Effects of Zinc Binding and Chelation on Structures and Dynamics in Amyloid-Beta Dimers

Abstract

Amyloid-β (Aβ) is a protein of 39-43 amino acids that self-associates into a diverse array of neurotoxic aggregates linked to Alzheimer's disease (AD). Recently, Aβ dimers and trimers have been shown to cause memory deficits when they are recruited to the synapse, perhaps due to zinc release during neurotransmission. We have employed single-molecule fluorescence methods to investigate dimer structures, to understand zinc-induced structural change and determine whether it may be reversed by metal chelation. Single-pair FRET measurements were performed using Aβ40 peptides labeled at the N-termini with donor and acceptor dyes; the donor peptide was additionally labeled at the C-terminus with a Lys-biotin moiety to permit tethering to a functionalized cover slip. Time-dependent FRET efficiencies (EFRET) were determined by measuring fluorescence from individual surface-tethered dimers, affording insight into dimer structures and structural dynamics. Characteristic EFRET values were determined for dimers in metal-free samples and in the presence of Zn2+ (1 equiv.), with and without excess amounts of the zinc chelator clioquinol (CQ, 10 equiv.). Under all sample conditions, dimers appear to exhibit at least two characteristic structures, as evidenced by at least two broad peaks in each of the EFRET ensemble histograms. Zinc binding causes a slight structural change that is not reversed by chelation. Strikingly, zinc also severely limits dimer structural dynamics: while 29% of dimers in metal-free samples visit two EFRET values over time, only 12% of dimers in the presence of zinc exhibit structural change. Chelation does not reverse this effect, as only 9% of dimers in the presence of CQ show structural dynamics. These results lend new insights into the role(s) of zinc in Aβ association and the use of metal chelators in AD prevention and treatment.