab3 (18-58), exhibited complete loss of binding to the Ab(1-40)-peptide, while the Z Ab3 (12-58) and Z Ab3 (15-58) Affibody molecules both displayed approximately one order of magnitude higher binding affinity to the Ab(1-40)-peptide compared to the full-length Affibody molecule. Nuclear magnetic resonance spectroscopy showed that the structure of Ab(1-40) in complex with the truncated Affibody dimers is very similar to the previously published solution structure of the Ab(1-40)-peptide in complex with the full-length Z Ab3 Affibody molecule. This indicates that the N-terminally truncated Affibody molecules Z Ab3 (12-58) and Z Ab3 (15-58) are highly promising for further engineering and future use as binding agents to monomeric Ab(1-40).