Mapping the pathway of membrane β-barrel protein folding by the Bam complex.

 描述(申请人提供)：通过BAM复合体定位膜β-Barrel蛋白折叠的路径。抗生素耐药性的上升是一个日益严重的人类健康问题，但针对这些病原体，特别是革兰氏阴性细菌的新抗生素的开发并没有跟上步伐。50多年来，没有发现一种针对革兰氏阴性菌的新抗生素，部分原因是未能确定抑制剂开发的新靶点。该建议旨在评估一种新的潜在靶点--基本BAM复合体，它将β-Barrel蛋白折叠并插入革兰氏阴性细菌的外膜(OM)中，从原核生物到真核生物都是如此。OM是革兰氏阴性菌的典型特征，缺陷会导致细胞膜通透性增加或细胞活力丧失。在该膜内存在完整的膜蛋白，它们完全采用β-Barrel结构。它们的组装机制目前尚不清楚，部分原因是在组装这些复杂的膜蛋白过程中，很难获得瞬时中间状态的高分辨率结构细节。为了研究这一过程，将采用化学和光交联策略来捕获和表征BAM复合体上的折叠中间体，使用缓慢折叠的必要外膜蛋白LptD。在这项提议中，我进一步减缓了LptD的组装，以捕获Bam复合体上折叠过程中的中间体。我证明了LptD桶的整个三级结构是在它的脂蛋白插头LptE周围形成的，在桶关闭和从Bam复合体释放之前。利用这个系统作为蛋白质折叠的模型，我将探索BAM亚基在桶组装过程中如何相互协调以及如何与其底物协调，方法是使用体内光交联策略绘制β链插入所需的相互作用表面和识别基序。我希望确定Bam复合体的两个基本成分BAMA和BAMD中的残基，这两个组分在促进桶折叠和插入方面至关重要。结果将用一份 Bam络合物活性的体外重建。最后，我将评估通过含有这些底物序列基序的多肽来抑制这种相互作用是否是一种可行的针对革兰氏阴性的策略。为了实现上述目标，我提出了以下具体目标：特定目标I：在体内表征BAM复合体与β桶底物之间的相互作用表面特定目标II：建立底物与BAM复合体结合的要求，并利用这些要求抑制LptD/E组装

 DESCRIPTION (provided by applicant): Mapping the pathway of membrane β-barrel protein folding by the Bam complex. The rise of antibiotic resistance is a growing human health concern, but the development of new antibiotics against these pathogens, particularly Gram-negative bacteria, has not kept pace. A new class of antibiotics against Gram-negatives has not been identified in over 50 years, partly due to failures to identify new targets for inhibitor development. This proposal aims to evaluate a new potential target, the essential Bam complex, which folds and inserts β-barrel proteins into the outer membrane (OM) of Gram-negative bacteria and is con- served from prokaryotes to eukaryotes. The OM is a defining characteristic of Gram-negatives and defects lead to increased membrane permeability or loss of cell viability. Within this membrane exist integral membrane proteins that exclusively adopt β-barrel structures. The mechanism of their assembly remains unclear partially because of difficulties in obtaining high- resolution structural details of transient intermediate states during the assembly of these complex membrane proteins. To study this process, chemical and photocrosslinking strategies will be employed to capture and characterize folding intermediates on the Bam complex using the slow folding essential outer membrane protein, LptD. In this proposal, I have further slowed the assembly of LptD to trap intermediates in the process of folding on the Bam complex. I show that the entire tertiary structure of the LptD barrel is formed around its lipoprotein plug, LptE, prior to barrel closure and release from the Bam complex. Using this system as a model for protein folding, I will explore how Bam subunits coordinate with each other and with their substrates during barrel assembly by mapping the interaction surfaces and recognition motifs required for β-strand insertion using an in vivo photocrosslinking strategy. I expect to identify residues in both essential components of the Bam complex, BamA and BamD, which are critical in facilitating barrel folding and insertion. Results will be recapitulated in a reconstitution of Bam complex activity in vitro. Lastly, I will evaluate if inhibition of such interactions by peptides containing these substrate sequence motifs is a viable strategy to target Gram-negatives. To achieve the outlined goals above, I propose the following Specific Aims: Specific Aim I: To characterize the interaction surfaces between the Bam complex and a β-barrel substrate in vivo Specific Aim II: To establish the requirements for substrate binding to th Bam complex and use these requirements to inhibit LptD/E assembly