High Resolution Analysis of Ribosome-Bound Nascent Polypeptides by NMR

描述(申请人提供)：这项研究的总体目标是通过利用最近开发的用于高灵敏数据收集的核磁共振脉冲序列和新可用的在溶液中制备稳定的核糖体结合的新生多肽的程序来进行核糖体-新生链(RNC)复合体的高分辨率核磁共振结构研究。几种作用于细菌翻译机制的抗生素，包括红霉素和泰利霉素，通过干扰核糖体出口通道的新生链条进程而发挥作用。然而，新生的链-核糖体复合体缺乏高分辨率的结构特征，阻碍了在理解这些抗生素发挥作用的三维环境方面亟需的进展。这种原子分辨率信息的缺乏减缓了设计新疗法的理性方法，并阻碍了对RNC在翻译机制中的结构作用的深入理解。基于RNC在翻译过程中经历分子内疏水崩溃的倾向和结合分子伴侣的倾向之间的竞争这一工作假设，我们将追求以下具体目标：(1)从天然展开的蛋白质PIR衍生的长度不断增加的核糖体结合新生链(RNC)的核磁共振结构表征。(2)脱脂蛋白序列衍生的RNC的核磁共振结构表征。拟议的调查将与从大肠杆菌和耐辐射球菌病原体中提纯的真细菌核糖体并行进行。在P.I.S实验室获得的初步结果支持了可行性，并为未来的工作奠定了基础。国家过敏和传染病研究所的主要目标之一是支持基础和应用研究，以更好地了解、治疗并最终预防细菌病原体引起的疾病。这项研究项目通过提供高分辨率的结构信息来实现该研究所的目标，这些信息将支持和促进未来针对大肠杆菌和耐辐射D菌感染的治疗策略的合理设计。项目简介本研究项目的目标是对来自大肠杆菌和耐辐射球菌的核糖体-肽-tRNA复合体的核磁共振结构进行研究。这项研究是一项先导性研究，目的是(A)产生获得有关核糖体结合的新生蛋白质的宝贵原子分辨率信息所需的结构数据，以及(B)产生高质量的结构洞察，这将有助于未来新型抗生素的合理设计。鉴于一些大肠杆菌菌株，例如O157：H7，会导致严重甚至有时致命的人类疾病[例如，2006年夏秋美国爆发的大肠杆菌，导致多人死亡]，建议的研究与人类健康直接相关。耐辐射球菌是世界上最耐紫外线辐射的细菌，因此，它是一种潜在的有害媒介，可用于开发致病菌株，可能被用作战剂。因此，拟议的工作与人类健康之间存在明显的联系。

DESCRIPTION (provided by applicant): The overall objective of this research is to perform high resolution NMR structural studies of ribosome-nascent chain (RNC) complexes by taking advantage of recently developed NMR pulse sequences for highly sensitive data collection and newly available procedures for the preparation of stable ribosome-bound nascent polypeptides in solution. Several antibiotics acting on the bacterial translation machinery, including erythromycin and telithromycin, act by interfering with the nascent chain progression through the ribosomal exit tunnel. Yet, the lack of high resolution structural characterization of nascent chain-ribosome complexes has prevented the urgently needed progress in understanding the three-dimensional environment upon which these antibiotics exert their action. This lack of atomic resolution information has slowed down rational approaches to the design of novel therapeutics and hampered a deep understanding of the structural role of RNCs within the translational machinery. Based on the working hypothesis that RNCs experience a competition between the tendency to experience intramolecular hydrophobic collapse and the tendency to bind molecular chaperones during translation, the following specific aims will be pursued: (1) NMR structural characterization of ribosome-bound nascent chains (RNCs) of increasing length derived from the natively unfolded protein PIR. (2) NMR structural characterization of RNCs derived from the apomyoglobin sequence in the absence and presence of selected cotranslationally active molecular chaperones. The proposed investigations will be carried out in parallel with purified eubacterial ribosomes from the Escherichia coli and Deinococcus radiodurans pathogens. Preliminary results obtained in the P.I.'s laboratory support feasibility and set the ground for future work. One of the main goals of the National Institute of Allergy and Infectious Diseases is to support basic and applied research to better understand, treat, and ultimately prevent disorders caused by bacterial pathogens. This research project embraces the objectives of this Institute by providing high resolution structural information that will enable and foster the future rational design of therapeutic strategies against infections by E. coli and D. radiodurans. Project Narrative This research project targets NMR structural studies on ribosome-peptidyl-tRNA complexes from Escherichia coli and Deinococcus radiodurans. This research is intended is a pilot study to (a) generate structural data required to gain precious atomic resolution information on ribosome-bound nascent proteins, and (b) to generate high quality structural insights that will aid the future rational design of novel antibiotics. Given that some Escherichia coli strains such as O157:H7 are responsible for serious and sometimes even lethal human disease [e.g., the Summer/Fall 2006 E. coli outbreak in the US, leading to several deaths], the proposed research is directly relevant to human health. Deinococcus radiodurans is the world's most resistant bacterium to UV radiation and, as such, a potentially harmful vector that could be employed for the development of pathogenic strains, possibly to be used as warfare agents. Hence, there are clear connections between the proposed work and human health.