PHYSICAL STUDIES OF RECOMBINANT PRION PROTEINS

重组朊病毒蛋白的物理研究

• 批准号: 6299220
• 负责人: SUSAN MARQUSEE
• 金额: $ 22.21万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6299220
• 关键词: affinity labeling; biophysics; cell aggregation; circular dichroism; conformation; fluorescence spectrometry; gene mutation; hydrogen ions; nuclear magnetic resonance spectroscopy; oligopeptides; prions; protein denaturation; protein folding; protein sequence; protein structure; recombinant proteins; scrapie; stop flow technique; thermodynamics; thermostability

Prion diseases are the result of a change in the folding and oligomerization of PrP, and the molecular mechanism of this transformation is at the heart of the mechanism of prion diseases. The goal of this proposal is to study the folding of the prion protein, and to identify and characterize any non-native conformers of rPrP. High energy partially folded forms, or folding intermediates of PrP present under so-called native conditions undoubtedly play an important role in this transformation from a globular protein in the scrapie form and ultimately aggregate. We plan to characterize the structure and energetics of folded conformers and any partially folded conformers of PrP with stabilities between the native conformers and the unfolded state. Such alternative conformers may be populated either transiently during the folding process or at equilibrium. Initially we will focus our studies on the wildtype PrP (29-231), the newly developed fragment PrP106, as well as variants containing endogenous mutations that lead to prion disease. The overall goal of this work will be to aid in the elucidation at the molecular level of the conversion from the cellular to the scrapie form of PrP. Specifically, the aims of this proposal are: 1. To determine the thermodynamics of PrPc conformation stability. 2. Identification of any rare partially folded conformations in equilibrium with the native state using native state hydrogen exchange. 3. Studies of the kinetic folding pathway of rPrP using stopped-flow CD and pulse-labeling hydrogen exchange. 4. Determination of the energy landscape of mutant PrPs.

政治疾病是PRP折叠和寡聚的变化的结果，这种转化的分子机制是prion疾病机制的核心。该提案的目的是研究prion蛋白的折叠，并识别和表征RPRP的任何非本地构象体。高能在所谓的天然条件下存在部分折叠的形式，或折叠PRP的中间体，无疑在这种转化中起着重要作用。我们计划表征折叠构象体的结构和能量学和任何部分折叠的PRP构象异构体，具有天然构象异构体和展开状态之间的稳定性。这种替代构象异构体可以在折叠过程中或平衡时暂时填充。最初，我们将研究将研究重点放在野生型PRP（29-231）上，即新开发的片段PRP106，以及含有导致病毒疾病的内源性突变的变体。这项工作的总体目标是帮助从细胞转换为PRP的crapie形式的分子水平的阐明。具体而言，该提案的目的是：1。确定PRPC构象稳定性的热力学。 2。使用天然状态氢交换鉴定与天然状态平衡的任何稀有构象。 3。使用停止流量CD和脉冲标记的氢交换对RPRP的动力学折叠途径的研究。 4。突变PRP的能量景观的确定。