Conformational conversions of prion protein

描述(申请人提供)：在这项申请中，我们建议继续我们的研究，旨在了解传染性海绵状脑病、一组致命的神经退行性疾病，如人类的克雅氏病和牛的海绵状脑病的致病过程的分子基础。据信，导致这些疾病的感染性病原体是PrPSc蛋白的一种错误折叠的、对蛋白酶有抵抗力的形式，PrPSc是由PrPC前体通过一种鲜为人知的机制而产生的。虽然PrPC的结构被很好地描述，但该领域最持久的谜团之一是具有感染性的PrPSc构象的分子结构。在这个项目中，我们建议做出重大努力来弥合在理解异常折叠的Prion蛋白聚集体的结构性质方面的差距，具体目标集中在：(I)通过脑PrPSc模板蛋白质错折叠循环扩增(PMCA)反应获得的重组叙利亚仓鼠PrPres蛋白的抗蛋白酶K形式的构象结构；(Ii)确定脑源性仓鼠PrPSc的中分辨结构(位点和区域特定的结构约束)；(Iii)阐明与TSE过度株和昏昏型菌株相对应的PrPSc结构差异的性质。这项研究将使用一些互补的生物物理方法，包括定点自旋标记、固态核磁共振光谱、氢/氢交换和羟基自由基介导的足迹结合质谱学。

DESCRIPTION (provided by applicant): In this application we propose to continue our studies aimed on understanding the molecular basis of the pathogenic process in transmissible spongiform encephalopathies, a group of fatal neurodegenerative diseases such as Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle. It is believed that the infectious pathogen responsible for these diseases is a misfolded, proteinase-resistant form of the prion protein, PrPSc, which is derived from a precursor PrPC by a poorly understood mechanism. While the structure of PrPC is well characterized, one of the most enduring mysteries in the field is the molecular structure of infectious PrPSc conformers. In this project, we propose to undertake a major effort to bridge the gap in understanding the structural properties of abnormally folded prion protein aggregates, with specific aims focused on: (i) determining the conformational structure of proteinase K- resistant form of the recombinant Syrian hamster prion protein, PrPres, obtained by brain PrPSc- templated protein misfolding cyclic amplification (PMCA) reaction; (ii) determining medium- resolution structure (site- and region-specific structural constraints) for brain-derived hamster PrPSc; (iii) elucidating the nature of structural differences between PrPSc corresponding to the hyper and drowsy strains of TSE. This study will employ a number of complementary biophysical methods including site-directed spin labeling, solid-state NMR spectroscopy, hydrogen/deuterium exchange, and hydroxyl radical-mediated footprinting coupled with mass spectrometry.