Single-molecule structural studies of unstable amyloidogenic protein oligomers

描述（由申请人提供）：蛋白质聚集与许多疾病有关，包括阿尔茨海默病、帕金森病、2型糖尿病、心脏和朊病毒疾病，并且越来越多地发现其功能意义。虽然淀粉样原纤维以前被认为是有毒的物种，但最近的数据表明，在原纤维形成过程中作为中间产物的较小的低聚物具有主要的疾病相关作用。由于淀粉样蛋白的形成发生在高维和复杂的反应环境中，反应中中间体的结构特征往往隐藏在标准的系综研究中。单分子研究对于揭示此类反应中复杂的低聚中间体混合物的结构性质的完整分布具有很大的希望。然而，由于单分子研究通常是在低浓度（nM及以下）和平衡条件下进行的，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低聚物不相容。本课题将利用单分子实验和微流控相结合的优势，开发一种新的快速稀释双色符合方法，用于研究这类弱寡聚中间体的寡聚化数分布。该方法背后的关键基本思想是，聚集反应的稀释将非常迅速地实现，随后立即进行单分子检测，从而允许在解离之前对弱低聚物进行单分子结构研究。该方法将使用模型DNA系统进行开发，该系统将提供一组具有定义良好和可调特性的低聚物，例如寡聚状态和稳定性，这对于我们探索该方法的优势以及开发实验方案和分析程序至关重要，以便从单分子荧光爆发中最佳地提取寡聚信息。所开发的符合方法将随后应用于研究蛋白质聚集早期阶段的低聚物分布。在这种情况下，该方法的最终目标是同时提供蛋白质寡聚化（通过符合测量）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的信息。所开发的方法对于细胞中广泛存在的弱配合物的结构和动力学的详细研究也将非常普遍有用。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过程中不稳定低聚中间体的分布。该方法有望提供此类不稳定低聚物结构特性的详细图谱，这对于了解其细胞功能和紊乱以及设计针对几种淀粉样蛋白疾病的治疗策略具有重要价值。

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.