The Second Cu(II)-Binding Site in a Proton-Rich Environment Interferes with the Aggregation of Amylold-β(1-40) into Amyloid Fibrils

The overall morphology and Cu(II) ion coordination for the aggregated amylold-beta(1-40) [A beta(1-40)] in N-ethylmorpholine (NEM) buffer are affected by Cu(II) ion concentration. This effect is Investigated by transmission electron microscopy (TEM), atomic force microscopy (AFM), and electron spin echo envelope modulation (ESEEM) spectroscopy. At lower than equimolar concentrations of Cu(II) ions, fibrillar aggregates of A beta(1-40) are observed. At these concentrations of Cu(II), the monomeric and fibrillar A beta(1-40) ESEEM data indicate that the Cu(II) ion is coordinated by histidine residues. For aggregated A beta(1-40) at a Cu(II):A beta molar ratio of 2:1, TEM and AFM images show both linear fibrils and granular amorphous aggregates. The ESEEM spectra show that the multi-histidine coordination for Cu(II) ion partially breaks up and becomes exposed to water or exchangeable protons of the peptide at a higher Cu(II) concentration. Since the continuous-wave electron spin resonance results also suggest two copper-binding sites in A beta(1-40), the proton ESEEM peak may arise from the second copper-binding site, which may be significantly involved in the formation of granular amorphous aggregates. Thioflavin T fluorescence and circular dichroism experiments also show that Cu(II) inhibits the formation of fibrils and induces a nonfibrillar beta-sheet coil formation. Therefore. we propose that A beta(1-40) has a second copper-binding site in a proton-rich environment and the second binding Cu(II) Ion interferes with a conformational transition into amyloid fibrils, Inducing the formation of granular amorphous aggregates.