Department of Biophysics, UT Southwestern Medical Center, Dallas, Texas 75390-8816, United States.
Center for Alzheimer's and Neurodegenerative Disease, UT Southwestern Medical Center, Dallas, Texas 75390, United States.
J Am Chem Soc. 2021 Nov 10;143(44):18454-18466. doi: 10.1021/jacs.1c06680. Epub 2021 Nov 1.
NMR has the resolution and specificity to determine atomic-level protein structures of isotopically labeled proteins in complex environments, and with the sensitivity gains conferred by dynamic nuclear polarization (DNP), NMR has the sensitivity to detect proteins at their endogenous concentrations. However, DNP sensitivity enhancements are critically dependent on experimental conditions and sample composition. While some of these conditions are theoretically compatible with cellular viability, the effects of others on cellular sample integrity are unknown. Uncertainty about the integrity of cellular samples limits the utility of experimental outputs of in-cell experiments. Using several measures, we establish conditions that support DNP enhancements that can enable detection of micromolar concentrations of proteins in experimentally tractable times that are compatible with cellular viability. Taken together, we establish DNP-assisted MAS NMR as a technique for structural investigations of biomolecules in intact viable cells that can be phenotyped both before and after NMR experiments.
NMR 具有分辨率和特异性，可以确定复杂环境中同位素标记蛋白质的原子水平结构，并且通过动态核极化 (DNP) 获得的灵敏度提高，NMR 具有在其内源性浓度下检测蛋白质的灵敏度。然而，DNP 灵敏度增强严重依赖于实验条件和样品组成。虽然其中一些条件在理论上与细胞活力兼容，但其他条件对细胞样品完整性的影响尚不清楚。对细胞样品完整性的不确定性限制了细胞内实验的实验结果的实用性。使用多种措施，我们确定了支持 DNP 增强的条件，这些条件可以在与细胞活力兼容的实验可处理时间内实现对微摩尔浓度蛋白质的检测。总之，我们建立了 DNP 辅助 MAS NMR 作为一种用于在完整存活细胞中对生物分子进行结构研究的技术，该技术可以在 NMR 实验之前和之后对其进行表型分析。
