High Resolution Analysis of Ribosome-Bound Nascent Polypeptides by NMR

描述（由申请人提供）：本研究的总体目标是利用最近开发的用于高灵敏度数据收集的核磁共振脉冲序列和在溶液中制备稳定的核糖体结合的新生多肽的新方法，对核糖体-新生链（RNC）复合物进行高分辨率核磁共振结构研究。几种作用于细菌转译机制的抗生素，包括红霉素和特利霉素，是通过干扰通过核糖体出口通道的新生链进展而起作用的。然而，缺乏对新生链状核糖体复合物的高分辨率结构表征，阻碍了了解这些抗生素发挥作用的三维环境的迫切需要的进展。原子分辨率信息的缺乏减缓了新疗法设计的理性途径，并阻碍了对rna在翻译机制中的结构作用的深入理解。基于rna在翻译过程中经历分子内疏水崩溃和结合分子伴蛋白趋势之间的竞争这一工作假设，我们将追求以下具体目标：(1)核糖体结合的新生链（rna）的核磁共振结构表征，这些新生链来自天然未折叠的蛋白PIR。(2)在缺少和存在选定的共翻译活性分子伴侣的情况下，无肌红蛋白序列衍生的rna的核磁共振结构表征。拟议的研究将与从大肠杆菌和耐辐射球菌病原体中纯化的真菌性核糖体同时进行。在P.I.中获得的初步结果为今后的工作奠定了基础。美国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、治疗并最终预防由细菌病原体引起的疾病。该研究项目通过提供高分辨率的结构信息来实现本研究所的目标，这些信息将使并促进未来合理设计对抗大肠杆菌和耐辐射菌感染的治疗策略。本研究项目针对大肠杆菌和耐辐射球菌核糖体-肽基- trna复合物进行核磁共振结构研究。本研究旨在作为一项先导研究(a)生成所需的结构数据，以获得核糖体结合新生蛋白的宝贵原子分辨率信息，以及(b)生成高质量的结构见解，这将有助于未来合理设计新型抗生素。鉴于某些大肠杆菌菌株，如O157:H7，会导致严重的、有时甚至是致命的人类疾病[例如，2006年夏秋季节在美国爆发的大肠杆菌疫情，导致数人死亡]，拟议的研究与人类健康直接相关。耐辐射球菌是世界上对紫外线辐射最具抵抗力的细菌，因此，它是一种潜在的有害载体，可用于开发致病性菌株，可能用作战剂。因此，拟议的工作与人类健康之间有明显的联系。

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.