Solid-State NMR Studies of Amyloid Materials: A Protocol to Define an Atomic Model of Aβ(1-42) in Amyloid Fibrils.
Solid-State NMR Studies of Amyloid Materials: A Protocol to Define an Atomic Model of Aβ(1-42) in Amyloid Fibrils.
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淀粉样蛋白材料的固态核磁共振研究： 定义淀粉样原纤维中 Aβ(1-42) 原子模型的协议。
DOI：
10.1007/978-1-4939-7811-3_26
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Ishii,Yoshitaka
中科院分区：
文献类型：
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作者：
Xiao,Yiling;McElheny,Dan;Hoshi,Minako;Ishii,Yoshitaka
为了深入了解阿尔茨海默病的病理机制，人们对错误折叠的β淀粉样蛋白（Aβ）的分子结构进行了大量的研究。固态核磁共振波谱（SSNMR）被认为是阐明不溶性和非晶体淀粉样蛋白原纤维和其他淀粉样蛋白组装结构的主要工具。在本章中，我们描述了一个详细的方案，从我们最近的SSNMR研究中获得结构均匀的淀粉样蛋白原纤维中42个残基的人a β肽a β(1-42)的第一个原子模型（Nat Struct Mol Biol 22:499-505, 2015）。尽管对Aβ(1-42)原纤维有很大的生物学和临床兴趣，但直到本研究之前，它们的结构细节一直难以捉摸。该协议分为四个部分。首先，描述了固相肽合成（SPPS）和纯化单体Aβ(1-42)。我们展示了一种受控的孵育方法，以碎片化的a β(1-42)原纤维作为种子，在水溶液中促使a β(1-42)错误折叠成均匀的淀粉样蛋白原纤维。接下来，我们通过SSNMR详细分析Aβ(1-42)原纤维以获得结构约束。最后，我们描述了基于SSNMR结果通过两阶段分子动力学计算构建Aβ(1-42)原纤维原子模型的方法。
Intense efforts have been made to understand the molecular structures of misfolded amyloid β (Aβ) in order to gain insight into the pathological mechanism of Alzheimer’s disease. Solid-state NMR spectroscopy (SSNMR) is considered a primary tool for elucidating the structures of insoluble and noncrystalline amyloid fibrils and other amyloid assemblies. In this chapter, we describe a detailed protocol to obtain the first atomic model of the 42-residue human Aβ peptide Aβ(1–42) in structurally homogeneous amyloid fibrils from our recent SSNMR study (Nat Struct Mol Biol 22:499–505, 2015). Despite great biological and clinical interest in Aβ(1–42) fibrils, their structural details have been long-elusive until this study. The protocol is divided into four sections. First, the solid-phase peptide synthesis (SPPS) and purification of monomeric Aβ(1–42) is described. We illustrate a controlled incubation method to prompt misfolding of Aβ(1–42) into homogeneous amyloid fibrils in an aqueous solution with fragmented Aβ(1–42) fibrils as seeds. Next, we detail analysis of Aβ(1–42) fibrils by SSNMR to obtain structural restraints. Finally, we describe methods to construct atomic models of Aβ(1–42) fibrils based on SSNMR results through two-stage molecular dynamics calculations.