SOLUTION SCATTERING OF PURIFIED GMPA OLIGOMERIC STRUCTURES

纯化 GMPA 寡聚结构的溶液散射

• 批准号: 7954469
• 负责人: Thomas N Earnest
• 金额: $ 0.02万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954469
• 关键词: Amino Acids; Architecture; Buffers; Caulobacter crescentus; Cell Polarity; Complex; Computer Retrieval of Information on Scientific Projects Database; Escherichia coli; Funding; Gel; Glutamic Acid; Grant; Homologous Protein; Institution; Maintenance; Molecular Sieve Chromatography; N-terminal; Pilot Projects; Proline; Proteins; Replication Origin; Research; Research Personnel; Resources; Solutions; Source; Structure; United States National Institutes of Health; in vivo; overexpression; protein complex; self assembly; structural biology; synchrotron radiation

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. GmpA is a recently discovered non-homologous protein from the assymetrically-dividing procaryote, Caulobacter crescentus, which has been shown to be a critical lynchpin in the establishment and maintenance of cell polarity though its ability to a localize a multi-protein complex which interacts with the origin of replication to maintain the proper subcellular architecture. Analysis of the primary sequence of GmpA shows a 177 amino acid protein with a proline content of over 15%, primarily in the N-terminal region, and a high glutamic acid content contributing to its low pI of ~4.0. Native gels and size exclusion chromatography indicate a higher-ordered oliomeric state when purified from overexpression in E. coli. Evidence of substates under the buffer conditions of purification is also present, though at a much lower level. We propose pilot studies of GmpA using solution x-ray scattering to analyse the oligomeric state of the protein under a wide range of conditions. This will assist in understanding the self-assembly of this complex, explore conditions of maximal protein/protein complex stabilization, and study interactions of GmpA with other proteins that we have identified as interaction partners in vivo.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 GmpA 是最近从不对称分裂的原核生物新月柄杆菌中发现的一种非同源蛋白，它已被证明是建立和维持细胞极性的关键关键，尽管它能够定位与复制起点相互作用的多蛋白复合物以维持适当的亚细胞结构。对 GmpA 一级序列的分析显示，这是一种由 177 个氨基酸组成的蛋白质，脯氨酸含量超过 15%（主要在 N 末端区域），并且高谷氨酸含量导致其 pI 较低，约为 4.0。当从大肠杆菌中的过度表达中纯化时，天然凝胶和尺寸排阻色谱显示出更高阶的寡聚状态。还存在纯化缓冲条件下亚状态的证据，尽管水平低得多。我们建议使用溶液 X 射线散射对 GmpA 进行初步研究，以分析多种条件下蛋白质的寡聚状态。这将有助于了解该复合物的自组装，探索最大蛋白质/蛋白质复合物稳定的条件，并研究 GmpA 与我们已确定为体内相互作用伙伴的其他蛋白质的相互作用。