BIOPHYSICAL PROPERTIES OF PRION PROTEIN OLIGOMERS

该计划项目的这一部分的长期目标是阐明 传染性海绵状脑病(TSE)的致病过程 折磨人类和动物的神经退行性疾病。纯蛋白质模型假定 导致这些疾病的感染性病原体是Pron蛋白的错误折叠形式， PrPSc通过与正常的普恩蛋白结合并催化其转化为 致病形式。继最近几年以来，TSE疾病已成为一个主要的公共卫生问题 牛海绵状脑病(BSE)的流行及BSE可能有的指征 跨越物种障碍，在人类中引起变异的克雅氏病。主要关注点 这个项目的重点是了解各种异常折叠形式的结构特性 重组普恩蛋白。第一个具体目标是确定淀粉样纤维的结构。 重组蛋白在体外以接近氨基酸残基的分辨率形成。这将是 使用许多新出现的生物物理技术来完成，这些技术提供了现场- 关于当地蛋白质迁移率和可及性的具体信息以及 蛋白淀粉样蛋白中特定位置之间的分子间和分子内距离。 另一个具体目标是扩展和优化最近开发的蛋白质技术 利用循环扩增(PMCA)实现有效的脑PrP^模板转换 将细菌表达的重组Prion蛋白转化为蛋白酶K-抗性形式，并对其 该产品在实验动物中的传染性。最后，在#年取得的经验的基础上 自发形成的蛋白淀粉样蛋白的结构研究(特定目标1)，我们将 确定在脑PrPSc中产生的重组PrP寡聚体的结构组织 模板PMCA反应。我们的假设是PrPSc的基本折叠基序类似于 PRP淀粉样纤维，虽然有些差异可能解释了特别高的抗性 预计大脑PrPSc将被蛋白质分解消化。

The long-term goal of this component of the Program Project is to elucidate the molecular basis of the pathogenic process in transmissible spongiform encephalopathies (TSEs), a group of fatal neurodegenerative diseases that afflict humans and animals. The protein-only model postulates that the infectious pathogen responsible for these diseases is a misfolded form of the prion protein, PrPSc, which self-propagates by binding to normal prion protein and catalyzing its conversion to the pathogenic form. TSE diseases have emerged as a major public health issue following recent epidemics of bovine spongiform encephalopathy (BSE) and indications that BSE might have crossed the species barrier to cause variant Creutzfeldt-Jakobdisease in humans. The main focus of this project is on understanding structural properties of various abnormally folded forms of the recombinaht prion protein. The first specific aim is to determine the structure of amyloid fibrils formed by the recombinant prion protein in vitro at a resolution close to amino acid residue. This will be accomplished using a number of newly emerged biophysical techniques that provide site- specific information about local protein mobility and accessibility and measurements of intermolecular and intramolecular distances between specific sites within the prion protein amyloid. Another specific aim is to expand and optimize the recently developed technique of protein misfolding by cyclic amplification (PMCA) to accomplish efficient brain PrP^-templated conversion of bacterially-expressed recombinant prion protein to proteinase K-resistantform, and assess the infectivity of this product in experimental animals. Finally, building on the experience gained in structural studies with spontaneously formed prion protein amyloid (Specific Aim 1), we will determine structural organization of the recombinant PrP oligomers generated in brain PrPSc- templated PMCA reaction. Our hypothesis is that the basic folding motif of PrPSc is similar to that of PrP amyloid fibrils, though some differences which may account for especially high resistance of brain PrPSc to proteolytic digestion are expected.