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The aggregation of amyloid-β (Aβ) 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 Aβ-peptides are important tools for investigating the aggregation process. An Aβ-binding Affibody molecule, ZAβ3 , has earlier been selected by phage display and shown to bind Aβ(1-40) with nanomolar affinity and to inhibit Aβ-peptide aggregation. In this study, we create truncated functional versions of the ZAβ3 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 ZAβ3 (12-58), ZAβ3 (15-58), and ZAβ3 (18-58) were produced in considerably higher synthetic yield than the corresponding full-length molecule ZAβ3 (1-58). Circular dichroism spectroscopy and surface plasmon resonance-based biosensor analysis showed that the shortest Affibody molecule, ZAβ3 (18-58), exhibited complete loss of binding to the Aβ(1-40)-peptide, while the ZAβ3 (12-58) and ZAβ3 (15-58) Affibody molecules both displayed approximately one order of magnitude higher binding affinity to the Aβ(1-40)-peptide compared to the full-length Affibody molecule. Nuclear magnetic resonance spectroscopy showed that the structure of Aβ(1-40) in complex with the truncated Affibody dimers is very similar to the previously published solution structure of the Aβ(1-40)-peptide in complex with the full-length ZAβ3 Affibody molecule. This indicates that the N-terminally truncated Affibody molecules ZAβ3 (12-58) and ZAβ3 (15-58) are highly promising for further engineering and future use as binding agents to monomeric Aβ(1-40).