Costello Whitney N, Xiao Yiling, Frederick Kendra K
UT Southwestern Medical Center, Dallas, TX, United States.
UT Southwestern Medical Center, Dallas, TX, United States.
Methods Enzymol. 2019;615:373-406. doi: 10.1016/bs.mie.2018.08.023. Epub 2018 Sep 18.
Structural investigations of biomolecules are typically confined to in vitro systems under extremely limited conditions. These investigations yield invaluable insights, but such experiments cannot capture important structural features imposed by cellular environments. Structural studies of proteins in their native contexts are not only possible using state-of-the-art sensitivity-enhanced (dynamic nuclear polarization, DNP) solid-state nuclear magnetic resonance (NMR) techniques, but these studies also demonstrate that the cellular context can and does have a dramatic influence on protein structure. In this chapter, we describe methods to prepare samples of isotopically labeled proteins at endogenous levels in cellular contexts alongside quality control methods to ensure that such samples accurately model important features of the cellular environment.
生物分子的结构研究通常局限于在极其有限的条件下的体外系统。这些研究产生了宝贵的见解，但此类实验无法捕捉细胞环境所施加的重要结构特征。利用最先进的灵敏度增强（动态核极化，DNP）固态核磁共振（NMR）技术，不仅可以在蛋白质的天然环境中进行结构研究，而且这些研究还表明，细胞环境能够且确实会对蛋白质结构产生巨大影响。在本章中，我们描述了在细胞环境中以内源性水平制备同位素标记蛋白质样品的方法，以及确保此类样品准确模拟细胞环境重要特征的质量控制方法。
