期刊
BIOCHEMISTRY
卷 48, 期 20, 页码 4388-4402出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi900254n
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资金
- Biotechnology and Biological Sciences Research Council
- Biotechnology and Biological Sciences Research Council [BB/D005027/1] Funding Source: researchfish
- BBSRC [BB/D005027/1] Funding Source: UKRI
Cu2+ ions are found concentrated within senile plaques of Alzheimer's disease patients directly bound to amyloid-beta peptide (A beta) and are linked to the neurotoxicity and self-association of A beta. The affinity of Cu2+ for monomeric A beta is highly disputed, and there have been no reports of affinity of Cu2+ for fibrillar A beta. We therefore measured the affinity of Cu2+ for both monomeric and fibrillar A beta(1-42) using two independent methods: fluorescence quenching and circular dichroism. The binding curves were almost identical for both fibrillar and monomeric forms. Competition studies with free glycine, L-histidme, and nitrilotriacetic acid (NTA) indicate an apparent (conditional) dissociation constant of 10(-11) M, at pH 7.4. Previous studies of Cu-A beta have typically found the affinity 2 or more orders of magnitude weaker, largely because the affinity of competing ligands or buffers has been underestimated. A beta fibers are able to bind a full stoichiometric complement of Cu2+ ions with little change in their secondary structure and have coordination geometry identical to that of monomeric A beta. Electron paramagnetic resonance studies (EPR) with A beta His/Ala analogues suggest a dynamic view of the tetragonal Cu2+ complex, with axial as well as equatorial coordination of imidazole nitrogens creating an ensemble of coordination geometries in exchange between each other. Furthermore, the N-terminal amino group is essential for the formation of high-pH complex II. The A beta(1-28) fragment binds an additional Cu2+ ion compared to full-length A beta, with appreciable affinity. This second binding site is revealed in A beta(1-42) upon addition of methanol, indicating hydrophobic interactions block the formation of this weaker carboxylate-rich complex. A Cu2+ affinity for A beta of 10(11) M-1 supports a modified amyloid cascade hypothesis in which Cu2+ is central to A beta neurotoxicity.
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