Unraveling the mechanism by which the BAM complex mediates OMP biogenesis

项目摘要 革兰氏阴性菌如E.大肠杆菌，有一个外膜，提供营养和添加 保护细胞。外膜还含有蛋白质，可以使病原菌 逃避宿主的免疫反应并引起许多严重的传染病。尽管 这些外膜蛋白（OMP）对细胞存活和毒力的重要性， 这些蛋白质是如何折叠并插入外膜的。然而，在E.我们知道， 200 kDa的五组分复合物称为β-桶组装机械（BAM）复合物介导了 这些膜蛋白的生物合成。在过去的十年中， BAM复合物已被报道具有BamA的结构，BamA是中心组分，提供了最多的功能。 BAM复合体如何运作的有用线索在这里，揭示了BamA可能会破坏 局部膜，从而促进蛋白质折叠和插入膜中。最近，我们的工作 和其他人已经确定了BAM复合物的结构，揭示了前所未有的构象 BamA桶域内的变化。我们在这里的工作将利用这些结构来研究 BAM复合物在OMP生物合成中的构象变化。此外，我们将 确定底物OMP如何被BAM复合物识别以及它们如何与BAM相互作用 在折叠成膜的过程中。最后，我们将使用结构生物学方法来研究BAM复合物 在脂质双层中和在底物存在下。我们提出的研究将提供关键的功能和 结构洞察力，以解开BAM复合物如何在细菌中发挥其重要作用。

Project Summary Gram-negative bacteria, such as E. coli, have an outer membrane which provides nutrients and added protection for the cell. The outer membrane also contains proteins that can enable pathogenic bacteria to evade host immune responses and cause a number of serious infectious diseases. Despite the importance of these outer membrane proteins (OMPs) for cell survival and virulence, little is known about how these proteins are folded and inserted into the outer membrane. However, in E. coli, we know that a 200 kDa five-component complex called the β-barrel assembly machinery (BAM) complex mediates the biogenesis of these membrane proteins. In the last decade, the individual structures of components of the BAM complex have been reported with the structure of BamA, the central component, providing the most useful clues to how the BAM complex may function. Here, it was revealed that BamA may destabilize the local membrane, thereby promoting protein folding and insertion into the membrane. Recently, our work and others’ have determined the structure of the BAM complex, revealing unprecedented conformational changes within the barrel domain of BamA. Our work here will utilize these structures to study the role of the conformational changes for the function of the BAM complex in OMP biogenesis. Additionally, we will determine how substrate OMPs are recognized by the BAM complex and how they interact with BAM during folding into the membrane. Lastly, we will use structural biology methods to study the BAM complex in a lipid bilayer and in the presence of substrates. Our proposed studies will provide crucial functional and structural insight towards unraveling how the BAM complex performs its essential role in bacteria.