NMR spectroscopic characterization of ribosomal complexes

核糖体是最复杂的生物机制之一，在原核和真核细胞中负责将遗传信息转化为功能蛋白质。核糖体是由核糖体RNA(rRNA，2/3)和核糖体蛋白(1/3)组成的大分子复合体。这些rRNA-蛋白质复合体由两个结构域组成，一个小的亚基确保解码的保真度，一个大的亚基包含核糖体的活性部位。单个亚基和完整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对蛋白质合成的一般分子和原子细节的感知。值得注意的是，50s亚基的结构显示了一个长100？，宽10-20？的隧道，新生的链在合成过程中穿过，然后离开核糖体。这种用于出口隧道的构象可以容纳大约30个延伸链中的残基或60个α-螺旋构象中的残基。许多生物物理和生化研究都集中在核糖体新生链上，并产生了不同的假说和模型，但新生链在翻译过程中是否采用隧道中的二级结构尚不清楚。核磁共振(NMR)在原子水平上研究生物分子的结构和动力学方面具有独特的能力，无论是在体外还是在体内。在过去的几年里，利用魔角旋转(MAS)技术对沉淀溶液样品进行大分子蛋白质复合体的结构分析已经取得了实质性的进展。此外，生物固体的质子探测技术与快速魔角旋转相结合，可以显著提高实验的灵敏度。这些发展为高分辨率研究复杂的生物组件开辟了道路，这些组件对于溶液状态的核磁共振来说太大了，使固态核磁共振成为结构生物学中的一种补充技术。在这个项目中，我们使用类似固体核磁共振的技术来研究核糖体复合体，包括新生核糖链(RNC)、特定重组的50和70年代核糖体复合体以及核糖体相关因子，以更好地了解新生核糖链的信号和通讯。这项拟议的工作不仅旨在表征结构细节，而且还旨在表征高分辨率X射线结晶学和低温电磁重建无法获得的动力学方面。由于共翻译过程中涉及的核糖体复合体的研究需要均一的RNC制备，而RNC迄今尚未结晶，因此固体核磁共振是获得这些复合体的原子分辨率数据的首选方法。

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.