PHYSICAL STUDIES OF RECOMBINANT PRION PROTEINS

Prion病是PrP折叠和寡聚改变的结果，而这种转变的分子机制是PrP病机制的核心。这项建议的目的是研究PrP蛋白的折叠，并鉴定和鉴定rPrP的任何非天然构象。高能的部分折叠形式，或在所谓的自然条件下存在的PrP的折叠中间体，无疑在这种由球状蛋白质以羊毛膜形式转化并最终聚集的过程中发挥着重要作用。我们计划对PrP的折叠构象和任何部分折叠构象的结构和能量进行表征，这些构象的稳定性介于天然构象和未折叠状态之间。这种替代构象可以在折叠过程中瞬时填充，也可以在平衡时填充。首先，我们将重点研究野生型PrP(29-231)，新开发的PrP106片段，以及包含导致PrP病的内源性突变的变体。这项工作的总体目标将是在分子水平上帮助阐明PrP从细胞形式到刮痕形式的转换。具体地说，本方案的目的是：1.确定PrPc构象稳定性的热力学。2.利用自然态氢交换确定与自然态平衡的任何稀有部分折叠构象。3.用停流CD和脉冲标记氢交换技术研究rPrP的动力学折叠途径。4.突变体pRPS能谱图的测定。

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.