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性蛋白质。核糖体是由核糖体RNA （rRNA, 2/3）和核糖体蛋白（1/3）组成的大型（bbb2mda或更多）大分子复合物。这些rna -蛋白复合物由两个结构域组成，一个小亚基确保解码的保真度，一个大亚基包含核糖体的活性位点。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，并为我们提供了对蛋白质合成的一般分子和原子细节的前所未有的认识。值得注意的是，50S亚基的结构显示出一条100 Å长，10-20 Å宽的隧道，新生链在合成过程中穿过该隧道，然后离开核糖体。出口通道的这种构象可容纳延伸链中的约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研究都集中在核糖体新生链上，并提出了不同的假设和模型，但新生链在翻译过程中是否在隧道中采用二级结构尚不清楚。核磁共振（NMR）在原子水平、体外或体内研究生物分子的结构和动力学方面具有独特的能力。在过去的几年中，利用魔角旋转（MAS）技术对沉积溶液样品进行大型蛋白质复合物的结构分析取得了实质性进展。此外，结合快速魔角旋转的生物固体质子检测技术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生物组件开辟了道路，这些组件对于溶液态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补充技术。在这个项目中，我们采用固态核磁共振技术来研究核糖体复合物，包括新生链（RNCs），特别是重建的50S和70S核糖体复合物和核糖体相关因子，以更好地理解新生链信号和通信。提出的工作旨在不仅表征结构细节，而且表征动力学方面，这是高分辨率x射线晶体学和低温电镜重建无法实现的。由于参与共翻译过程的核糖体复合物的研究需要均匀的rna制备，迄今为止尚未结晶，因此固态核磁共振是获得这些复合物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.