In-Cell NMR of Intact Mammalian Cells Preserved with the Cryoprotectants DMSO and Glycerol Have Similar DNP Performance.

Kendra K Frederick,Rupam Ghosh,Yiling Xiao

doi:10.3389/fmolb.2021.789478

Abstract

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.

Highlights

Human embryonic kidney 293 (HEK293) Cells Cryopreserved With 10% DMSO Remain Viable During dynamic nuclear polarization (DNP) MAS Nuclear Magnetic Resonance (NMR)
Cells Cryopreserved With 10% DMSO Retain High Viability After DNP MAS NMR To determine whether any of the manipulations required for DNP MAS NMR sample preparation compromise cellular viability when 10% DMSO is used as a cryoprotectant, we assessed trypan blue membrane permeability at several steps of our sample preparation workflow (Figure 1A, arrows)
Incubation of cells cryoprotected 15% glycerol with AMUPol resulted in an inhomogeneous distribution of the polarization agent, AMUPol, through the cellular biomass, which will result in a spatial bias of the NMR peak intensities

Summary

Introduction

In-cell structural biology enables the study of protein conformation in environments that maintain the identity, stoichiometry, concentrations and organization of the myriad of biomolecules that can interact with a protein of interest. (Frederick et al, 2015; Theillet et al, 2016; Burmann et al, 2020; Luchinat et al, 2020) Capturing the effect of these complicated environments on biomolecular conformation is of particular importance for proteins that have more than one stable conformation, interact with cellular components or contain regions of intrinsic disorder. We established methods that maintained cellular viability throughout the DNP NMR experiments and found that the magnitude of the sensitivity enhancements for such samples were high enough to enable detection of a protein at micromolar concentrations inside intact cells in experimentally tractable experimental times. The handful of studies that examine preparations of mammalian cells using DNP NMR use DMSO (Albert et al, 2018; Narasimhan et al, 2019; Schlagnitweit et al, 2019; Overall et al, 2020), the sample composition—including the choice of cryoprotectant—and post-experiment cellular viability, have only very recently been considered (Ghosh et al, 2020; Ghosh et al, 2021; Overall and Barnes, 2021). Given the widespread preference for 10% DMSO over 15% glycerol as the cryoprotectant for cellular cryopreservation, we sought to determine if cryoprotection using 10% DMSO could support cellular viability throughout the DNP NMR experiments

Methods

Results

Conclusion