A Solid-State NMR Study Reveals Structure and Dynamics in Copper(ii)-Binding to Alzheimer's Beta-Amyloid Fibrils

Abstract

β-amyloid (Aβ) peptide associated with Alzheimer's diseases exhibit neural toxicity upon aggregation. One of the most widespread hypotheses on the origin of the toxicity of Aβ aggregates is the binding of Cu2+ ions to Aβ fibrils and subsequent generation of reactive oxygen species (ROS) such as H2O2 by Cu2+-bound Aβ. Although a variety of studies have been performed on Cu2+-binding to Aβ, the proposed binding sites or models have been controversial partly because non-crystalline and insoluble nature of Aβ fibrils have limited access to site-specific structure and dynamic properties on Cu2+-bound Aβ fibrils. Here, we examine the effect of Cu2+ binding to amyloid fibrils of 40-residue Aβ(1-40) by UV-VIS spectroscopy and solid-state NMR (SSNMR). Specifically, we will answer the following questions (i) Is the Cu2+ binding is site specific? (ii) If so, which sites are involved in binding? (iii) Are there any major structural changes introduced by Cu2+ binding or oxidization due to Cu2+?UV-VIS spectroscopy showed that Cu2+ binds to Aβ(1-40) fibrils almost completely when the ratio of Cu2+ to Aβ(1-40) is less than 1. Based on the result, we performed high-resolution SSNMR experiments on Cu2+-bound Aβ(1-40) fibrils. First, the 13C T1 paramagnetic relaxation enhancement (PRE) due to Cu2+ binding on Aβ was measured for different residues; the PRE data highlight possible binding sites, where the relaxation enhancements are notable. The analysis indicates that the binding is specific, and Cu2+ most likely binds to His-13/14 and His-6. Second, the comparison of 2D 13C/13C correlation spectra of Aβ fibrils with and without Cu2+ revealed that the secondary structure of Aβ(1-40) fibrils is largely unaltered by Cu2+ binding. Third, we tested a previously proposed hypothesis that Met-35/Gly-33 can be oxidized[4] by Cu2+ to produce ROS by SSNMR.