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生物体细胞中其功能和故障的关键。蛋白质折叠发生在高维复杂表面上，其特征往往隐藏在蛋白质折叠的标准集合研究中。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。我们将重点了解两种淀粉样蛋白的折叠特性，Sup 35和a-synuclein。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（a-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是非常重要的。我们将在生物折叠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，极化和相关光谱，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，还是在聚集过程的早期阶段。我们的研究将揭示这些单体蛋白（都被认为是内在无序的）是否具有残余结构的元素，重复序列如何影响它们的折叠和动力学，以及其他关键的细胞因子，如伴侣蛋白（Sup 35）和与膜的结合（a-synuclein）如何影响这些蛋白质的折叠和结构动力学。此外，通过在聚集的早期阶段监测它们的折叠特性，我们的目标是了解这两个过程是如何在这种高度复杂和异质性的低聚物种混合物的背景下耦合的，这将对理解蛋白质淀粉样变性的分子和结构机制有价值。最后，这些见解预计在设计对抗淀粉样蛋白疾病的治疗策略方面是非常有价值的。公共卫生相关性：本项目旨在开发和应用新的单分子荧光方法来探测淀粉样蛋白的复杂折叠特征。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，这与帕金森病和朊病毒病等疾病有关。在设计预防或逆转这类疾病的治疗策略时，预计获得的见解将很有价值，从而有助于改善公众健康。

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.