Single-molecule structural studies of unstable amyloidogenic protein oligomers

描述（由申请人提供）：蛋白质聚集体与包括阿尔茨海默氏病、帕金森氏病、II型糖尿病、心脏病和朊病毒疾病在内的许多疾病有关，并且还越来越多地发现其具有功能重要性。虽然淀粉样蛋白原纤维以前被认为是有毒物质，但最近的数据表明，作为原纤维形成中间体的较小寡聚体具有重大的疾病相关作用。由于淀粉样蛋白的形成发生在一个高维和复杂的反应景观，在反应中的中间体的结构特征往往隐藏在标准的合奏研究。单分子研究对于揭示此类反应中低聚中间体的复杂混合物的结构性质的全部分布具有很大的希望。然而，由于单分子研究通常在低浓度（nM及以下）和平衡条件下进行，因此它们通常与淀粉样蛋白形成过程中遇到的高浓度、非平衡条件和不稳定寡聚体不相容。在这个项目中，我们将专注于开发一种新的快速稀释双色符合方法，用于研究这种弱低聚中间体的低聚数分布，通过使用单分子实验和微流体的综合优势。该方法背后的关键基本思想是，将非常迅速地实现来自聚集反应的稀释，随后立即进行单分子检测，从而允许在解离之前对弱寡聚体进行单分子结构研究。该方法将使用模型DNA系统开发，该系统将提供一组具有明确定义和可调特性的低聚物，例如低聚状态和稳定性，这对于让我们探索该方法的优势至关重要，以及开发实验方案和分析程序，用于从单分子荧光猝发中最佳提取低聚信息。开发的符合方法，然后将被应用于研究蛋白质聚集的早期阶段的寡聚体分布。在这种情况下，该方法的最终目标是同时提供关于蛋白质寡聚化（通过重合测量）和构象分布（例如，使用FRET）作为淀粉样蛋白形成早期阶段时间的函数。所开发的方法也将是非常普遍的有用的结构和动力学的详细研究广泛流行的细胞中的弱复合物。公共卫生相关性本项目旨在开发和应用一种新的单分子荧光方法来探测淀粉样蛋白形成中不稳定的寡聚中间体的分布。这种方法预计将提供一个详细的映射这种不稳定的低聚物的结构特性，有价值的基本了解他们的细胞功能和疾病，并在设计治疗策略对几种淀粉样蛋白疾病。

DESCRIPTION (provided by applicant): Protein aggregates are implicated in a host of diseases including Alzheimer's, Parkinson's, Type II diabetes, heart and prion diseases, and are also being increasingly found to be of functional significance. While amyloid fibrils were previously believed to be the toxic species, more recent data suggest a major disease-associated role for smaller oligomers that are intermediates in fibril formation. Because amyloid formation occurs on a high-dimensionality and complex reaction landscape, structural features of intermediates in the reaction are often hidden in standard ensemble studies. Single molecule studies hold great promise for uncovering the full distributions of structural properties for the intricate mixture of oligomeric intermediates in such reactions. However, because single molecule studies are typically carried out at low concentrations (nM and below) and under equilibrium conditions, they are often not compatible with the high-concentrations, nonequilibrium conditions and unstable oligomers encountered during amyloid formation. In this project, we will focus on developing a novel rapid-dilution dual-color coincidence method for studying oligomerization-number distributions of such weak oligomeric intermediates, by using the combined strengths of single molecule experiments and microfluidics. The key underlying idea behind the methodology is that dilution from the aggregation reaction will be achieved very rapidly, followed immediately by single molecule detection, thus allowing single molecule structural studies of the weak oligomers prior to dissociation. The method will be developed using a model DNA system, which will provide a set of oligomers with well-defined and tunable properties such as oligomerization state and stability, essential in allowing us to explore the strengths of the method, as well as to develop experimental protocols and analysis routines for optimally extracting oligomerization information from single molecule fluorescence bursts. The developed coincidence method will then be applied to study oligomer distributions during the early phases of protein aggregation. The ultimate goal of the method in this context is to simultaneously provide information about protein oligomerization (via coincidence measurements) and conformational distributions (e.g., using FRET) as a function of time during the early stages of amyloid formation. The developed method will also be very generally useful for the detailed study of structure and dynamics of weak complexes widely prevalent in the cell. PUBLIC HEALTH RELEVANCE This project aims to develop and apply a novel single-molecule fluorescence methodology to probe distributions of unstable oligomeric intermediates in amyloid formation. This methodology is anticipated to provide a detailed mapping of the structural properties of such unstable oligomers, valuable for a basic understanding of their cellular functions and disorders, and during the design of therapeutic strategies against several amyloid diseases.