BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF PRION PROTEIN 2D CRYSTALS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 普恩病毒会导致可传染的神经退行性疾病，而且总是会导致致命的进展。普恩是一种具有感染性的粒子，完全由普恩蛋白组成。可区分两种形式：良性的细胞型PrPC和致病的“瘙痒病”亚型PrPSc，后者被证实重新排列。由于PrPC和PrPSc在结构上不同，所以对每种构象的研究是必要的。我们的研究重点是PrPSc。我们开发了一种高产的蛋白质纯化方案。我们的制剂含有蛋白2D晶体，用标准和低剂量冷冻电子显微镜对晶体进行了结构分析。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识别污染物/共价蛋白质，以便系统地去除它们。这就是质谱学的任务。在合作的基础上，通过对考马斯染色的聚丙烯酰胺凝胶的条带进行分析来鉴定相互复制的蛋白质。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.