Nascent Dynamic Intermediates in Amyloid Aggregation

描述（由申请人提供）：本提案的长期目标是开发淀粉样蛋白聚集起始的详细动力学描述，淀粉样蛋白聚集是多种破坏性疾病的共同特征，其特征是错误折叠蛋白质的纤维聚集体沉积。重点是对新生中间分子物种的身份和功能的描述，作为识别靶蛋白物种的手段，用于开发控制聚集途径的方法。具体提出的假设是，非纤维寡聚蛋白物种与天然蛋白的构象变化一起在淀粉样蛋白聚集的起始中起着至关重要的作用。这一假设是基于以下证据：1)已知淀粉样蛋白的可溶性低聚物具有细胞毒性；2)影响淀粉样蛋白二级结构的溶液会影响其聚集动力学。实验策略依赖于基于多光子显微镜和荧光相关光谱的现代生物物理仪器来表征三种关键淀粉样蛋白的溶液和脂质相关行为：α -突触核蛋白，a- β和无肌红蛋白。两个具体目标是：1。定量和理解与聚集起始相关的构象变化，利用荧光相关光谱来表征蛋白质构象波动和折叠/错误折叠相关的无肌红蛋白、α -突触核蛋白和a- β构象变化。2. 由α - Synuclein和a- β形成的可溶性低聚物的生物物理特性，包括(i)它们的溶液平衡特性，以及（ii）与脂质膜相互作用的性质，特别是膜电荷和脂质有序相形成的作用，以了解细胞膜相互作用如何影响聚集。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is development of a detailed kinetic description of the initiation of amyloid aggregation, a common feature of the diverse group of devastating diseases characterized by the deposition of fibrillar aggregates of misfolded proteins. The focus is a description of the identities and functions of the nascent intermediate molecular species as a means for identifying target protein species for the development of methods of controlling the aggregation pathway. The specific proposed hypothesis is that non-fibrillar oligomeric protein species in conjunction with conformational changes in the native proteins play the crucial role in the initiation of aggregation of amyloid-forming proteins. This hypothesis is based on evidence that 1) soluble oligomers of amyloid proteins are known to be cytotoxic and 2) solutions influencing protein secondary structure of amyloid proteins affect their aggregation kinetics. The experimental strategies rely on modern biophysical instrumentations based on multiphoton microscopy and fluorescence correlation spectroscopies to characterize the solution and lipid-associated behavior of three key amyloid-forming proteins: alpha-Synuclein, A-beta, and apomyoglobin. Two specific aims are: 1. Quantification and understanding of the conformational changes associated with the initiation of aggregation utilizing adaptations of fluorescence correlation spectroscopy to characterize protein conformational fluctuations and folding/misfolding associated conformational changes in apomyoglobin, alpha-Synuclein, and A-beta. 2. Biophysical characterization of the soluble oligomers formed by alpha- Synuclein and A-beta, including (i) their solution equilibrium properties, and (ii) the nature of the interactions with lipid membranes, in particular the role of membrane charge and lipid-ordered phase formation, to understand how cell membrane interactions affect aggregation.