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NMR spectroscopic characterization of ribosomal complexes

核糖体复合物的核磁共振波谱表征

基本信息

批准号：
289622162
负责人：
Professor Dr. Bernd Reif
金额：
--
依托单位：
Professur für Festkörper-NMR-Spektroskopie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2016
资助国家：
德国
起止时间：
2015-12-31 至 2020-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/289622162?language=en
关键词：
NMR spectroscopic characterization ribosomal complexes

项目摘要

Ribosomes are amongst the most complex biological machineries, being responsible for the conversion of genetic information into functional proteins in both prokaryotic and eukaryotic cells. Ribosomes are large (> 2 MDa or more) macromolecular complexes composed of ribosomal RNA (rRNA, 2/3) and ribosomal proteins (1/3). These rRNA-protein complexes are constituted of two domains, a small subunit ensuring the fidelity of decoding, and a large subunit containing the active site of the ribosome. Crystal structures of individual subunits and complete ribosome particles have elucidated the architecture of these sophisticated macromolecular machines and provided us with unprecedented perception of the general molecular and atomic details of protein synthesis. Notably, the structure of the 50S subunit revealed a tunnel 100 Å long and 10-20 Å wide, through which the nascent chains transit during synthesis before exiting the ribosome. Such a conformation for the exit tunnel can accommodate about 30 residues in an extended chain or 60 residues in alpha-helix conformation. Many biophysical and biochemical studies have focused on ribosomal nascent chains and produced different hypotheses and models, but whether nascent chains adopt secondary structure in the tunnel during translation is not clearly understood.Nuclear Magnetic Resonance (NMR) has the unique ability to study both structural and dynamical aspects in biomolecules at the atomic level, in vitro or in vivo. Substantial progress has been made in the past few years for the structural analysis of large protein complexes using magic-angle-spinning (MAS) techniques on sedimented solution samples. Moreover, proton detection techniques for biological solids in combination with fast magic angle spinning allows to significantly increase the sensitivity of the experiment. These developments open the way to high-resolution investigations of complex biological assemblies that are too large for solution-state NMR, making solid-state NMR a complementary technique in structural biology. Within this project, we employ solid-state NMR like techniques to study ribosomal complexes, including nascent chains (RNCs), specifically reconstituted 50S and 70S ribosomal complexes and ribosome associated factors to better understand nascent chain signalling and communication. The proposed work aims at characterizing not only structural details but also dynamical aspects, which are inaccessible to high resolution X-ray crystallography and cryo-EM reconstruction. Since studies of ribosome complexes involved in co-translational processes require homogeneous RNCs preparations, which have not been crystallized to date, solid-state NMR is the method of choice to obtain atomic-resolution data on these complexes.

核糖体是最复杂的生物机器之一，负责在原核和真核细胞中将遗传信息转化为功能蛋白。核糖体是由核糖体RNA（rRNA，2/3）和核糖体蛋白（1/3）组成的大分子复合物（> 2 MDa或更大）。这些rRNA-蛋白质复合物由两个结构域组成，一个是确保解码保真度的小亚基，另一个是含有核糖体活性位点的大亚基。单个亚基和完整的核糖体颗粒的晶体结构阐明了这些复杂的大分子机器的结构，并为我们提供了前所未有的蛋白质合成的一般分子和原子细节的感知。值得注意的是，50 S亚基的结构显示了一个100 nm长，10-20 nm宽的隧道，新生链在合成过程中通过该隧道运输，然后离开核糖体。出口通道的这种构象可以容纳延伸链中的约30个残基或α-螺旋构象中的60个残基。许多生物物理和生物化学的研究都集中在核糖体新生链上，并提出了不同的假说和模型，但新生链在翻译过程中是否采用二级结构还不清楚，核磁共振（NMR）具有独特的能力，可以在体外或体内从原子水平研究生物分子的结构和动力学方面。在过去的几年里，利用魔角旋转（MAS）技术对沉积溶液样品进行大蛋白质复合物的结构分析取得了实质性进展。此外，生物固体的质子检测技术与快速魔角旋转相结合，可以显着提高实验的灵敏度。这些发展为高分辨率研究溶液态NMR太大的复杂生物组装体开辟了道路，使固态NMR成为结构生物学的补充技术。在这个项目中，我们采用固态NMR等技术来研究核糖体复合物，包括新生链（RNC），特别是重组的50 S和70 S核糖体复合物和核糖体相关因子，以更好地了解新生链信号传导和通信。所提出的工作旨在表征不仅结构细节，而且动态方面，这是无法访问的高分辨率X射线晶体学和cryo-EM重建。由于参与共翻译过程的核糖体复合物的研究需要均匀的RNC制剂，迄今尚未结晶，固态NMR是获得这些复合物的原子分辨率数据的首选方法。