Engineered non-fluorescent Affibody molecules facilitate studies of the amyloid-beta (Aβ) peptide in monomeric form: low pH was found to reduce Aβ/Cu(II) binding affinity

Abstract

Aggregation of amyloid-beta (Aβ) peptides into oligomers and amyloid plaques in the human brain is considered a causative factor in Alzheimer's disease (AD). As metal ions are over-represented in AD patient brains, and as distinct Aβ aggregation pathways in presence of Cu(II) have been demonstrated, metal binding to Aβ likely affects AD progression. Aβ aggregation is moreover pH-dependent, and AD appears to involve inflammatory conditions leading to physiological acidosis. Although metal binding specificity to Aβ varies at different pH's, metal binding affinity to Aβ has so far not been quantitatively investigated at sub-neutral pH levels. This may be explained by the difficulties involved in studying monomeric peptide properties under aggregation-promoting conditions. We have recently devised a modified Affibody molecule, Z(Aβ3)(12-58), that binds Aβ with sub-nanomolar affinity, thereby locking the peptide in monomeric form without affecting the N-terminal region where metal ions bind. Here, we introduce non-fluorescent Aβ-binding Affibody variants that keep Aβ monomeric while only slightly affecting the Aβ peptide's metal binding properties. Using fluorescence spectroscopy, we demonstrate that Cu(II)/Aβ(1-40) binding is almost two orders of magnitude weaker at pH 5.0 (apparent K(D)=51 μM) than at pH 7.3 (apparent K(D)=0.86 μM). This effect is arguably caused by protonation of the histidines involved in the metal ligandation. Our results indicate that engineered variants of Affibody molecules are useful for studying metal-binding and other properties of monomeric Aβ under various physiological conditions, which will improve our understanding of the molecular mechanisms involved in AD.