PHYSICAL STUDIES OF RECOMBINANT PrPs

朊病毒疾病是PrP折叠和寡聚化变化的结果，这种转化的机制是朊病毒病理学的核心。本提案的目标是表征朊病毒蛋白的能量景观。我们建议使用基于溶剂可及性的生物物理探针，如酰胺氢交换或侧链硫醇交换来阐明这些蛋白质的不同构象。这种技术非常适合这些非传统的蛋白质构象：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，大多数研究都集中在蛋白质的可溶性细胞形式上。未来的工作重点是探索其他区域的结构和稳定性的新技术-特别是该反应的两端，PrP c和低聚物。我们建议

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.