Characterizing Alzheimer's Amyloid-beta Oligomer Structures by Solid-State NMR and Cryo-Electron Microscopy

项目总结 在阿尔茨海默病(AD)的研究中，大部分注意力都集中在淀粉样蛋白-β(Aβ)的寡聚体上 多肽(一种小蛋白质)。齐聚物是由50个或更少的多肽聚集而成的小纳米颗粒 分子。多肽聚集最容易产生淀粉样纤维，这是一个谜，是什么阻止了一些 低聚物不会进一步聚集成纤维。即使在低聚物和原纤维聚集体中，多肽 已经检测到具有一系列结构的聚集体。然而，结构生物学方法需要 稳定和结构均匀的样品，以实现高分辨率的信息。为了阐明什么 齐聚物结构是可能的，这些结构中的哪些应该作为AD治疗的靶点，实验 旨在了解β程序集的一般术语。初步数据显示，有可能生产出 150Aβ齐聚物(32Aβ多肽分子)的稳定均相样品及其结构研究 固体核磁共振(核磁共振)和电子显微镜(EM)。值得注意的是，150 kDa的低聚物 结构不同于之前所理解的任何β组件，这一观察提供了一个独特的机会 探讨齐聚物和原纤维的结构有何不同。该项目将追求高分辨率的原子级 通过整合固态核磁共振、低温电子显微镜和计算模型来表征150 kDa低聚物。 这项工作进一步试图通过测试假设来推广对β装配机制的理解 受150 kDa齐聚物启发的组装路径。拟议的工作将：实现三维图像 使用冷冻-EM(目标1)测量150 kDa低聚物；测量互补的原子级结构约束 固态核磁共振(目标2)；并测试机械假说，以产生与EM和 核磁共振工作流程(目标3)。了解β可能的低聚物结构(以及如何分离特定的 结构)对于开发新的阿尔茨海默氏症疗法、实施早期检测策略、 以及在疾病模型中设计机制研究。

PROJECT SUMMARY In Alzheimer's disease (AD) research, much focus has been on oligomeric assemblies of the amyloid-β (Aβ) peptide (a small protein). Oligomers are small nanoparticles produced by the aggregation of 50 or fewer peptide molecules. Peptide aggregation most readily produces amyloid fibrils, and it is a mystery what prevents some oligomers from aggregating further into fibrils. Even within oligomeric and fibrillar aggregate classes, peptide aggregates with a range of structures have been detected. Nevertheless, structural biology methods require stable and structurally homogeneous samples to achieve high-resolution information. To elucidate what oligomer structures are possible and which of these structures should be targeted by AD therapies, experiments are designed to understand Aβ assembly in general terms. Preliminary data show that it is possible to produce stable homogeneous samples of a 150 Aβ oligomer (32 Aβ peptide molecules) and study its structure using solid-state nuclear magnetic resonance (NMR) and electron microscopy (EM). Notably, the 150 kDa oligomer structure is unlike any previously understood Aβ assembly, and this observation presents a unique opportunity to probe how oligomer and fibril structures may differ. The project will pursue high-resolution atomic-level characterization of the 150 kDa oligomer by integrating solid-state NMR, cryo-EM, and computational modeling. The work further seeks to generalize understanding of Aβ assembly mechanisms by testing hypothesized assembly pathways inspired by the 150 kDa oligomer. The proposed work will: achieve a 3-dimensional image of the 150 kDa oligomer using cryo-EM (Aim 1); measure complementary atomic-level structural constraints with solid-state NMR (Aim 2); and test mechanistic hypotheses to produce new oligomers compatible with EM and NMR workflows (Aim 3). Knowledge of what oligomer structures are possible for Aβ (and how to isolate specific structures) is critical for developing new Alzheimer's therapeutics, implementing strategies for early detection, and designing mechanistic studies in disease models.