Crystallization of outer membrane proteins for export of polysaccharide capsule

描述(申请人提供)：细菌经常输出高分子量的多糖，这些多糖可以形成与细菌表面相关的胶囊，或者以胞外多糖的形式分泌到周围环境中。胶囊可以帮助细菌逃避宿主免疫系统，促进与宿主细胞的黏附，并形成生物膜。与脂多糖(LPS)一样，第1组和第4组胶囊由短的寡糖重复单位通过类似Wzy的糖基转移酶组装而成，但通常要大得多(&gt；500 kDa)。革兰氏阴性菌需要额外的分子机制来运输胶囊多糖通过外膜，在外膜上它以一种未知的机制与内毒素结合。目前的模型是，Wza是一种保守的外膜辅助蛋白，是生产胶囊所必需的，它在多糖链退出的外膜上形成一个具有螺旋孔的八聚体。但有没有其他蛋白质可以在这个角色中发挥作用呢？肠源性和肠出血性大肠埃希氏菌需要GFC操纵子的7个基因来产生由O-抗原重复单位组成的第4组胶囊。与第一组系统不同，在gfcE(Wza同源基因)上游有四个必需基因(GfcABCD)编码未知功能的周质或外膜蛋白。有趣的是，大肠杆菌和许多其他革兰氏阴性菌编码一个类似的yjbEFGH操纵子，与生物膜的形成和第一组囊膜的表达有关。GfcD和YjbH被预测为大的外膜2桶蛋白，也可能被酰化。总体假设是，这种蛋白质是一个更复杂的分泌系统的一部分，该系统为多糖本身或通过外膜的辅助分子提供了一条退出路线。为了阐明GfcD在多糖出口中的结构和作用，我们为R21提出了两个目标。在具体目标1中，实验将确认GfcD的膜位置，表征gfcD突变对体内囊膜表达的影响程度，并确定哪些其他蛋白质与GfcD相互作用。在具体目标2中，我们将准备GfcD和三个同源物的表达构建体，并评估哪一个最适合于大规模纯化。蛋白质将用各种洗涤剂进行筛选，以获得最佳的溶解度和单分散性。纯蛋白将被用来筛选结晶条件。标记的蛋白质也将被准备用于固体核磁共振实验，以检查蛋白质的取向、二级结构和是否存在溶剂可访问的通道。这个短期项目的结果将为一个长期项目提供基础，以确定GfcD结构，阐明其他GFC蛋白在多糖输出中的功能，并评估该输出系统是否可以成为旨在抑制胶囊表达的广谱药物的靶点。

DESCRIPTION (provided by applicant): Bacteria often export high molecular weight polysaccharides that can form a capsule associated with the bacterial surface, or be secreted into the surrounding environment as exopolysaccharide. Capsules can help bacteria evade host immune systems, promote adhesion to host cells, and form biofilms. Like lipopolysaccharide (LPS), group 1 and 4 capsules are assembled from short oligosaccharide repeat units by a Wzy-like glycosyltransferase, but are typically much larger (>500 kDa). Gram-negative bacteria require additional molecular machinery to transport the capsule polysaccharide through the outer membrane where it associates with the LPS by an unknown mechanism. The current model is that Wza, a conserved outer membrane auxiliary protein necessary for capsule production, forms an octamer with a helical pore in the outer membrane from which the polysaccharide chain exits. But are there other proteins that could function in this role? Enteropathogenic and enterohaemorrhagic Escherichia coli require the seven genes of the gfc operon to produce a group 4 capsule comprised of O-antigen repeating units. Unlike the group 1 system, there are four essential genes (gfcABCD) upstream of gfcE (Wza homolog) that encode periplasmic or outer membrane proteins of unknown function. Interestingly, E. coli and many other Gram-negative species encode a similar yjbEFGH operon implicated in biofilm formation and group 1 capsule expression. GfcD and YjbH are predicted to be large outer membrane 2-barrel proteins that also may be acylated. The overall hypothesis is that this protein is part of a more complex secretion system that provides an exit route for the polysaccharide itself or for an accessory molecule through the outer membrane. To clarify the structure and role of GfcD in polysaccharide export we propose two aims for this R21. In Specific Aim 1, experiments will confirm the membrane location of GfcD, characterize the degree to which a gfcD- mutant affects capsule expression in vivo, and identify what other proteins interact with GfcD. In Specific Aim 2, we will prepare expression constructs of GfcD and three homologs and evaluate which will be best suited to for large scale purification. Proteins will be screened with a variety of detergents for optimal solubility and monodispersity. Pure protein will be used to screen for crystallization conditions. Labeled protein will also be prepared for solid-state NMR experiments to examine protein orientation, secondary structure and for the presence of a solvent accessible channel. Results from this short-term project will provide the foundation for a longer term project to determine the GfcD structure, elucidate the function of the other Gfc proteins in polysaccharide export, and evaluate if the export system could be a target for broad spectrum drugs aimed to suppress capsule expression.