Structure and conformational dynamics of alpha-synuclein

描述（由申请人提供）：帕金森病（PD）是一种常见的神经系统疾病，从轻微的肌肉协调丧失发展为严重的身体和精神残疾，最终死亡。没有治愈方法，也没有治疗方法来减缓疾病的进展。目前，美国有约100万PD患者，每年花费约250亿美元。随着老年人口的迅速增加，预计到2050年，这一数字将增加两倍。因此，迫切需要有效的疾病修饰治疗。为了有助于药物开发工作，本项目的目标是确定o-突触核蛋白（<$-syn）的结构，以深入了解其在帕金森病发病机制中的功能及其机制，这对于合理的药物设计至关重要。戊-syn的聚集导致所谓的路易体（LB）的出现，作为帕金森病的定义性病理标志。类似的聚合物-syn还涉及其他神经退行性疾病，包括多系统萎缩（MSA）、路易体痴呆（DLB）和其他统称为突触核蛋白病的疾病。Syn-Syn在大脑中含量很高，似乎对学习和记忆很重要;然而，它的确切功能仍然未知。它没有已知的酶活性，因此其未知的生物学功能取决于其物理结构;此外，它通过与其结构转化为高度有序的原纤维相关的毒性获得功能引起疾病。因此，详细的结构信息是了解其功能和深入了解其转化为致病实体的关键。然而，这些结构细节目前还无法获得，主要是因为直到最近，<$-syn在溶液中缺乏持久的结构，并且被认为是一种天然未折叠的蛋白质，不适合结构测定。然而，我们最近在溶液中分离出了一种有序的四聚体形式的<$-syn。我们通过开发一种表达和纯化程序来实现这一点，目的是在整个纯化过程中保留其天然结构。大约在同一时间，哈佛医学院的丹尼斯·塞尔科（Dennis Selkoe）的研究小组独立地证明，从大鼠大脑和活的人类细胞中分离出的<$-syn也是天然折叠和四聚体的，与我们的重组<$-syn惊人地相似。此外，我们进一步证明了我们的<$-syn制剂对聚集有抗性，不损害脂质体膜，并且对细胞无毒，因此强烈支持有序的四聚体<$-syn是生理学相关的。现在我们有了一种适合于传统生物物理工具的有序形式的<$-syn，这个提议的具体目标是：1）确定<$-syn的原子结构; 2）确定其四级结构的动力学。我们将结合使用X射线晶体学方法、核磁共振（NMR）方法和单分子荧光方法来进行研究。

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a common neurological condition that progresses from subtle loss of muscular coordination to severe physical and mental disability and eventual death. There is no cure and no treatments to slow the disease progression. Currently there are ~1 million PD sufferers in the US, which costs about $25 billion USD per year. These numbers are expected to triple by 2050 as the population of the elderly is increasing rapidly. Thus, effective disease-modifying treatments are desperately needed. To contribute to the drug development effort, the goal of this project is to determine the structure o ¿-synuclein (¿-syn) to gain insights into its function and its mechanisms in the pathogenesis of Parkinson's disease, which are critical for rational drug design. The aggregation of ¿-syn leads to appearance of so-called Lewy bodies (LBs) as a defining pathological hallmark of Parkinson's disease. Similar aggregates of ¿-syn are also implicated in other neurodegenerative diseases, including multiple system atrophy (MSA), dementia with Lewy bodies (DLB), and others collectively known as synucleinopathies. ¿-Syn is highly abundant in the brain and it appears to be important for learning and memory; however, its precise function is still unknown. It has no known enzymatic activity, thus its unknown biological function depends on its physical structure; moreover, it causes disease via a gain-of-toxic function that is associated with its structural transformation into highly ordered fibrils. Thus, detailed structural information of ¿-syn is crucil for understanding its function and to gain insights into its transformation into a disease-causing entity. However, such structural details are currently unavailable mainly because, until recently, ¿-syn lacked a persistent structure in solution and was thought to be a natively unfolded protein, which is not amenable for structure determination. However, we have recently isolated an ordered tetrameric form of ¿-syn in solution. We did so by developing an expression and purification procedure aimed at preserving its native structure throughout the purification process. At about the same time, Dennis Selkoe's group at Harvard Medical School independently demonstrated that ¿-syn isolated from rat brains and living human cells are also natively folded and tetrameric with striking resemblance to our recombinant ¿-syn. Moreover, we further demonstrated that our ¿-syn preparation was resistant to aggregation, did not compromise liposome membranes, and non-toxic to cells, thus strongly supporting that the ordered tetrameric ¿-syn is physiologically relevant. Now that we have an ordered form of ¿-syn that is amenable to traditional biophysical tools, the specific aims of this proposal are; 1) To determine the atomic structure of ¿-syn; and 2) To determine the dynamics of its quaternary structure. We will do so by using a combination of X-ray crystallographic methods, nuclear magnetic resonance (NMR) methods, and single-molecular fluorescence methods.