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通过利用最近开发的用于高灵敏度数据收集的NMR脉冲序列和用于在溶液中制备稳定的核糖体结合的新生多肽的新可用程序，对核糖体新生链（RNC）复合物进行高分辨率NMR结构研究。作用于细菌翻译机制的几种抗生素，包括红霉素和泰利霉素，通过干扰新生链通过核糖体出口通道的进展来起作用。然而，缺乏新生链-核糖体复合物的高分辨率结构表征，阻碍了在理解这些抗生素发挥作用的三维环境方面迫切需要的进展。这种原子分辨率信息的缺乏减缓了设计新疗法的合理方法，并阻碍了对RNCs在翻译机制中的结构作用的深入理解。基于RNCs在翻译过程中经历分子内疏水性塌陷的倾向和结合分子伴侣的倾向之间的竞争的工作假设，将追求以下具体目标：（1）来自天然未折叠蛋白PIR的长度增加的核糖体结合的新生链（RNCs）的NMR结构表征。(2)在不存在和存在选定的协同活性分子伴侣的情况下，源自脱辅基肌红蛋白序列的RNC的NMR结构表征。拟议的调查将平行进行纯化的真细菌核糖体从大肠杆菌和耐辐射异常球菌病原体。在P.I.的实验室支持可行性，并为今后的工作奠定基础。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，治疗和最终预防细菌病原体引起的疾病。本研究项目通过提供高分辨率的结构信息，实现了本研究所的目标，这些信息将促进未来针对E. coli和D.耐辐射的本研究项目的目标是对大肠杆菌和耐辐射奇球菌的核糖体-肽基-tRNA复合物进行NMR结构研究。这项研究的目的是一个试点研究，（a）产生所需的结构数据，以获得宝贵的原子分辨率信息的核糖体结合的新生蛋白质，和（B）产生高质量的结构见解，这将有助于未来的合理设计的新型抗生素。考虑到一些大肠杆菌菌株如O 157：H7是严重的，有时甚至是致命的人类疾病的原因[例如，2006年夏季/秋季E.大肠杆菌在美国爆发，导致数人死亡]，拟议的研究是直接关系到人类健康。耐辐射异常球菌是世界上对紫外线辐射抵抗力最强的细菌，因此是一种可能有害的载体，可用于发展致病菌株，可能用作战剂。因此，拟议的工作与人类健康之间存在着明显的联系。

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.