Loquet Antoine, Tolchard James, Berbon Melanie, Martinez Denis, Habenstein Birgit
Institute of Chemistry, Biology of Membranes, Nanoobjects, UMR5248 CNRS, Université de Bordeaux;
Institute of Chemistry, Biology of Membranes, Nanoobjects, UMR5248 CNRS, Université de Bordeaux.
J Vis Exp. 2017 Sep 17(127):55779. doi: 10.3791/55779.
Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of C/N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.
超分子蛋白质组装体在从宿主-病原体相互作用、病毒感染到神经退行性疾病传播等生物过程中发挥着基础作用。这类组装体由多个以非共价方式组织的蛋白质亚基组成，形成能够执行多种细胞功能或产生有害后果的大型大分子物体。由于其固有的不溶性和非结晶性常常大幅降低从结构生物学中使用的大多数技术（如X射线晶体学和溶液核磁共振（NMR））获得的数据质量，因此对这些大分子组装体的组装机制和功能的原子层面见解往往很少。我们在此介绍魔角旋转固态核磁共振光谱（SSNMR），这是一种在原子分辨率下研究大分子组装体结构的强大方法。SSNMR可以揭示组装复合物的原子细节，而不受尺寸和溶解度的限制。这里介绍的方案描述了从生产碳/氮同位素标记的大分子蛋白质组装体到获取标准SSNMR光谱及其分析和解释的基本步骤。作为一个例子，我们展示了丝状蛋白质组装体的SSNMR结构分析流程。
