Identifying differences in dynamics and residual structure of intrinsically disordered domains between monomer and fibers: using alpha-synuclein as a model

项目总结/摘要 许多无法治愈的神经退行性疾病，包括阿尔茨海默氏症，帕金森氏症和亨廷顿氏症， 有毒错误折叠蛋白的存在。这些不溶性蛋白质聚集体，淀粉样纤维（AF），已经被 广泛的研究和结构表征表明，它们主要由β-折叠和 有序的交叉β纤维核心;然而，它们通常被N中的固有无序结构域（IDD）框定。 C终端增加AF复杂性的是发现许多AF，包括α-突触核蛋白， （α-syn）、淀粉样蛋白-β和tau被发现为多晶型物。在这种情况下，多态性使蛋白质能够折叠 并自我传播到不同的AF结构中，通常伴随着不同的神经病理学。高 IDDs的灵活性使得很难拍摄它们的分子照片，从而限制了我们对IDDs如何作用的理解。 它们影响疾病中的AF特性。最近的研究表明， AF，因为已发现它们在介导纤维毒性、聚集和相互作用中起重要作用 有细胞成分。α-Syn是天然存在的可溶性内在无序蛋白（IDP），但在 帕金森氏病，它错误折叠并聚集成路易体中发现的AF。α-syn在疾病中的重要性 背景导致了对其单体和纤维形式的大量研究，因此α-syn的纤维核心是很好的- 表征，并有几个高分辨率结构，包括至少四个多晶型 结构.这使得α-syn成为确定结构和动力学差异的理想候选者。 IDDs在单体和纤维状状态和多晶型物之间。为了实现这一点，我将使用固态 核磁共振和电子顺磁共振波谱结合分子 动力学模拟，以开发两个全长α-syn模型，将描述动态和结构 IDDs在纤维中的性质，它将提供对IDDs的多态性的影响的见解。此外，本发明还提供了一种方法， 将在神经细胞培养模型中测试多晶型物的毒性。这项研究将提供基本的 对AF的了解可以帮助未来的研究，重点是了解IDDs如何介导毒性，纤维- 细胞相互作用和播种特性。

PROJECT SUMMARY/ ABSTRACT Many incurable neurodegenerative diseases including Alzheimer's, Parkinson's, and Huntingtin's feature the presence of toxic misfolded proteins. These insoluble protein aggregates, amyloid fibrils (AF), have been extensively studied and structural characterization has shown that they are largely composed of β-sheets and an ordered cross-β fibril core; however, they are often framed by intrinsically disordered domains (IDDs) in the N and C-terminal. Adding to the complex nature of AFs is the finding that many of them, including α-synuclein (α-syn), amyloid-β, and tau, are found as polymorphs. Polymorphism, in this context, enables a protein to fold and self-propagate into different AF structures often accompanied by varying neuropathology. The high flexibility of the IDDs has made it difficult to take their molecular picture thus limiting our understanding of how they affect AF properties in disease. Recent studies have demonstrated the importance of studying the IDDs of AF as they have been found to play an important role in mediating fibril toxicity, aggregation, and interactions with cellular components. α-Syn is natively found as a soluble intrinsically disordered protein (IDP), but in Parkinson's disease it misfolds and aggregates into AF found in Lewy bodies. The importance of α-syn in disease context has resulted in numerous studies of its monomeric and fibrillar form, thus the fibril core of α-syn is well- characterized and there are several high-resolution structures available including at least four polymorph structures. This makes α-syn an ideal candidate to determine the differences in structure and dynamics of the IDDs in the monomeric and fibrillar state and between polymorphs. To accomplish this, I will use solid-state nuclear magnetic resonance and electron paramagnetic resonance spectroscopy in combination with molecular dynamics simulations to develop two full-length α-syn models that will describe the dynamic and structural properties of IDDs in fibrils and it will provide insight on the effects of polymorphism on the IDDs. In addition, polymorphs will be tested for toxicity in a neural cell culture model. This research will provide fundamental understanding of AF that can help future studies that focus on understanding how IDDs mediate toxicity, fiber- cell interactions, and seeding properties.