Folding and chaperone interactions of multi-domain proteins

多结构域蛋白质的折叠和分子伴侣相互作用

• 批准号: 9217889
• 负责人: Christian Kaiser
• 金额: $ 30.74万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9217889
• 关键词: Alzheimer' s Disease; Binding; Binding Sites; Biological; Biological Models; Biotin; Cells; Complement; Complex; Coupled; Defect; Disease; Environment; Eukaryota; Goals; In Vitro; Individual; Label; Lead; Ligase; Malignant Neoplasms; Measures; Methods; Modeling; Molecular Chaperones; Molecular Conformation; Neurodegenerative Disorders; Organism; Orthologous Gene; Parkinson Disease; Pathway interactions; Peptide Elongation Factor G; Proteins; Proteome; Research; Resolution; Ribosomes; Route; Shapes; Spectrum Analysis; Structure; System; Tertiary Protein Structure; Testing; Therapeutic Intervention; Translations; Work; biophysical properties; experimental study; human disease; in vivo; insight; laser tweezer; misfolded protein; novel; novel therapeutics; polypeptide; protein aggregation; protein folding; protein misfolding; proteostasis; public health relevance; single molecule; three dimensional structure

ABSTRACT Many large, multi-domain proteins are inherently prone to misfolding and aggregation. As soon as they emerge from the ribosome during translation, they are met by molecular chaperones that help them fold into their native structures. While it is clear that molecular chaperones are essential for correct folding in vivo, we know very little about the underlying mechanisms. The long-term goal of our studies is to define the folding mechanisms of complex, multi-domain proteins in the cell. The aim of this proposal is to determine the function of two nascent chain-binding chaperones, Trigger factor and the DnaK system, in the folding of a model multi-domain protein, EF-G. We will study how these chaperones guide the folding of nascent multi- domain proteins using single-molecule force spectroscopy with optical tweezers. This approach is uniquely suited to manipulate and observe the folding of individual nascent polypeptides in the complex environment of the ribosome and molecular chaperones. We will first measure folding transitions of nascent proteins on the ribosome to define their folding energy landscapes. Then, we will determine how Trigger factor and DnaK change these energy landscapes. Our in vitro experiments will reveal in mechanistic detail and with single- molecule resolution how these chaperones contribute to efficient folding. In the cell, the nascent polypeptide interacts with a network of molecular chaperones and other factors that influence its folding and processing. To complement our single-molecule experiments, we will carry out experiments in live cells to determine folding waypoints of multi-domain proteins in vivo. In addition, we will define how chaperones engage their substrates in living cells. Together, these studies will establish a framework for mechanistically understanding protein folding in vivo. Protein folding is of key importance for cellular protein homeostasis. Protein misfolding and aggregation are a hallmark of many diseases, including neurodegenerative diseases and cancer. The research proposed here may ultimately lead to a better understanding and possible treatments for these diseases.

抽象的 许多大型多域蛋白质本质上容易折叠和聚集。他们一开始 从翻译过程中的核糖体中，它们被分子伴侣遇到，可以帮助它们折叠到自己的 天然结构。虽然很明显，分子伴侣对于体内正确折叠至关重要，但我们知道 关于基本机制的很少。我们研究的长期目标是定义折叠 细胞中复合物多域蛋白的机制。该提议的目的是确定 折叠的两个新生链结合伴侣，触发因子和DNAK系统的功能 模型多域蛋白EF-G。我们将研究这些伴侣如何指导新生多的折叠 使用单分子力光谱与光学镊子使用单分子力光谱。这种方法是独特的 适合在复杂环境中操纵和观察单个新生多肽的折叠 核糖体和分子伴侣。我们将首先测量新生蛋白的折叠过渡 核糖体定义其折叠能量景观。然后，我们将确定如何触发因素和DNAK 改变这些能源景观。我们的体外实验将以机械细节和单一的方式揭示 分子分辨率这些伴侣如何促进有效折叠。在细胞中，新生的多肽 与分子伴侣网络以及影响其折叠和加工的其他因素相互作用。到 补充我们的单分子实验，我们将在活细胞中进行实验以确定折叠 体内多域蛋白的路点。此外，我们将定义伴侣如何与他们的底物互动 在活细胞中。这些研究将共同​​建立一个框架，以理解蛋白质 在体内折叠。蛋白质折叠对于细胞蛋白稳态至关重要。蛋白质错误折叠和 聚集是许多疾病的标志，包括神经退行性疾病和癌症。研究 在这里提出的建议最终可能导致对这些疾病的更好理解和可能的治疗。