Conformational conversions of prion protein

描述（由申请人提供）：在本申请中，我们建议继续我们的研究，旨在了解传染性海绵状脑病的致病过程的分子基础，这是一组致命的神经退行性疾病，如人类的克雅氏病和牛的牛海绵状脑病。人们认为，导致这些疾病的传染性病原体是朊病毒蛋白PrPSc的一种错误折叠的蛋白酶抗性形式，PrPSc由前体PrPC衍生而来，其机制尚不清楚。虽然PrPC的结构已被很好地表征，但该领域最持久的谜团之一是感染性PrPSc构象的分子结构。在这个项目中，我们建议承担主要的努力来弥补在理解异常折叠的朊病毒蛋白聚集体的结构特性方面的差距，具体目标集中在：(i)确定重组叙利亚仓鼠朊病毒蛋白PrPres的蛋白酶K抗性形式的构象结构，PrPSc-模板化蛋白错误折叠循环扩增（PMCA）反应获得；（ii）确定脑源性仓鼠PrPSc的中分辨率结构（地点和区域特异性结构限制）；（iii）阐明TSE的亢奋和困倦菌株对应的PrPSc的结构差异性质。本研究将采用许多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/氘交换、羟基自由基介导的足迹与质谱结合。

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.