Structures of Protein Aggregates by Solid-State NMR

描述：本研究项目的长期目标是开发一个详细的原子分辨率 对与人类疾病相关蛋白质聚集和纤维化的结构和功能的理解， 主要是关于α-突触核蛋白的研究野生型α-突触核蛋白及其突变体（A30 P、E46 K和E46 K） A53 T）与早发性帕金森病（PD）相关的几种结构状态， 包括原纤维、原纤维和膜相关复合物。我们建议，化学细节的 这些结构决定了α-突触核蛋白的天然生理功能， 环境压力，有助于PD病理学。此外，我们建议，转换之间的几个 结构状态是突触核蛋白的一个基本特征;这种结构可塑性可能是突触核蛋白形成所必需的。 神经元的发育和维持。为了研究这些结构，魔角旋转（MAS）固体- 将采用固态NMR（SSNMR）实验，阐明共价化学、构象 和其他实验技术无法达到的动力学。补充分析和 表征技术将包括动力学测量（通过硫磺素T荧光、光散射 和沉降分析），高分辨率质谱，原子力和电子显微镜， 溶液NMR和噬菌体展示。我们将利用这些技术沿着进行样品制备， 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学原理 聚集和膜缔合，（2）使合理设计成为可能的特定结构信息 用于调节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-突触核蛋白和其他脑蛋白之间的特定结构相互作用。 与公共卫生的相关性：α-突触核蛋白是帕金森病的核心参与者。的精确 α-突触核蛋白结构和这种疾病之间的关系还没有很好的理解。我们提出的 研究旨在推进这一基础知识，这将有助于更广泛的研究社区， 开发改进的帕金森病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.