The role of BamA in the biogenesis of beta-barrel membrane proteins

描述（由申请人提供）：传染病每年在世界各地引起广泛的疾病，是第二大死亡原因，特别是在不发达国家。随着多药耐药菌株的出现，需要新的，更有效的，更可持续的治疗方法是立即和至关重要的，以防止任何未来的流行病。我的研究将提供至关重要的深入了解表面受体和蛋白质的生物起源，帮助致病菌的毒力。这些知识将大大有助于开发更好的治疗这些感染性疾病的方法。 剂.革兰氏阴性菌含有内膜和外膜。外膜含有通常称为外膜蛋白（OMP）的β桶蛋白，其在货物运输，信号传导和细菌毒力中发挥重要作用。在革兰氏阴性细菌中，已知OMP在细胞质中合成，然后经由Sec易位子穿过内膜转运到周质中。一旦在周质中，伴侣蛋白引导新生的OMP穿过周质和肽聚糖到达外膜的内表面。在这里，新生的OMPs被称为BAM复合物的复合物识别，该复合物折叠并将新的OMPs插入外膜。BAM复合体究竟是如何实现其功能的仍然是未知的。然而，我们知道BAM复合物由五个亚基组成，分别为BamA（本身是一种OMP）、BamB、BamC、BamD和BamE，它们都是脂蛋白。研究表明，BamA和BamD对细胞活力和OMP生物合成是绝对必要的。我们的实验室和其他人已经报道了BamB，BamC，BamD，BamE和BamA的大部分周质结构域的结构，为BAM复合物如何发挥功能提供了初步的见解。然而，即使这些结构是已知的，BAM复合物如何识别，折叠，并插入新生OMP到外膜的机制仍然难以捉摸，主要是由于缺乏全长BamA结构和BamA复合物。最近，我确定了从杜克雷嗜血杆菌到2.9的截短BamA构建体和从淋病奈瑟菌到3.2的全长BamA构建体的晶体结构。在我提出的研究中，我的目标是在最近的成功的基础上，使用X射线晶体学来确定BamA与BamB-E复合物的结构，使用DEER光谱和交联来表征BamA的构象动力学，并使用交联来探索新生OMPs与BamA和其他BAM组分之间的相互作用，我的目标是了解BamA在BAM复合物中的功能作用。我在X射线晶体学方面有很强的背景，在我的博士后研究期间，我增加了使用最新技术（如新的稳定洗涤剂，bicelles和无规立方相（LCP）方法）处理和结晶膜蛋白的强大背景。最近，我解决了致病性奈瑟氏菌的两个重要表面蛋白的结构，这项工作作为完整的研究文章发表在Nature上（2012年3月）。对于这篇手稿，我还解决了研究小组几十年来一直试图解决的二铁人转铁蛋白的结构，但没有成功。最近，我还解决了激动剂结合神经降压素受体NTSR 1的晶体结构，NTSR 1是一种负责结合神经降压素和其他神经递质的GPCR。这项工作也于2012年10月发表在《自然》杂志上（研究文章）。我目前的工作重点是研究BAM复合体的BamA，我将把这个项目作为学术界的终身教职员工。有了这个，我最近的结果，BamA（H。ducreyi和N.淋病），最近也在《自然》杂志上发表了一篇完整的研究文章（2013年9月）。我的长期目标是拥有自己的研究实验室，作为研究I学术机构的教员，在那里我可以继续我的研究兴趣。这一直是我一生的抱负，虽然学术研究的资金现在比以往任何时候都更具竞争力，但我仍然致力于研究事业，我希望能够建立一个研究小组，显着推进我们目前对细菌和人类β桶膜蛋白的理解。

DESCRIPTION (provided by applicant): Infectious diseases cause widespread sickness throughout the world each year and are the second leading cause of death, particularly in underdeveloped countries. And with the emergence of multi-drug resistance strains, the necessity for new, more effective, and more sustainable therapies is immediate and vital to protect against any future pandemics. My studies will provide crucial insight into the biogenesis of surface receptors and proteins that assist pathogenic bacteria in their virulence. This knowledge will significantly assist in the development of better therapies against these infectious agents. Gram-negative bacteria contain an inner and outer membrane. The outer membrane contains a host of beta-barrel proteins commonly called outer membrane proteins (OMPs), which serve essential functions in cargo transport, signaling, and bacterial virulence. In Gram-negative bacteria, it is known that OMPs are synthesized in the cytoplasm and then transported across the inner membrane into the periplasm via a Sec translocon. Once in the periplasm, chaperones guide the nascent OMPs across the periplasm and peptidoglycan to the inner surface of the outer membrane. Here, the nascent OMPs are recognized by a complex known as the BAM complex which folds and inserts the new OMPs into the outer membrane. Exactly how the BAM complex is able to accomplish its function remains unknown. However, we do know that the BAM complex consists of five subunits named BamA (an OMP itself), BamB, BamC, BamD, and BamE, which are all lipoproteins. Studies have shown that BamA and BamD are absolutely essential for cell viability and OMP biogenesis. Our lab and others have reported the structures of BamB, BamC, BamD, BamE and a large portion of the periplasmic domain of BamA, providing initial insight into how the BAM complex may function. However, even with these structures being known, the mechanism for how the BAM complex recognizes, folds, and inserts nascent OMPs into the outer membrane remains elusive, largely due to the lack of a full length BamA structure and complexes with BamA. Recently, I determined the crystal structures of a truncated BamA construct from Haemophilus ducreyi to 2.9 ¿ and of a full length BamA construct from Neisseria gonorrhea to 3.2 ¿. In my proposed studies, I aim to build on this recent success to use X-ray crystallography to determine structures of BamA in complex with BamB-E, to use DEER spectroscopy and crosslinking to characterize the conformational dynamics of BamA, and to use crosslinking to explore the interactions between nascent OMPs and BamA and the other BAM components, with my goal being understand the functional role of BamA within the BAM complex. I have a strong background in X-ray crystallography and during my postdoctoral studies, have added a strong background in working with and crystallizing membrane proteins using the latest technologies such as new stabilizing detergents, bicelles, and lipidic cubic phase (LCP) methods. Recently, I solved the structures of two important surface proteins from pathogenic Neisseria and this work was published in Nature as a full research article (March 2012). For this manuscript, I also solved the structure of diferric human transferrin which research groups have been trying to solve for decades without success. More recently, I also solved the crystal structure of the agonist bound neurotensin receptor NTSR1, a GPCR responsible for binding neurotensin and other neurotransmitters. This work was published in Nature as well in October 2012 (Research Article). My current efforts are focused on studying BamA of the BAM complex and I will take this project with me as a tenure-track faculty member in academia. And with this, my most recent results, the crystal structures of BamA (H. ducreyi and N. gonorrhea), were also recently published in Nature as a full research article as well (Sept 2013). My long-term goal is to have my own research lab as a faculty member at a Research I academic institution where I can continue my research interests. This has been my lifelong ambition and while funding for academic research is more competitive now than ever, I remain dedicated to a career in research with aspirations that I will be able to establish a research group that significantly advances our current understanding of beta-barrel membrane proteins in both bacteria and humans.