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

 描述（由申请人提供）：通过BAM复合物绘制膜β-桶蛋白折叠的途径。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，特别是革兰氏阴性菌的新抗生素的开发并没有跟上步伐。针对革兰氏阴性菌的一类新抗生素在50多年来一直没有被发现，部分原因是未能确定抑制剂开发的新靶点。该提议旨在评估一种新的潜在靶点，必需的Bam复合物，其折叠并将β-桶蛋白插入革兰氏阴性菌的外膜（OM）中，并且从原核生物到真核生物都是保守的。OM是革兰氏阴性菌的定义特征，缺陷导致膜渗透性增加或细胞活力丧失。在该膜内存在完全采用β-桶结构的整合膜蛋白。它们的组装机制仍然不清楚，部分原因是难以获得这些复杂膜蛋白组装过程中瞬时中间状态的高分辨率结构细节。为了研究这一过程，化学和光交联策略将被用来捕获和表征的BAM复杂的折叠中间体，使用缓慢折叠的基本外膜蛋白，LptD。在这个提议中，我进一步减慢了LptD的组装，以捕获在BAM复合物上折叠过程中的中间体。我表明，整个三级结构的LptD桶周围形成的脂蛋白塞，LptE，桶关闭和释放之前，从BAM复杂。使用这个系统作为蛋白质折叠的模型，我将探索如何Bam亚基相互协调，并与他们的基板在桶组装映射的相互作用表面和识别基序所需的β-链插入使用体内光交联策略。我希望能确定残基的两个重要组成部分的BAM复合物，BamA和BamD，这是至关重要的，在促进桶折叠和插入。结果将在 在体外重建BAM复合物活性。最后，我将评估通过含有这些底物序列基序的肽抑制这种相互作用是否是靶向革兰氏阴性菌的可行策略。为了实现上述目标，我提出了以下具体目标：具体目标I：在体内表征Bam复合物与β-桶底物之间的相互作用表面具体目标II：建立与Bam复合物结合的底物要求，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