Journal
BIOCHEMISTRY
Volume 44, Issue 14, Pages 5478-5487Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi047611e
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Copper is implicated in the in vitro formation and toxicity of Alzheimer's disease amyloid plaques containing the beta-amyloid (A beta) peptide (Bush, A. I., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 11934). By low temperature electron paramagnetic resonance (EPR) spectroscopy, the importance of the N-terminus in creating the Cu2+ binding site in native A beta has been examined. Peptides that contain the proposed binding site for Cu2+-three histidines (H6, H13, and H14) and a tyrosine (Y10)-but lack one to three N-terminal amino acids, do not bind Cu2+ in the same coordination environment as the native peptide. EPR spectra of soluble A beta with stoichiometric amounts of Cu2+ show type 2 Cu2+ EPR spectra for all peptides. The ligand donor atoms to Cu2+ are 3N1O when Cu2+ is bound to any of the A beta peptides (A beta 16, A beta 28, A beta 40, and A beta 42) that contain the first 16 amino acids of full-length A beta. When a Y10F mutant of A beta is used, the coordination environment for Cu2+ remains 3N1O and Cu2+ EPR spectra of this mutant are identical to the wild-type spectra. Isotopic labeling experiments show that water is not the O-atom donor to Cu2+ in A beta fibrils or in the Y10F mutant. Further, we find that Cu2+ cannot be removed from Cu2+-containing fibrils by washing with buffer, but that Cu2+ binds to fibrils initially assembled without Cu2+ in the same coordination environment as in fibrils assembled with Cu2+. Together, these results indicate (1) that the O-atom donor ligand to Cu2+ in A beta is not tyrosine, (2) that the native Cu2+ binding site in A beta is sensitive to small changes at the N-terminus, and (3) that Cu2+ binds to A beta fibrils in a manner that permits exchange of Cu2+ into and out of the fibrillar architecture.
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