PHYSICAL STUDIES OF RECOMBINANT PrPs

重组 PrP 的物理研究

• 批准号: 6742808
• 负责人: SUSAN MARQUSEE
• 金额: $ 21万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6742808
• 关键词: alkylation; biophysics; biotechnology; chemical stability; conformation; fungal proteins; prions; protein binding; protein folding; protein structure; recombinant proteins; thermodynamics; yeasts

Prion diseases are the result of a change in the folding and oligomerization of PrP, and the mechanism of this transformation is at the heart of prion pathology. The goal of this proposal is to characterize the energy landscape of prion proteins. We propose to use biophysical probes based on solvent accessibility such as amide hydrogen exchange or side chain thiol exchange to elucidate the different conformations of these proteins. Such techniques are ideally suited for these non-traditional protein conformations: they are not limited by solubility, size or physical state of the protein. To date most work has focused on the soluble, cellular form of the protein. Future work focuses on new techniques to probe the structure and stability of other regions of the landscape - particularly the two ends of this reaction, PrP c and oligomers. We propose to obtain structural information on the amyloid state of prion proteins monitoring protection of cysteine residues to chemical alkylation. We will initially carry out these experiments using the yeast prion Sup35 and then tackle the more challenging PrP. We also propose to exploit the thermodynamic linkage between binding and stability to identify ligands that bind and stabilize the cellular form of the prion protein. Specifically, the aims of this project are: 1. Characterize the fibril form of a prion protein based on its residue accessibility to solvent. 2. Determine the structural differences between different fiber types of the yeast prion protein. 3. Characterize the energy landscape of the prion protein in vitro using thiol exchange. Develop a screen for ligand binding to the cellular form of the prion protein based on the thermodynamic lingage between binding and protein stability.

王室疾病是PRP折叠和低聚的变化的结果，这种转化的机理是prion病理学的核心。该提案的目的是表征prion蛋白的能量景观。我们建议使用基于溶剂可及性（例如酰胺氢交换或侧链硫醇兑换）的生物物理探针来阐明这些蛋白质的不同构象。这样的技术非常适合这些非传统蛋白构象：它们不受蛋白质的溶解度，大小或物理状态的限制。迄今为止，大多数工作都集中在蛋白质的可溶性细胞形式上。未来的工作着重于探测景观其他区域的结构和稳定性的新技术，尤其是该反应的两端PRP C和低聚物。我们建议 获取有关淀粉样蛋白蛋白淀粉样蛋白的结构信息，监测半胱氨酸残基对化学烷基化的保护。最初，我们将使用酵母菌sup35进行这些实验，然后解决更具挑战性的PRP。我们还建议利用结合和稳定性之间的热力学联系，以鉴定结合和稳定prion蛋白细胞形式的配体。具体而言，该项目的目的是： 1。根据prion蛋白的原纤维形式，基于其残基的溶剂可及性。 2。确定酵母菌prion蛋白不同纤维类型之间的结构差异。 3。使用硫醇交换在体外表征prion蛋白的能量景观。 基于结合和蛋白质稳定性之间的热力学lingage，开发了配体结合prion蛋白的细胞形式的筛选。