Substrate Recognition by the Bam Complex

描述（由申请人提供）：病原微生物对抗生素产生耐药性是人类健康的一个基本问题。解决这一日益严重的流行病需要共同努力，以揭示对细胞活力至关重要的过程，并设计对这些过程中的离散组分具有选择性的分子。Bam复合物促进了桶蛋白的折叠和插入到革兰氏阴性细菌的外膜中。这是一个重要的过程，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，因为这将提供重要的洞察到一个关键的细胞过程。此外，对这一过程的理解将使抑制剂的合理发展，作为未来的抗生素对革兰氏阴性菌的使用。 因此，本研究研究了蛋白质-蛋白质相互作用，这是必要的底物被识别的巴姆在E。杆菌我们将开始探索未折叠外膜蛋白的哪些特征对于通过使用未折叠BamA作为底物招募到机器中至关重要。亲和共纯化和荧光测定将是用于该分析的主要工具。为了表征Bam复合物的相关脂蛋白组分内的结合位点，我们将对该蛋白进行突变分析并测定结合亲和力。 为了验证从我们的生化和生物物理数据研究BamA作为底物得出的结论，我们将寻找识别必需的外膜蛋白LptD内的BAM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，因为我们试图提高天然和非天然外膜蛋白在大肠杆菌中的表达效率。杆菌反过来，这一技术成就将证明对研究人员非常有利，因为我们寻求开发新的抗生素来治疗病原体感染。 根据上述项目纲要，我们提出以下具体目标：具体目标一。描述未折叠BamA和BamD之间相互作用的要求。 具体目标二。评估<$ss基序的一般性，并确定其是否影响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.