Xiao Yiling, Ghosh Rupam, Frederick Kendra K
Department of Biophysics, UT Southwestern Medical Center, Dallas, TX, United States.
Center for Alzheimer's and Neurodegenerative Disease, UT Southwestern Medical Center, Dallas, TX, United States.
Front Mol Biosci. 2022 Jan 25;8:789478. doi: 10.3389/fmolb.2021.789478. eCollection 2021.
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. Prior work established that DNP MAS NMR is compatible with cellular viability. However, in that work, 15% glycerol, rather than the more commonly used 10% DMSO, was used as the cellular cryoprotectant. Moreover, incubation of cells cryoprotected 15% glycerol with the polarization agent, AMUPol, resulted in an inhomogeneous distribution of AMUPol through the cellular biomass, which resulted in a spatial bias of the NMR peak intensities. Because 10% DMSO is not only the most used cryoprotectant for mammalian cells, but also because DMSO is often used to improve delivery of molecules to cells, we sought to characterize the DNP performance of cells that were incubated with AMUPol and cryoprotected with 10% DMSO. We found that, like cells preserved with 15% glycerol, cells preserved with 10% DMSO retain high viability during DNP MAS NMR experiments if they are frozen at a controlled rate. However, DMSO did not improve the dispersion of AMUPol throughout the cellular biomass. Cells preserved with 15% glycerol and with 10% DMSO had similar DNP performance for both the maximal DNP enhancements as well as the inhomogeneous dispersion of AMUPol throughout the cellular biomass. Therefore, 10% DMSO and 15% glycerol are both appropriate cryoprotectant systems for DNP-assisted MAS NMR of intact viable mammalian cells.
核磁共振（NMR）具有分辨率和特异性，能够在复杂环境中确定同位素标记蛋白质的原子级结构，并且借助动态核极化（DNP）带来的灵敏度提升，NMR具备在内源浓度下检测蛋白质的灵敏度。先前的研究表明，DNP MAS NMR与细胞活力兼容。然而，在该项研究中，使用的细胞冷冻保护剂是15%的甘油，而非更为常用的10%二甲基亚砜（DMSO）。此外，用极化剂AMUPol处理经15%甘油冷冻保护的细胞后，AMUPol在整个细胞生物质中的分布并不均匀，这导致了NMR峰强度出现空间偏差。由于10% DMSO不仅是哺乳动物细胞最常用的冷冻保护剂，而且DMSO常被用于改善分子向细胞的递送，因此我们试图表征用AMUPol处理并用10% DMSO冷冻保护的细胞的DNP性能。我们发现，与用15%甘油保存的细胞一样，如果以可控速率冷冻，用10% DMSO保存的细胞在DNP MAS NMR实验过程中仍保持高活力。然而，DMSO并未改善AMUPol在整个细胞生物质中的分散情况。用15%甘油和10% DMSO保存的细胞在最大DNP增强以及AMUPol在整个细胞生物质中的不均匀分散方面具有相似的DNP性能。因此，10% DMSO和15%甘油都是完整活哺乳动物细胞DNP辅助MAS NMR合适的冷冻保护剂系统。
