Single Molecule Studies of Protein Folding Mechanisms

总结： 蛋白质向其天然结构的折叠是它们在细胞中的功能和故障的关键， 活的有机体蛋白质折叠发生在高维复杂的表面上，其特征在于 通常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以探测这些复杂的特征 蛋白质折叠的过程。我们将重点了解两种淀粉样蛋白的折叠特性， Sup 35和突触核蛋白。这种淀粉样蛋白的错误折叠和聚集与 包括疯牛病和帕金森氏症在内的一系列疾病。此外，越来越多的证据 最近暗示这些蛋白质的聚集在生物建设性的作用，如作为一个 酵母中的仅蛋白质遗传载体（Sup 35）。因此，详细了解折叠和 从人类健康的观点来看，这些蛋白质的动力学是非常重要的。我们将建立在我们的 生物折叠的单分子研究的坚实基础，以进一步开发和应用 一套单分子荧光方法，包括单分子FRET，偏振和 相关光谱学，结合新的和强大的微流体方法和蛋白质 工程，以了解这些蛋白质的折叠，无论是作为单体物种，以及 在聚合过程的早期阶段。我们的研究将揭示这些单体是否 蛋白质（两者都被认为是内在无序的）具有残留结构的元素，如何重复 序列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， (for Sup 35）和与膜的结合（对于<$-突触核蛋白）会影响蛋白的折叠和结构动力学。 这样的蛋白质。此外，通过在聚集的早期阶段监测它们的折叠性质， 我们的目标是了解这两个过程是如何耦合在这个高度复杂的背景下， 低聚物种的异质混合物，洞察力，这将是有价值的理解， 蛋白质淀粉样变性的分子和结构机制。最后，这些见解预计将成为 在对抗淀粉样疾病的治疗策略的设计中非常有价值。

Summary: 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 ¿-synuclein. The misfolding and aggregation of such amyloidogenic proteins are implicated in a host of diseases including Mad Cow and Parkinson's (¿-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 ¿-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 heterogenous 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.