Conformational conversions of prion protein

描述（由申请人提供）：在本申请中，我们建议继续开展旨在了解传染性海绵状脑病（一组致死性神经退行性疾病，如人类克雅氏病和牛海绵状脑病）致病过程的分子基础的研究。据信，导致这些疾病的感染性病原体是朊病毒蛋白PrPSc的错误折叠的蛋白酶抗性形式，其通过知之甚少的机制衍生自前体PrPC。虽然PrPC的结构是很好的特点，在该领域最持久的奥秘之一是感染性PrPSc构象的分子结构。在本项目中，我们提出了一个主要的努力，以弥补差距在理解异常折叠的朊病毒蛋白聚集体的结构特性，具体目标集中在：（i）确定蛋白酶K-抗性形式的重组叙利亚仓鼠朊病毒蛋白，PrPres的构象结构，通过脑PrPSc模板蛋白错误折叠循环扩增（PMCA）反应获得;（ii）确定脑源性仓鼠PrPSc的中等分辨率结构（位点和区域特异性结构限制）;（iii）阐明对应于TSE的高和嗜睡株的PrPSc之间的结构差异的性质。这项研究将采用一些互补的生物物理方法，包括定点自旋标记，固态NMR光谱，氢/氘交换，羟基自由基介导的足迹结合质谱。

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.