Deprotonation of the Asp1-Ala2 Peptide Bond Induces Modification of the Dynamic Copper(II) Environment in the Amyloid-β Peptide near Physiological pH
Deprotonation of the Asp1-Ala2 Peptide Bond Induces Modification of the Dynamic Copper(II) Environment in the Amyloid-β Peptide near Physiological pH
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DOI：
10.1002/anie.200904512
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发表时间：
2009-01-01
影响因子：
16.6
通讯作者：
Faller, Peter
中科院分区：
文献类型：
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作者：
Hureau, Christelle;Coppel, Yannick;Faller, Peter
淀粉样蛋白-β（Aβ）肽的聚集和由聚集体产生的活性氧是阿尔茨海默病的两个关键特征。[1]铜离子与这两个事件有关，[2，3]因此确定Cu和Aβ的基本相互作用对于了解其在病理学发展中的作用至关重要。天然Aβ肽由39至43个氨基酸残基组成，并已显示出强烈的聚集倾向（从几μm浓度）。然而，CuII结合位点位于肽的N-末端部分，包括前16个氨基酸残基（参见肽序列支持信息中的方案S1），[4，5]是一种高度可溶的截短肽。因此，这种缩短的肽被认为是CuII与全长Aβ配位的有价值的模型，其高溶解度允许使用经典的光谱方法，例如本研究的方法。虽然大多数技术旨在鉴定CuII配体，（综述参见参考文献[6]，最近的报道参见参考文献[7，8]），NMR光谱是少数几种还能够揭示Cu II与Aβ配位的动力学过程的方法之一。实际上，Cu II离子的顺磁性诱导肽核的弛豫速率的增强，该效应根据原子间距离的六次方倒数而减小（综述参见参考文献[9，10]）。因此，观察到空间上接近金属离子结合位点的核的NMR信号的选择性加宽。在CuII的情况下，线加宽是严重的，并且在顺磁性离子的快速交换的情况下，可以检测到顺磁性离子的大部分亚化学计量比的影响。这也是真实的13 C NMR信号，尽管较低的灵敏度为该核的增宽效应，由于其较低的旋磁比相比，质子。关于CuII与Aβ的配位，仅报道了少数NMR研究，并且它们限于1H NMR [11，12]或1H-15 N杂原子单量子相关（HSQC）实验。[13在后一种情况下，快速酰胺质子交换导致apo-Aβ肽中几个氨基酸（包括Asp 1和三个His残基）的信号丢失，这种效应排除了CuII诱导的信号增宽的分析。出于这些原因，本文中我们关注13 C {1H} NMR光谱，这是检查CuII对Aβ肽信号的影响的直接方法。此外，已知在接近生理pH时，Aβ的两种CuII复合物共存，其在肽的质子化状态及其光谱特征方面不同。[6，15]它们在下文中被称为“低pH”和“高pH”物种。我们确定了参与铜II结合的氨基酸残基，并给出了明确的证据，在这两种形式的不同配体之间的平衡的存在。我们还提供了新的见解之间的pH值约6.6和8.7，这是由于Asp 1丙氨酸2肽键酰胺的去质子化和结合的Aβ中的铜II结合位点所经历的戏剧性变化。图1显示了在pH 6.6和8.7下加入0.1当量CuII后Aβ肽（序列DAEFRHDSGYEVHHQK）的13 C {1H} NMR光谱的变化（另见关于His残基光谱域的支持信息中的图S4）。[16加入CuII导致几种信号的加宽，其在高pH下更具选择性（图1中的右侧光谱），其中仅Asp 1、Ala 2和His的侧链主要受影响。更准确地说，在pH 6.6时，羧酸根基团的峰……
Aggregation of the amyloid-β (Aβ) 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 Aβ is essential for understanding its roles in the development of the pathology. The native Aβ peptides consist of 39 to 43 amino acid residues and have been shown to be strongly prone to aggregation (from a few μm concentration). However, the CuII 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 CuII coordination to full-length Aβ 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 CuII 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 CuII to Aβ.Indeed, the paramagnetism of the CuII 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 CuII, 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 13C 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 CuII coordination to Aβ, only a few NMR studies have been reported and they are limited to 1H NMR [11, 12] or 1H–15N 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–Aβ peptide in the latter cases, an effect that precludes the analysis of CuII-induced signal broadening. For those reasons, herein we focus on 13C {1H} NMR spectroscopy, which is a straightforward way to inspect the effect of CuII on Aβ peptide signals. Furthermore, it is known that near physiological pH, two CuII complexes of Aβ 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 CuII 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 the CuII binding sites in Aβ between pH values of about 6.6 and 8.7, which arises from the deprotonation and binding of the Asp1ÀAla2 peptide bond amide. Figure 1 shows the evolution of the 13C {1H} NMR spectra of the Aβ peptide (sequence DAEFRHDSGYEVHHQK) upon addition of 0.1 equivalents of CuII at pH 6.6 and 8.7 (see also FigureS4 in the Supporting Information for spectral domains that concern His residues).[16, 17] Addition of CuII leads to broadening of several signals that is more selective at high pH (right-hand spectra in Figure 1) with only Asp1, Ala2, and the side chain of His mainly affected. More precisely, at pH 6.6 peaks of the carboxylate groups …