Substrate Recognition by the Bam Complex

描述(申请人提供)：病原体对抗生素产生抗药性是一个基本的人类健康问题。针对这种日益严重的流行病的解决方案涉及到协同努力，既要发现对细胞生存至关重要的过程，又要设计对这些过程中的离散成分具有选择性的分子。Bam复合体促进了桶蛋白的折叠和插入革兰氏阴性细菌的外膜。这是一个必不可少的过程，但人们对此知之甚少。我们试图探索折叠机制的早期步骤-底物识别，因为这将为关键的细胞过程提供重要的见解。此外，对这一过程的了解将有助于合理开发抑制剂，作为未来对抗革兰氏阴性菌的抗生素。因此，这项研究研究了在大肠杆菌中Bam识别底物所必需的蛋白质-蛋白质相互作用。我们将首先探索通过使用未折叠的BAMA作为底物，未折叠的外膜蛋白的哪些特征对机器的招募至关重要。亲和共纯化和荧光分析将是用于这一分析的主要工具。为了确定BAM复合体中隐含的脂蛋白成分中的结合部位，我们将对该蛋白质进行突变分析并测定结合亲和力。为了验证从我们以BAMA为底物的生化和生物物理数据中得出的结论，我们将寻找在必要的外膜蛋白LptD中识别BAM所需的类似信号序列。一旦我们的结论在这种新底物的背景下被概括，我们将探索底物对BAM的亲和力是否与OM中发现的折叠桶蛋白的数量相关。了解底物-Bam识别事件将被证明是有价值的，因为我们寻求提高天然和非天然外膜蛋白在大肠杆菌中的表达效率。反过来，这一技术成就将为研究人员提供非常有利的条件，因为我们正在寻求开发新的抗生素来治疗病原体感染。根据上述项目大纲，我们提出了以下具体目标：具体目标一、描述展开的BAMA和BAMD之间相互作用的要求。具体目的II.评估SS基序的一般性，并确定它是否影响OM中的蛋白质水平。

DESCRIPTION (provided by applicant): The emergence of resistance to antibiotics by pathogenic organisms is a fundamental human health concern. The solution to this growing epidemic involves a concerted effort to both uncover processes that are critical to cellular viability, and design molecules selective for discrete components within these processes. The Bam complex facilitates the folding and insertion of ¿-barrel proteins into the outer membrane of Gram-negative bacteria. This is an essential but poorly understood process. We seek to explore an early step of the folding mechanism, substrate recognition, because this will provide important insight into a critical cellular process. Further, an understanding of this process will enable the rational development of inhibitors for use as future antibiotics against Gram-negative bacteria. Accordingly, this research studies a protein-protein interaction that is necessary for substrates to be recognized by Bam in E. coli. We will begin by exploring what features of unfolded outer membrane proteins are critical for recruitment to the machine through the use of unfolded BamA as a substrate. Affinity co- purifications and a fluorescence assay will be the principle tools used for this analysis. To characterize a binding site within an implicated lipoprotein component of the Bam complex we will perform mutational analysis of this protein and assay for binding affinity. To verify the conclusions drawn from our biochemical and biophysical data studying BamA as substrate, we will look to identify an analogous signal sequence necessary for recognition by Bam within the essential outer membrane protein LptD. Once our conclusions are recapitulated within the context of this new substrate, we will explore whether the affinity of a substrate for Bam correlates with the amount of folded ¿-barrel proteins found in the OM. Understanding of the substrate-Bam recognition event will prove valuable as we seek to increase efficiency in the expression of native and non-native outer membrane proteins in E. coli. In turn, this technological achievement will prove very enabling for researchers as we seek to develop new antibiotics for treatment of infection by pathogenic organisms. In accordance to the project outline above, we propose the following Specific Aims: Specific Aim I. To characterize requirements for interaction between unfolded BamA and BamD. Specific Aim II. To assess the generality of the ¿ ss motif and determine whether it influences protein levels in the OM.