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

BamA 在 β-桶膜蛋白生物发生中的作用

基本信息

批准号：
9110832
负责人：
Nicholas Noinaj
金额：
$ 10.8万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-15 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9110832
关键词：
Academia Agonist Bacteria Binding Biochemical Biogenesis Cause of Death Cell Survival Cessation of life Communicable Diseases Complex Country Cytoplasm Cytoprotection Detergents Development Electron Microscopy Faculty Funding Future G-Protein-Coupled Receptors Goals Gram-Negative Bacteria Health Hemophilus ducreyi Human Immune response Infectious Agent Institution Knowledge Label Learning Length Lipoproteins Manuscripts Mediating Membrane Membrane Proteins Methods Molecular Molecular Chaperones Multi-Drug Resistance Names Nature Neisseria Neisseria gonorrhoeae Neurotensin Neurotensin Receptors Neurotransmitters Nutrient Peptidoglycan Phase Proteins Publishing Reporting Research Role Route Signal Transduction Site Spectrum Analysis Structure Surface Technology Transferrin Virulence Work X-Ray Crystallography base beta barrel career crosslink fascinate insight interest member membrane biogenesis pandemic disease pathogenic bacteria peptidomimetics periplasm protein folding receptor resistant strain success tenure track

项目摘要

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.

描述（由适用提供）：传染病每年在全世界引起宽广的疾病，是死亡的第二大原因，尤其是在欠发达国家。随着多药抵抗菌株的出现，新的，更有效，更可持续的疗法的必要条件对于防止未来的大流行者而言是直接和至关重要的。我的研究将为有助于病毒病原细菌的表面受体和蛋白质的生物发生提供至关重要的见解。这些知识将极大地帮助开发更好的疗法来针对这些感染力代理商。革兰氏阴性细菌含有内膜和外膜。外膜包含通常称为外膜蛋白（OMP）的β-桶蛋白的宿主，它们在货物运输，信号传导和细菌病毒中起着重要的功能。在革兰氏阴性细菌中，众所周知，OMP在细胞质中合成，然后通过SEC易位将内膜转运到周期。一旦进入周质，伴侣引导新生的周围跨外周和辣椒粉到外膜的内表面。在这里，新生的OMP被称为BAM复合物的复合物识别，该复合物将新的OMP折叠并插入外膜。 BAM复合物如何实现其功能仍然未知。但是，我们确实知道，BAM综合体由五个名为Bama的亚基（一个本身），BAMB，BAMC，BAMD和BAME，它们都是脂蛋白。研究表明，巴马和BAMD对于细胞生存能力和生物发生绝对必不可少。我们的实验室和其他人报告了BAMB，BAMC，BAMD，BAME和BAMA周围域的很大一部分的结构，从而提供了有关BAM复合物如何起作用的初步见解。然而，即使知道这些结构，BAM复合物如何识别，折叠和插入新生的机制中的机制仍然难以捉摸，这在很大程度上是由于缺乏全长的巴马结构和与巴马的复合物。最近，我确定了截短的巴马构建体的晶体结构，从杜卡里（Ducreyi）到2.9 - 和从淋病奈瑟氏菌（Neisseria Gonorrhea）到3.2的全长bama构建体的晶体结构。在我提出的研究中，我旨在以最近的成功来建立使用X射线晶体学来确定与BAMB-E复杂化的Bama结构，使用鹿光谱和交联来表征Bama的构象动态，并使用交联用来探索Nascent Omps和Bama和BAM Comploctional Comploctional Comploctional in Comploct BAMA的相互作用。我在X射线晶体学方面具有很强的背景，在我的博士后研究期间，我使用最新的技术（例如新稳定的确定剂，双皮克和脂质立方阶段（LCP）方法）在与膜蛋白合作和结晶的膜蛋白方面增加了强大的背景。最近，我解决了致病性奈瑟氏菌的两种重要表面蛋白质的结构，这项工作在自然界中作为完整的研究文章发表（2012年3月）。对于此手稿，我还解决了研究小组几十年来未能成功解决的差异人转铁蛋白的结构。最近，我还解决了负责结合神经素和其他神经递质结合的GPCR激动剂结合神经素受体NTSR1的晶体结构。这项工作也在2012年10月发表（研究文章）。我目前的努力专注于研究BAM综合体的巴马，我将把这个项目作为学术界的终身教师。因此，我最近的结果是，巴马（H. ducreyi和N. gonorrhea）的晶体结构最近也作为完整的研究文章发表（2013年9月）。我的长期目标是在研究I学术机构中拥有自己的研究实验室作为教职员工，在那里我可以继续我的研究兴趣。这一直是我一生的野心，尽管现在的学术研究资金比以往任何时候都更具竞争力，但我仍然致力于志向研究的职业，我将能够建立一个研究小组，从而显着提高我们对细菌和人类中β-桶膜蛋白的理解。