Insertion and folding of beta-barrel membrane proteins into lipid bilayers - Functions of BamA (YaeT) and of membrane anchored Bam lipoproteins in membrane protein folding.

尽管跨膜蛋白的插入和折叠过程对膜生物发生和细胞生长很重要，但这些过程还不清楚。对于外膜蛋白（OMP）在细菌和真核细胞线粒体中的插入和折叠，桶装配机械（BAM）复合物是至关重要的。基于我们以前对OmpA和FomA等外膜蛋白折叠和插入机制的研究，我们正在研究大肠杆菌BAM蛋白的生物物理和生化功能。这种BAM复合物由必需的跨膜蛋白BamA（也称为YaeT或Omp 85）和四种外周脂蛋白BamB、C、D和E组成。只有BamD（Yf 10）是必需的，但同时缺失BamB和BamE也是致命的。在之前的资助期间，我们发现BamA、BamD和BamB独立地提高了OmpA从在8 M尿素中制备的未折叠形式到脂质双层的折叠速率。BamA还促进了OmpA与其周质伴侣Skp的复合物的折叠。分析进一步表明BamA的周质结构域（PD）对观察到的折叠速率具有主要贡献。脂质、脂蛋白的脂质锚或结合的客户OMP在BAM复合物的任何已知结构中均未解析，这是复合物的结构-功能关系未被理解的原因之一。本项目的两个目的是研究BAM的蛋白质如何在脂质环境中与OmpA和OmpG等外膜蛋白相互作用，以及这些相互作用如何促进折叠和膜插入。折叠和插入的外膜蛋白的速率可以通过温度和膜的脂质组成来调节，并且我们最近确定了与含有BamA的二月桂酰磷脂双层结合的OmpA的折叠中间体。在BAM蛋白存在下，较慢的折叠过程和捕获的客户端OMP折叠中间体将使我们能够研究BAM蛋白如何发挥功能。我们将使用定点光谱来确定BAM蛋白在OmpA折叠中的相互作用，并研究相关的蛋白质-蛋白质和蛋白质-脂质相互作用。BamA和BamD的可溶性PD在周质中形成大的环状结构，并且我们可以证明两者都与脂质膜结合。定点荧光方法和荧光淬灭与脂质结合的淬灭剂将被用来调查哪些区域的PD BamA和BamD与脂质相互作用。我们还将研究这两种蛋白质的脂质选择性和在其附近可能形成的脂质微区。这项工作的结果将大大推进我们的理解的原则，蛋白质插入机制如何在膜蛋白折叠。由于细菌和真核细胞的蛋白质插入机制存在差异，这些基础知识也可能对抗生素的未来发展有用。

Insertion and folding of transmembrane proteins are not well understood, although these processes are important for membrane biogenesis and cell growth. For insertion and folding of outer membrane proteins (OMPs) in bacteria and mitochondria of eukaryotic cells, the barrel assembly machinery (BAM) complex is vital. Based on our previous work on the mechanism of folding and insertion of OMPs like OmpA and FomA, we are investigating the biophysical and biochemical functions of the BAM proteins of Escherichia coli. This BAM complex is composed of the essential transmembrane protein BamA (also called YaeT or Omp85) and of four peripheral lipoproteins, BamB, C, D, and E. Only BamD (YfiO) is essential, but the simultaneous deletion of BamB and BamE is also lethal. In the previous funding period, we found that BamA, BamD, and BamB independently improve the folding rates of OmpA into lipid bilayers from an unfolded form prepared in 8 M urea. BamA also facilitated folding of OmpA from a complex with its periplasmic chaperone Skp. The analysis indicated further that the periplasmic domain (PD) of BamA has a principal contribution to the observed folding rates. Lipids, the lipid anchors of the lipoproteins, or bound client OMPs are not resolved in any of the known structures of the BAM complex, which is one of the reasons why the structure-function relationships of the complex are not understood. Two aims of the present project are to examine how proteins of the BAM interact with OMPs like OmpA and OmpG in a lipid environment and how these interactions facilitate folding and membrane insertion. The rates of folding and insertion of OMPs can be modulated by temperature and by the lipid composition of the membrane and we recently identified a folding intermediate of OmpA bound to bilayers of dilauroyl phospholipids containing BamA. A slower folding process and trapped folding intermediates of client OMPs in the presence of BAM proteins will now allow us to investigate how BAM proteins function. We will use site-directed spectroscopy to determine interactions of the BAM proteins in the folding of OmpA and investigate the relevant protein-protein and protein-lipid interactions. The soluble PD of BamA and BamD form a large ring-like structure in the periplasm and we could demonstrate that both bind to lipid membranes. Site-directed fluorescence methods and fluorescence quenching with lipid-bound quenchers will be used to investigate which regions of the PD of BamA and of BamD interact with lipids. We will also examine the lipid-selectivity of both proteins and a possible formation of a lipid microdomain in their vicinity. The results of this work will greatly advance our understanding of the principles how protein insertion machinery works in membrane protein folding. Since there are differences in the protein insertion machineries of bacteria und eukaryotic cells, this basic knowledge could also be useful for future developments of antibiotics.