Capturing a Reactive State of Amyloid Aggregates: NMR-based Characterization of Cu-ion Bound Alzheimer’s Amyloid {beta} Fibrils in a Redox Cycle [Molecular Bases of Disease]

February 12th, 2014 by Parthasarathy, S., Yoo, B., McElheny, D., Tay, W., Ishii, Y.

The interaction of redox-active Cu ions with misfolded amyloid β (Aβ) is linked to production of reactive oxygen species (ROS), which has been associated with oxidative stress and neuronal damages in Alzheimer′s disease (AD). Despite intensive studies, it is still not conclusive how the interaction of Cu+/Cu2+ with Aβ aggregates leads to ROS production even at the in-vitro level. In this study, we examined the interaction between Cu+/Cu2+ and Aβ fibrils by solid-state NMR (SSNMR) and other spectroscopic methods. Our photometric studies confirmed the production of ~60 μM hydrogen peroxide (H2O2) from a solution of 20 μM Cu2+ ions in complex with Aβ(1-40) in fibrils ([Cu2+]/[Aβ] = 0.4) within 2 hours of incubation after addition of biological ascorbate at the physiological concentration (~1 mM). Furthermore, SSNMR 1H T1 measurements demonstrated that during ROS production, conversion of paramagnetic Cu2+ into diamagnetic Cu+ occurs while the reactive Cu+ ions remain bound to the amyloid fibrils. The results also suggest that O2 is required for rapid recycling of Cu+ bound to Aβ back to Cu2+, which allows for continuous production of H2O2. Both 13C and 15N SSNMR results show that Cu+ coordinates to Aβ(1-40) fibrils primarily through the side chain Nδ of both His-13 and His-14, suggesting major rearrangements from the Cu2+ coordination via Nϵ in the redox cycle. 13C SSNMR chemical-shifts analysis suggests that the overall Aβ conformations are largely unaffected by Cu+-binding. These results present crucial site-specific evidence of how the full-length Aβ in amyloid fibrils offers catalytic Cu+-centers.
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