BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF PRION PROTEIN 2D CRYSTALS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 子项目的主要研究者可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 朊病毒可引起传染性神经退行性疾病，其进展总是致命的。朊病毒是一种只由朊病毒蛋白组成的感染性颗粒。两种形式的区别：良性的，细胞形式PrPC，和致病的“羊瘙痒症”亚型PrPSc，这是确认重排。由于PrPC和PrPSc在结构上不同，因此必须对每种构象进行研究。我们的研究重点是PrPSc。我们开发了一种高产量的蛋白质纯化方案。我们的制剂含有朊病毒蛋白2D晶体，其结构用标准和低剂量冷冻电子显微镜分析。 然而，为了进一步提高我们样品的质量和纯度，我们需要从我们的样品中鉴定污染物/共纯化蛋白质，以便系统地去除它们。这就是质谱分析的任务。 在合作的基础上，通过分析考马斯染色聚丙烯酰胺凝胶的条带来鉴定共纯化蛋白。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Prions cause transmissible, neurodegenerative diseases with invariably fatal progression. A prion is an infective particle consisting exclusively of the prion protein. Two forms are discriminated: the benign, cellular form PrPC, and the disease-causing "scrapie" isoform PrPSc, which is confirmationally rearranged. Since PrPC and PrPSc differ in structure, studies of each conformation are essential. Our apporach focuses on PrPSc. We developed a high-yield protein purification protocol. Our preparations contain prion protein 2D crystals, which are structurally analyzed with standard and low-dose cryo electron microscopy. However, to further the quality and purity of our samples, we need to identify contaminants/copurified proteins from our samples for their systematic removal. This is the task for mass spectrometry. On a collaborative basis, copurified proteins are to be identified by analysis of bands derived from Coomassie-stained polyacrylamide gels.