Deprotonation of the Asp1Ala2 Peptide Bond Induces Modification of the Dynamic Copper(II) Environment in the Amyloid‐β Peptide near Physiological pH

Abstract

Aggregation of the amyloid-b (Ab) peptide and the production of reactive oxygen species by aggregates are two key features in Alzheimer's disease. [1] Copper ions have been linked to both of these events, [2,3] and hence determination of the basic interaction of Cu and Ab is essential for understanding its roles in the development of the pathology. The native Ab peptides consist of 39 to 43 amino acid residues and have been shown to be strongly prone to aggregation (from a few mm concentration). However, the Cu II binding site has been localized in the N-terminal part of the peptide encompassing the first 16 amino acid residues (see Scheme S1 in the Supporting Information for the peptide sequence), [4,5] a truncated peptide that is highly soluble. Hence, this shortened peptide is accepted as a valuable model of Cu II coordination to full-length Ab and its high solubility allows classical spectroscopic methods, such as those of the present study, to be used. While most techniques aim at identifying the Cu II ligands (for a review, see reference [6] and for very recent reports, see references [7,8]), NMR spectroscopy is among the few methods also able to reveal dynamical processes in the coordination of Cu II to Ab.Indeed, the paramagnetism of the Cu II ion induces an enhancement of the relaxation rate of the peptide nuclei, this effect diminishing according to the inverse sixth power of the interatomic distance (for reviews, see references [9, 10]). Consequently, selective broadening of the NMR signals of nuclei spatially close to the metal-ion binding site(s) is observed. In the case of Cu II , the line broadening is severe and the effect of the largely substoichiometric ratio of the paramagnetic ion is detectable in the case of fast exchange of the paramagnet between sites. This is also true for 13 C NMR signals despite the lower sensitivity to broadening effects for this nucleus as a result of its lower gyromagnetic ratio compared to that of the proton.As concerns Cu II coordination to Ab, only a few NMR studies have been reported and they are limited to 1 H NMR [11,12] or 1 H-15 N heteronuclear single quantum correlation (HSQC) experiments. [13,14] Fast amide proton exchanges are responsible for the loss of the signals of several amino acids (including Asp1 and the three His residues) in apo-Ab peptide in the latter cases, an effect that precludes the analysis of Cu II -induced signal broadening. For those reasons, herein we focus on 13 C{ 1 H} NMR spectroscopy, which is a straightforward way to inspect the effect of Cu II on Ab peptide signals. Furthermore, it is known that near physiological pH, two Cu II complexes of Ab coexist, which differ in the protonation state of the peptide and their spectroscopic signatures. [6,15] They are referred to below as "low-pH" and "high-pH" species. We identify the amino acid residues involved in Cu II binding, and give clear-cut evidence for the presence of equilibria between different ligands in both forms. We also give new insights into the dramatic change undergone by ...

Copper(II) Coordination to Amyloid β: Murine versus Human Peptide

“…[25] The results obtained were in line with most previous studies [12,19,22,26,27] and showed that the equatorial binding site of component I is formed by the NH 2 group of Asp1, two of the three imidazole rings of His6, His13, and His14, and a CO function. At higher pH values, deprotonation of the Asp1ÀAla2 peptide bond leads to the replacement of one imidazole ring with the Asp1ÀAla2 deprotonated amide (amidyl) ligand.…”

“…Gaggelli et al did not propose coordination of the amidyl function on the basis of their NMR spectroscopic data obtained at pH 7.5 in a micellar solution. [21] The most plausible reason for this discrepancy with our results is that, as we previously detailed, [25] broadening of the side-chain signals as a result of Cu II binding is more important than the broadening of backbone signals.…”

“…[25] This difference is even more evident from the 13 C NMR spectra in Figure 2, in which the effect of Cu II on the C a regions of hAb and mAb peptides are shown for I and II. For I, there is no significant difference between hAb and mAb in the broadening of the signals for the His, Asp, and Ala2 residues upon Cu II binding.…”

“…[30] Cu II ions bound to both Asp1 and Ala2 in the hAb peptide might enhance the formation of p3E-hAb by assisting with the hydrolysis of the Ala2ÀGlu3 bond. [25,27] Such an effect cannot apply in the case of the mAb peptide.…”

CuII Binding to Various Forms of Amyloid‐β Peptides: Are They Friends or Foes?

“…The apical position may be occupied by an oxygen atom that comes either from a water molecule, [70] or from carboxylate groups from the side chains of Asp or Glu. [71] In component II, the main equatorial Cu II ligands are the N-terminal amine, the amidyl function from the Asp1-Ala2 peptide bond, the adjacent CO from the Ala2-Glu3 peptide bond, and one N atom from the imidazole ring of one of the three His.…”