Nascent Dynamic Intermediates in Amyloid Aggregation

淀粉样蛋白聚集中的新生动态中间体

• 批准号: 6963283
• 负责人: WATT W WEBB
• 金额: $ 20.15万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-15 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6963283
• 关键词: alpha synuclein; amyloid proteins; conformation; cytotoxicity; electrical property; fluorescence microscopy; fluorescence polarization; fluorescence spectrometry; intermolecular interaction; lipid bilayer membrane; myoglobin; phase change; protein folding; protein protein interaction; protein structure function; sedimentation equilibrium

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.

描述（由申请人提供）：该提案的长期目标是对淀粉样蛋白聚集的起始进行详细的动力学描述，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的纤维状聚集体的沉积。重点是描述新生中间分子种类的身份和功能，作为识别目标蛋白种类的手段，以开发控制聚集途径的方法。具体提出的假设是，非纤维状寡聚蛋白种类与天然蛋白的构象变化相结合，在淀粉样蛋白形成蛋白聚集的启动中发挥着至关重要的作用。该假设基于以下证据：1) 已知淀粉样蛋白的可溶性寡聚物具有细胞毒性；2) 影响淀粉样蛋白的蛋白质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形成蛋白：α-突触核蛋白、A-β和脱肌红蛋白的溶液和脂质相关行为。两个具体目标是： 1. 利用荧光相关光谱的适应性来量化和理解与聚集起始相关的构象变化，以表征脱辅基红蛋白、α-突触核蛋白和 A-β 中的蛋白质构象波动和折叠/错误折叠相关的构象变化。 2. α-突触核蛋白和 A-β 形成的可溶性低聚物的生物物理表征，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。