There is now direct evidence that copper is bound to amyloid-␤ peptide (A␤) in senile plaque of Alzheimer's disease. Copper is also linked with the neurotoxicity of A␤ and free radical damage, and Cu 2؉ chelators represent a possible therapy for Alzheimer's disease. We have therefore used a range of complementary spectroscopies to characterize the coordination of Cu 2؉ to A␤ in solution. The mode of copper binding is highly pH-dependent. EPR spectroscopy indicates that both coppers have axial, Type II coordination geometry, square-planar or square-pyramidal, with nitrogen and oxygen ligands. Circular dichroism studies indicate that copper chelation causes a structural transition of A␤. Competition studies with glycine and L-histidine indicate that copper binds to A␤-(1-28) at pH 7.4 with an affinity of K a ϳ10 M؊1 .

Cu(2+) ions are found concentrated within senile plaques of Alzheimer's disease patients directly bound to amyloid-beta peptide (Abeta) and are linked to the neurotoxicity and self-association of Abeta. The affinity of Cu(2+) for monomeric Abeta is highly disputed, and there have been no reports of affinity of Cu(2+) for fibrillar Abeta. We therefore measured the affinity of Cu(2+) for both monomeric and fibrillar Abeta(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-histidine, and nitrilotriacetic acid (NTA) indicate an apparent (conditional) dissociation constant of 10(-11) M, at pH 7.4. Previous studies of Cu-Abeta have typically found the affinity 2 or more orders of magnitude weaker, largely because the affinity of competing ligands or buffers has been underestimated. Abeta fibers are able to bind a full stoichiometric complement of Cu(2+) ions with little change in their secondary structure and have coordination geometry identical to that of monomeric Abeta. Electron paramagnetic resonance studies (EPR) with Abeta His/Ala analogues suggest a dynamic view of the tetragonal Cu(2+) 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 Abeta(1-28) fragment binds an additional Cu(2+) ion compared to full-length Abeta, with appreciable affinity. This second binding site is revealed in Abeta(1-42) upon addition of methanol, indicating hydrophobic interactions block the formation of this weaker carboxylate-rich complex. A Cu(2+) affinity for Abeta of 10(11) M(-1) supports a modified amyloid cascade hypothesis in which Cu(2+) is central to Abeta neurotoxicity.

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The casein milk proteins and the brain proteins a-synuclein and tau have been described as natively unfolded with random coil structures, which, in the case of a-synuclein and tau, have a propensity to form the ®brils found in a number of neurodegenerative diseases. New insight into the structures of these proteins has been provided by a Raman optical activity study, supplemented with dierential scanning calorimetry, of bovine b-and j-casein, recombinant human a-, b-and c-synuclein, together with the A30P and A53T mutants of a-synuclein associated with familial cases of Parkinson's disease, and recombinant human tau46 together with the tau46 P301L mutant associated with inherited frontotemporal dementia. The Raman optical activity spectra of all these proteins are very similar, being dominated by a strong positive band centred at » 1318 cm )1 that may be due to the poly(L-proline) II (PPII) helical conformation.There are no Raman optical activity bands characteristic of extended secondary structure, although some unassociated b strand may be present. Dierential scanning calorimetry revealed no thermal transitions for these proteins in the range 15±110°C, suggesting that the structures are loose and noncooperative. As it is extended,¯exible, lacks intrachain hydrogen bonds and is hydrated in aqueous solution, PPII helix may impart a rheomorphic (¯owing shape) character to the structure of these proteins that could be essential for their native function but which may, in the case of a-synuclein and tau, result in a propensity for pathological ®bril formation due to particular residue properties.

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