Cook Erik C, Usher Grace A, Showalter Scott A
Department of Chemistry, The Pennsylvania State University, University Park, PA, United States.
Department of Biochemistry and Molecular Biology, Center for Eukaryotic Gene Regulation, The Pennsylvania State University, University Park, PA, United States.
Methods Enzymol. 2018;611:81-100. doi: 10.1016/bs.mie.2018.08.025. Epub 2018 Sep 25.
NMR spectroscopy remains the only experimental technique that provides (near) atomistic structural information for intrinsically disordered proteins (IDPs), but their sequence and structure characteristics still pose major challenges for high-resolution spectroscopy. Carbon-13 direct-detect NMR spectroscopy can overcome poor spectral dispersion and other difficulties associated with traditional H-detected NMR of nonaggregating disordered proteins. This chapter presents spectroscopic protocols suitable for complete characterization of IDPs that rely exclusively on C direct-detect experiments. The protocols described span initial characterization and chemical shift assignment; structure constraint through residual dipolar coupling and paramagnetic relaxation enhancement measurements; and assessment of intramolecular dynamics through N spin relaxation. The experiments described empower investigators to establish molecular mechanisms and structure-function relationships for IDPs and other proteins characterized by high internal flexibility.
核磁共振光谱仍然是唯一能为内在无序蛋白质（IDP）提供（近乎）原子水平结构信息的实验技术，但其序列和结构特征仍给高分辨率光谱带来重大挑战。碳-13直接检测核磁共振光谱可以克服与非聚集无序蛋白质传统氢检测核磁共振相关的光谱分散性差等困难。本章介绍了仅依靠碳直接检测实验对IDP进行完整表征的光谱分析方案。所描述的方案涵盖初始表征和化学位移归属；通过残余偶极耦合和顺磁弛豫增强测量进行结构约束；以及通过氮自旋弛豫评估分子内动力学。所描述的实验使研究人员能够为IDP和其他具有高内部灵活性的蛋白质建立分子机制和结构-功能关系。
