Single Molecule Studies of Protein Folding Mechanisms

描述（由申请人提供）：蛋白质折叠成其天然结构是其在活生物体细胞中的功能和故障的关键。蛋白质折叠发生在一个高维复杂的表面上，其特征往往被隐藏在标准的蛋白质折叠系综研究中。在这个项目中，我们将继续改进和开发新的单分子荧光方法来探测蛋白质折叠的复杂特征。我们将重点了解两个淀粉样蛋白，Sup 35和α-突触核蛋白的折叠特性。这种淀粉样蛋白的错误折叠和聚集与许多疾病有关，包括疯牛病和帕金森病（α-突触核蛋白）。此外，最近越来越多的证据表明，这些蛋白质的聚集在生物学上具有建设性作用，例如在酵母中作为蛋白质的遗传载体（Sup 35）。因此，从人类健康的角度来看，详细了解这些蛋白质的折叠和动力学非常重要。我们将建立在生物折叠的单分子研究的坚实基础上，进一步开发和应用一套单分子荧光方法，包括单分子FRET，偏振和相关光谱，结合新颖而强大的微流体方法和蛋白质工程，以深入了解这些蛋白质的折叠，无论是作为单体物种，以及在聚集过程的早期阶段。我们的研究将揭示这些单体蛋白质（两者都被认为是内在无序的）是否具有残留结构的元素，重复序列如何影响它们的折叠和动力学，以及其他关键细胞因子如伴侣蛋白（Sup 35）和与膜结合（a-突触核蛋白）如何影响这些蛋白质的折叠和结构动力学。此外，通过监测它们的折叠特性在早期阶段的聚集，我们的目标是了解这两个过程是如何耦合的背景下，这种高度复杂和异质性的混合物的低聚物物种，洞察力，将是有价值的蛋白质淀粉样变性的分子和结构机制的理解。最后，这些见解预计将是非常有价值的治疗策略，以打击淀粉样疾病的设计。公共卫生相关性：本计画旨在发展及应用新颖的单分子萤光方法来侦测淀粉样蛋白质的复杂折叠特徴。在这些研究中获得的机制见解将是理解淀粉样蛋白和蛋白质聚集的结构生物学的关键，这些蛋白质聚集与帕金森病和朊病毒病等疾病有关。在设计预防或扭转这些疾病的治疗战略时，预计所获得的见解是有价值的，从而有助于改善公共卫生。

DESCRIPTION (provided by applicant): The folding of proteins to their native structures is key to their function and malfunction in the cells of living organisms. Protein folding occurs on a high dimensionality and complex surface, whose features are often hidden in standard ensemble studies of protein folding. In this project, we will continue to improve and develop novel single molecule fluorescence methodologies to probe such complex features of protein folding. We will focus on understanding the folding properties of two amyloidogenic proteins, Sup 35 and a-synuclein. The misfolding and aggregation of such amyloidogenic proteins are implicated in a host of diseases including Mad Cow and Parkinson's (a-synuclein). Additionally, mounting evidence recently implicates the aggregation of these proteins in biologically constructive roles, such as acting as a protein-only genetic vehicle in yeast (Sup 35). Hence, a detailed understanding of the folding and dynamics of such proteins is very important from the point of view of human health. We will build on our strong foundation of single molecule investigations of biological folding to further develop and apply a suite of single molecule fluorescence methods, including single molecule FRET, polarization and correlation spectroscopy, in combination with novel and powerful microfluidic methods and protein engineering to gain insights into the folding of these proteins, both as monomeric species, as well as during the early stages of the aggregation process. Our studies will uncover whether these monomeric proteins (both understood to be intrinsically disordered) have elements of residual structure, how repeat sequences influence their folding and dynamics, and how other key cellular factors such as chaperones (for Sup 35) and binding to membranes (for a-synuclein) influence the folding and structural dynamics of such proteins. Furthermore, by monitoring their folding properties during the early stages of aggregation, we aim to understand how these two processes are coupled within the context of this highly complex and heterogeneous mixture of oligomeric species, insight that will be valuable in the understanding of the molecular and structural mechanisms of protein amyloidosis. Finally, these insights are anticipated to be extremely valuable in the design of therapeutic strategies to combat amyloid diseases. PUBLIC HEALTH RELEVANCE: This project aims to develop and apply novel single molecule fluorescence methodologies to probe complex folding features of amyloidogenic proteins. Mechanistic insights gained in these studies will be key in understanding the structural biology of amyloidogenic proteins and protein aggregation, which are implicated in diseases such as Parkinson's and Prion diseases. Insights obtained are expected to be valuable during the design of therapeutic strategies to prevent or reverse such diseases, thus contributing to improving public health.