SAXS CHARACTERIZATION OF PRION FORMATION

朊病毒形成的 SAXS 表征

• 批准号: 8362408
• 负责人: LESTER G CARTER
• 金额: $ 0.27万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362408
• 关键词: Amyloid Fibrils; Amyloid fibers; Biological Models; Bovine Spongiform Encephalopathy; Buffers; Cell Adhesion; Disease; Exclusion; Funding; Grant; Hamsters; High Pressure Liquid Chromatography; Infectious Agent; Link; Membrane Proteins; Molecular Sieve Chromatography; National Center for Research Resources; Physiological; Polymers; PrP; PrPSc Proteins; Principal Investigator; Prions; Protein Analysis; Protein Isoforms; Proteins; Protocols documentation; Radiation; Research; Research Infrastructure; Resources; Signal Transduction; Solutions; Source; United States National Institutes of Health; beamline; cost; instrument; recombinant PrP; structural biology

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. PrPC is a membrane-associated protein believed to be involved in cellular adhesion and signaling. Furthermore, the protein also exists in an infectious form that is linked to diseases such as Bovine Spongiform Encephalopathy ("mad cow disease") and Creutzfeldt?Jakob disease. This infectious isoform, termed PrPSc, is also known as a prion. PrPSc is capable of acting as an infectious agent through templating of the misfolded state onto PrPC. The precise conversion mechanism how PrPC is converted to PrPSc, and how subsequently amyloid fibrils are formed, are currently unknown. Several potential intermediates have been identified, including a mainly ?-sheeted oligomer and several polymers. Using a recombinantly expressed hamster PrP as a model system, it has been shown that some of these multimers can be separated by size exclusion chromatography (SEC). However, due to the tendency of PrP to rapidly aggregate, these different forms have yet to be characterized structurally. We propose to characterize these isoforms using solution x-ray scattering at bio-SAXS beamline BL4-2. We have developed a new protocol for connecting a HPLC size exclusion column (SEC) column directly to the BL4-2 scattering instrument, allowing rapid analysis of the protein fractions as they are eluted. We have already succeeded in characterizing the normal physiological form of recombinant PrP using this HPLC setup (preliminary results discussed below), and we now propose to refine our protocol to analyze the other isoforms and study initial oligomeric assembly intermediates leading to the formation of infectious amyloid fibers. Additionally we will also explore the effects of varying the protein buffer and the addition of small compounds on PrPC using the BL4.2 high-throughput facilities.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 PRPC是一种与膜相关的蛋白质，据信与细胞粘附和信号传导有关。 此外，该蛋白质还以感染性形式存在，与牛海绵状脑病（“疯牛病”）和克鲁特兹菲尔德（Creutzfeldt？Jakob）病有关。 这种称为PRPSC的传染性同工型也被称为prion。 PRPSC能够通过将错误折叠状态的PRPC模板模板化为感染剂。 当前未知的精确转化机制如何转换为PRPC转换为PRPSC，以及随后的淀粉样蛋白原纤维的形成方式。 已经确定了几种潜在的中间体，其中包括主要的？ - 表演的低聚物和几种聚合物。 使用重组表达的仓鼠PRP作为模型系统，已经证明其中一些可以通过尺寸排除色谱法（SEC）进行分离。 但是，由于PRP趋于汇总的趋势，这些不同的形式尚未在结构上进行表征。 我们建议使用溶液X射线散射在Bio-Saxs Beamine BL4-2上表征这些同工型。 我们已经开发了一种新的协议，该方案将HPLC尺寸的排除柱（SEC）直接连接到BL4-2散射仪器，从而可以在洗脱过程中快速分析蛋白质分数。 我们已经成功地使用此HPLC设置来表征重组PRP的正常生理形式（下面讨论的初步结果），现在我们建议完善我们的方案，以分析其他同工型并研究最初的寡聚组装中间体，从而导致感染性淀粉样纤维的形成。 此外，我们还将探索使用BL4.2高通量设施在PRPC上添加蛋白质缓冲液的影响。