ESR Spectroscopy Provides Direct Evidence of Cu$^{2+}$ Coordination by Three Histidine Residues in A$\beta _{1-16}$

We provide direct evidence that all three histidine residues in amyloid-$\beta _{1-16}$ (A$\beta _{1-16})$ coordinate to Cu$^{2+}$. In our approach, we generate three A$\beta _{1-16}$ analogues, in each of which a selected histidine residue is isotopically enriched with $^{15}$N. Pulsed electron spin resonance (ESR) experiments such as electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation (HYSCORE) clearly show that each of the three histidine imidazole rings at position 6, 13, and 14 in A$\beta _{1-16}$ binds to Cu$^{2+}$ as each of the three Cu$^{2+}$--$^{15}$N-labeled A$\beta _{1-16 }$complexes displays ESEEM and HYSCORE spectra which are distinctively different from those of the Cu$^{2+}$--nonlabeled A$\beta _{1-16}$ complex. The method employed here does not require either chemical side-chain modification or amino acid residue replacement, each of which is traditionally used to determine whether an amino acid residue in a protein binds to a metal ion. We also find that the histidine coordination in A$\beta _{1-16}$ is independent of the Cu$^{2+}$-to-peptide ratio, which is in contrast to the case of A$\beta _{1-40}$. The ESR results suggest tight binding between the histidine residues and the Cu$^{2+}$ ion, which is likely the reason of the high binding affinity of A$\beta $ peptide to Cu$^{2+}$.