SOLUTION SCATTERING OF PURIFIED GMPA OLIGOMERIC STRUCTURES

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。 GMPA是最近发现的一种非同源蛋白，它来自不对称分裂的原核生物新月弯杆菌，它是细胞极性建立和维持的关键，因为它能够定位与复制起点相互作用的多蛋白复合体，以维持适当的亚细胞结构。对该蛋白一级序列的分析表明，该蛋白由177个氨基酸组成，主要位于N-末端，其脯氨酸含量超过15%，并含有较高的谷氨酸含量，其等电点为~4.0。天然凝胶和大小排除层析表明，当从大肠杆菌的过度表达中纯化时，具有更高的有序橄榄状状态。在纯化的缓冲条件下也存在亚态的证据，尽管水平要低得多。我们建议对GMPA进行初步研究，使用溶液X射线散射来分析广泛条件下蛋白质的低聚状态。这将有助于理解该复合体的自组装，探索最大限度稳定蛋白质/蛋白质复合体的条件，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。

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.