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.

摘要 许多大的多结构域蛋白质固有地易于错误折叠和聚集。一旦它们出现 在翻译过程中，它们从核糖体中分离出来时，会遇到分子伴侣，帮助它们折叠成 原生结构虽然很明显，分子伴侣是必要的正确折叠在体内，我们知道， 对潜在机制知之甚少。我们研究的长期目标是确定折叠 细胞中复杂的多结构域蛋白的机制。本提案的目的是确定 两个新生的链结合分子伴侣，触发因子和DnaK系统，在折叠的功能， 模型多结构域蛋白EF-G。我们将研究这些分子伴侣是如何引导新生的多- 结构域蛋白质使用单分子力光谱与光镊。这种方法是独一无二的。 适合于操纵和观察单个新生多肽在复杂环境中的折叠， 核糖体和分子伴侣。我们将首先测量新生蛋白质的折叠转变， 核糖体来定义它们的折叠能量景观。然后，我们将确定触发因子和DnaK 改变这些能源格局。我们的体外实验将揭示机制细节，并与单一- 分子解析这些分子伴侣如何有助于有效折叠。在细胞中，新生多肽 与分子伴侣网络和影响其折叠和加工的其他因素相互作用。到 作为单分子实验的补充，我们将在活细胞中进行实验以确定折叠 多结构域蛋白在体内的路径点。此外，我们将定义如何伴侣从事他们的基板 在活细胞中。总之，这些研究将建立一个框架，从机制上理解蛋白质 体内折叠。蛋白质折叠对于细胞内蛋白质的稳态具有关键的重要性。蛋白质错误折叠和 聚集是许多疾病的标志，包括神经变性疾病和癌症。研究 在此提出的建议可能最终导致对这些疾病的更好理解和可能的治疗。