Cu(II) binding to monomeric, oligomeric, and fibrillar forms of the Alzheimer's disease amyloid-β peptide

Copper has been proposed to play a role in Alzheimer's disease through interactions with the amyoid-beta (A beta) peptide. The coordination environment of bound copper as a function of Cu:A beta stoichiometry and A beta oligomerization state are particularly contentious. Using low-temperature electron paramagnetic resonance (EPR) spectroscopy, we spectroscopically distinguish two Cu(II) binding sites on both soluble and fibrillar A beta (for site 1, A(parallel to) = 168 +/- 1 G and g(parallel to) = 2.268; for site 2, A(parallel to) = 157 +/- 2 G and g(parallel to) = 2.303). When fibrils that have been incubated with more than 1 equiv of Cu(II) are washed, the second Cu(II) ion is removed, indicating that it is only weakly bound to the fibrils. No change in the Cu(II) coordination environment is detected by EPR spectroscopy of Cu(II) with A beta (1: 1 ratio) collected as a function of A beta fibrillization time, which indicates that the Cu(II) environment is independent of A beta oligomeric state. The initial Cu(II)-A beta complexes go on to form Cu(II)-containing A beta fibrils. Transmission electron microscopy images of A beta fibrils before and after Cu(II) addition are the same, showing that once incorporated, Cu(II) does not affect fibrillar structure; however, the presence of Cu(II) appears to induce fibril-fibril association. On the basis of our results, we propose a model for Cu(II) binding to A beta during fibrillization that is independent of peptide oligomeric state.