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.

β-桶膜蛋白插入和折叠到脂质双层中 - BamA (YaeT) 和膜锚定的 Bam 脂蛋白在膜蛋白折叠中的功能。

• 批准号: 88778146
• 负责人: Professor Dr. Jörg H. Kleinschmidt
• 金额: --
• 依托单位: Abteilung Biophysik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2008
• 资助国家: 德国
• 起止时间: 2007-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/88778146?language=en
• 关键词: Insertion; folding; beta; barrel; membrane

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.

跨膜蛋白的插入和折叠还不是很清楚，尽管这些过程对于膜的生物发生和细胞生长是重要的。在细菌和真核细胞线粒体中，外膜蛋白(OMP)的插入和折叠过程中，桶组装机械(BAM)复合体是至关重要的。在前期对ompA和FOMA等OMPS折叠和插入机制研究的基础上，我们对大肠杆菌BAM蛋白的生物物理和生化功能进行了研究。这种BAM复合体由必需的跨膜蛋白BAMA(也称为YaeT或Omp85)和四种外周脂蛋白Bamb、C、D和E组成，只有BAMD(YfiO)是必需的，但Bamb和BAME的同时缺失也是致命的。在之前的资助期间，我们发现BAMA、BAMD和BAMB独立地提高了OmpA在8M尿素中制备的未折叠形式到脂质双层中的折叠率。BAMA还促进了OmpA与其周质伴侣Skp的复合体的折叠。分析进一步表明，BAMA的周质结构域(PD)对观察到的折叠速度有主要贡献。脂类、脂蛋白的脂锚或结合的客户OMP在BAM复合体的任何已知结构中都不能分解，这是为什么人们不了解复合体的结构-功能关系的原因之一。本项目的两个目标是研究BAM的蛋白质如何在脂质环境中与OmpA和OmpG等OMP相互作用，以及这些相互作用如何促进折叠和膜插入。OMPS的折叠和插入速度可以受到温度和膜脂组成的调节，我们最近发现了一种结合到含有BAMA的双月桂酰磷脂双层上的OmpA折叠中间体。在BAM蛋白存在的情况下，较慢的折叠过程和捕获的客户OMP的折叠中间产物现在将使我们能够研究BAM蛋白是如何发挥作用的。我们将使用定点定向光谱来确定OmpA折叠过程中BAM蛋白的相互作用，并研究相关的蛋白质-蛋白质和蛋白质-脂相互作用。BAMA和BAMD的可溶性Pd在周质中形成一个大的环状结构，我们可以证明两者都与脂膜结合。我们将使用定点荧光方法和用脂质结合的猝灭剂猝灭荧光来研究BAMA和BAMD的PD的哪些区域与脂质相互作用。我们还将检查这两种蛋白质的脂类选择性，以及它们附近可能形成的脂类微域。这项工作的结果将极大地促进我们对蛋白质插入机制如何在膜蛋白质折叠中工作的原理的理解。由于细菌和真核细胞的蛋白质插入机制不同，这些基本知识也可能对未来抗生素的发展有用。