PHYSICAL STUDIES OF RECOMBINANT PRION PROTEINS

朊病毒疾病是PrP折叠和寡聚化变化的结果，这种转化的分子机制是朊病毒疾病机制的核心。本提案的目的是研究朊病毒蛋白的折叠，并确定和表征rPrP的任何非天然构象。在所谓的天然条件下存在的PrP的高能量部分折叠形式或折叠中间体无疑在从羊瘙痒症形式的球状蛋白转化并最终聚集中起重要作用。我们计划的结构和能量的折叠构象和任何部分折叠构象的PrP与本地构象和未折叠状态之间的稳定性。这种替代构象可以在折叠过程中或在平衡时瞬时填充。最初，我们将集中我们的研究野生型PrP（29-231），新开发的片段PrP 106，以及含有导致朊病毒疾病的内源性突变的变体。这项工作的总体目标将是帮助阐明在分子水平上的转换从细胞的羊瘙痒症形式的朊蛋白。具体而言，本提案的目的是：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罕见的部分折叠构象。3.利用停流CD和脉冲标记氢交换研究rPrP的动力学折叠途径。4.突变PrPs的能量景观的确定。

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.