Structures of Protein Aggregates by Solid-State NMR

描述：该研究项目的长期目标是开发一种详细的原子分辨率 了解与人类疾病相关的蛋白质聚集和纤颤的结构和功能， 主要是对α-突触核蛋白的研究。野生型α-突触核蛋白及其突变体(A30P、E46K和 与早发性帕金森病(PD)相关的A53T)将在几种结构状态下进行检查， 包括原纤维、原纤维和膜结合复合体。我们认为，化学物质的细节 这些结构决定了α-突触核蛋白的自然生理功能，在突变体或突变体中 环境应激，参与帕金森病的病理过程。此外，我们还提出了几个之间的转换 结构状态是突触核蛋白的一个基本特征；这种结构可塑性很可能是 神经元的发育和维持。为了检查这些结构，魔角旋转(MAS)固体- 将采用状态核磁共振(SSNMR)实验，阐明共价化学、构象的细节 以及其他实验技术所无法获得的动力学。互补性分析和 表征技术将包括动力学测量(通过硫代黄素T荧光、光散射 和沉积分析)、高分辨率质谱学、原子力和电子显微镜， 溶液核磁共振和噬菌体展示。我们将利用这些技术以及样品制备和 同位素标记策略以提高我们对与疾病相关的蛋白质聚集的理解 α-突触核蛋白的具体情况，包括：(1)α-突触核蛋白的基本生物物理原理 聚集和膜结合，(2)使合理设计成为可能的特定结构信息 调节α-突触核蛋白功能和/或作为诊断工具的小分子的策略 PD，以及(3)阐明α-突触核蛋白与其他脑蛋白质之间的特殊结构相互作用。 与公共健康相关：α-突触核蛋白是帕金森氏症的核心因素。精准的 α-突触核蛋白结构与本病的关系尚不清楚。我们的建议 研究的目的是促进这一基本知识，这将有助于更广泛的研究界 开发改进的帕金森氏症诊断工具和治疗方法。

Description: The long-term objective of this research project is to develop a detailed atomic-resolution structural and functional understanding of protein aggregation and fibrillation relevant to human disease, principally with studies of alpha-synuclein. Wild type alpha-synuclein and its mutants (A30P, E46K and A53T) associated with early-onset Parkinson's disease (PD) will be examined in several structural states, including protofibrils, fibrils and membrane-associatedcomplexes. We propose that the chemical details of these structures determine the natural physiological function of alpha-synuclein and, in the mutants or under environmental stress, contribute to PD pathology. Moreover, we propose that conversion among several structural states is an essential feature of synucleins; this structural plasticity is likely to be required for neuronal development and maintenance. To examine these structures, magic-angle spinning (MAS) solid- state NMR (SSNMR) experiments will be employed, elucidating details of covalent chemistry, conformation and dynamics that are inaccessible to other experimental techniques. Complementary analysis and characterization techniques will include kinetic measurements (by thioflavin T fluorescence, light scattering and sedimentation analysis), high-resolution mass spectrometry, atomic force and electron microscopy, solution NMR and phage display. We will leverage these technologies along with sample preparation and isotopic labeling strategies to improve our understanding of protein aggregation relevant to disease in the specific case of alpha-synuclein, including: (1) the fundamental biophysical principles of alpha-synuclein aggregation and membrane association, (2) specific structural information that will enable rational design strategies for small molecules that modulate alpha-synuclein function and/or serve as diagnostic tools for PD, and (3) elucidation of specific structural interactions between alpha-synuclein and other brain proteins. Relevance to public health: Alpha-synuclein is a central player in Parkinson's disease. The precise relationships between alpha-synuclein structure and this disease are not yet well understood. Our proposed studies aim to advance this fundamental knowledge, which will assist the broader research community in developing improved diagnostic tools and therapies for Parkinson's disease.