SAXS STUDY ON THE CYTOPLASMIC SUBCOMPLEX FROM THE VIBRIO CHOLERAE TYPE 2 SECRETI

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者的研究机构。 致病菌如霍乱弧菌采用II型分泌系统通过外膜输出其感染因子如霍乱毒素。当孔位于外膜时，系统由细胞质分泌ATP酶（EpsE）供能。EpsE通过双位内膜蛋白EpsL连接到系统的其余部分。来自相关系统（例如III型分泌、IV型皮利）的各种报道表明，核苷酸结合对EpsE的寡聚状态具有显著影响，并且寡聚状态对ATP酶活性具有影响。然而，有迹象表明，与EpsL的胞质结构域（cyto-EpsL）复合的EpsE的寡聚化模式不同于目前的教条。我们有证据表明，EpsE（E）和cyto-EpsL（cL）形成寡聚体组装在最低E2 cL 2，并可能E6 cL 6，在核苷酸的存在下。我们已经开始进行溶液X射线散射研究，以调查这些组件作为不同的核苷酸的功能，并补充我们正在进行的结晶学研究，这个系统。我们在两种情况下测量了E-cL复合物，并且获得了略微不同的大小：Rg~ 43 A为500 kDa，Rg~ 38 A为400 kDa。电子对分布函数在相同的原子间距离处具有峰位置，表明两种形式的复合物具有共同的组装单元，可能是E-cL二聚体。 我们研究了ATP对来自不同生物体的E-cL复合物的影响，因为它们被认为在结晶筛选中表现得更好。在加入ATP后，来自副溶血性弧菌（Vpo）复合物的复合物没有改变其寡聚化状态：Rg 44.4 vs 43.8A（+ATP）。创伤弧菌（Vibriovulnificus，VV）的另一种复合物也显示出类似的微小变化：Rg 38.8 vs 39.8A（+ATP）。虽然我们迄今为止尚未观察到寡聚体组装中的任何实质性ATP依赖性变化，但这些结果表明存在两种不同形式的寡聚体组装，一种是Rg~43- 44 A，另一种是38- 39 A。

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. Pathogenic bacteria such as Vibrio cholerae employ Type II Secretion Systems for the export of their infectious agents such as cholera toxin through the outer membrane. While the pore is located in the outer membrane, the system is energized by a cytoplasmic secretion ATPase (EpsE). EpsE is linked to the rest of the system via the bitopic inner membrane protein EpsL. Various reports from related systems, e.g. Type III Secretion, Type IV Pili, indicate that nucleotide binding has a dramatic effect on the oligomeric state of EpsE, and that the oligomeric state has an effect on the ATPase activity. However, there are indications that the oligomerization pattern of EpsE in complex with the cytoplasmic domain of EpsL (cyto-EpsL) differs from the current dogma. We have evidence that EpsE (E) and cyto-EpsL (cL) form oligomeric assemblies at minimum E2cL2, and likely E6cL6, in the presence of nucleotides. We have begun conducting solution x-ray scattering studies to investigate these assemblies as a function of different nucleotides and complement our on-going crystallographic studies on this system. We have measured E-cL complex in two occasions, and obtained slightly different sizes: 500kDa with Rg~43A and 400kDa with Rg~38A. The electron pair distribution functions have the peak position at the identical inter-atomic distance, suggesting both forms of complex have common assembly unit, likely E-cL dimer. We studied the effects of ATP on E-cL complex from different organisms as they are thought to behave better in crystallization screening. Upon addition of ATP, the complex from Vibrio parahaemolyticus (Vpo) complex did not change its oligomerization state: Rg 44.4 vs 43.8A (+ATP). The other complex from vibrio vulnificus (VV) showed similarly minute change: Rg 38.8 vs 39.8A (+ATP). Although we have not so far observed any substantial ATP-dependent change in oligomer assembly, these results indicate that there are two distinctive forms of oligomer assembly, one with Rg~43-44A and the other with 38-39A.