Single Molecule Studies of Protein Folding Mechanisms

蛋白质折叠机制的单分子研究

• 批准号: 8121134
• 负责人: Ashok A Deniz
• 金额: $ 9.97万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8121134
• 关键词: Abbreviations; Address; Affect; Amyloid; Amyloidosis; Anisotropy; Binding; Biological; Cattle; Cells; Charge; Complex; Computer Analysis; Coupled; Coupling; Detection; Diffuse; Disease; Elements; Equilibrium; Fluorescence; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Foundations; Genetic; Health; Human; In Situ; Investigation; Kinetics; Life; Ligand Binding; Light; Lipid Bilayers; Lipids; Maps; Measurement; Membrane; Methodology; Methods; Microfluidics; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Monitor; Oligopeptides; Organism; Parkinson Disease; Pathway interactions; Point Mutation; Population; Prion Diseases; Prions; Process; Property; Protein Engineering; Proteins; Protocols documentation; Public Health; Reaction; Residual state; Resolution; Role; Seeds; Spectrum Analysis; Staging; Structural Protein; Structure; Surface; Technology; Tertiary Protein Structure; Therapeutic; Time; Yeasts; aggregation pathway; alpha synuclein; amyloid formation; combat; conformational conversion; design; dimer; human disease; improved; insight; intermolecular interaction; monomer; novel; prevent; protein aggregation; protein folding; public health relevance; research study; single molecule; single-molecule FRET; structural biology; sup35; synuclein; yeast prion

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.

描述(申请人提供)：蛋白质折叠到它们的天然结构是它们在活的有机体细胞中功能和故障的关键。蛋白质折叠发生在高维和复杂的表面上，其特征往往隐藏在蛋白质折叠的标准系综研究中。在这个项目中，我们将继续改进和发展新的单分子荧光方法来探索蛋白质折叠的这种复杂特征。我们将重点了解两个淀粉样蛋白，Sup 35和a-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包括疯牛和帕金森氏症(a-突触核蛋白)在内的一系列疾病有关。此外，最近越来越多的证据表明，这些蛋白质的聚集在生物上具有建设性的作用，例如在酵母中作为仅有蛋白质的遗传载体(Sup 35)。因此，从人类健康的角度出发，详细了解这些蛋白质的折叠和动态是非常重要的。我们将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，结合新的和强大的微流控方法和蛋白质工程，深入了解这些蛋白质作为单体物种的折叠，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(都被认为是内在无序的)是否具有残基结构元素，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素，如伴侣(对于Sup 35)和膜结合(对于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，通过监测它们在聚集早期阶段的折叠特性，我们旨在了解这两个过程是如何在这种高度复杂和不同种类的寡聚物种混合物的背景下耦合的，这一见解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，这些见解有望在设计抗击淀粉样蛋白疾病的治疗策略方面具有极其重要的价值。公共卫生相关性：该项目旨在开发和应用新的单分子荧光方法来探索淀粉样蛋白的复杂折叠特征。在这些研究中获得的机械洞察力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氏症和Prion病等疾病有关。在设计预防或逆转这类疾病的治疗战略时，所获得的见解可望有价值，从而有助于改善公共卫生。