TY - JOUR
T1 - Stability of the nitroxide biradical AMUPol in intact and lysed mammalian cells
AU - Ghosh, Rupam
AU - Dumarieh, Rania
AU - Xiao, Yiling
AU - Frederick, Kendra K.
N1 - Publisher Copyright:
© 2022
PY - 2022/3
Y1 - 2022/3
N2 - Dynamic Nuclear Polarization (DNP) enhanced solid state NMR increases experimental sensitivity, potentially enabling detection of biomolecules at their physiological concentrations. The sensitivity of DNP experiments is due to the transfer of polarization from electron spins of free radicals to the nuclear spins of interest. Here, we investigate the reduction of AMUPol in both lysed and intact HEK293 cells. We find that nitroxide radicals are reduced with first order reduction kinetics by cell lysates at a rate of ∼ 12% of the added nitroxide radical concentration per hour. We also found that electroporation delivered a consistent amount of AMUPol to intact cells and that nitroxide radicals are reduced just slightly more rapidly (∼15% per hour) by intact cells than by cell lysates. The two nitroxide radicals of AMUPol are reduced independently and this leads to considerable accumulation of the DNP-silent monoradical form of AMUPol, particularly in preparations of intact cells where nearly half of the AMUPol is already reduced to the DNP silent monoradical form at the earliest experimental time points. This confirms that the loss of the DNP-active biradical form of AMUPol is faster than the nitroxide reduction rate. Finally, we investigate the effect of adding N-ethyl maleimide, a well-known inhibitor of thiol (-SH) group-based reduction of nitroxide biradicals in cells, on AMUPol reduction, cellular viability, and DNP performance. Although pre-treatment of cells with NEM effectively inhibited the reduction of AMUPol, exposure to NEM compromised cellular viability and, surprisingly, did not improve DNP performance. Collectively, these results indicate that, currently, the most effective strategy to obtain high DNP enhancements for DNP-assisted in-cell NMR is to minimize room temperature contact times with cellular constituents and suggest that the development of bio-resistant polarization agents for DNP could considerably increase the sensitivity of DNP-assisted in-cell NMR experiments.
AB - Dynamic Nuclear Polarization (DNP) enhanced solid state NMR increases experimental sensitivity, potentially enabling detection of biomolecules at their physiological concentrations. The sensitivity of DNP experiments is due to the transfer of polarization from electron spins of free radicals to the nuclear spins of interest. Here, we investigate the reduction of AMUPol in both lysed and intact HEK293 cells. We find that nitroxide radicals are reduced with first order reduction kinetics by cell lysates at a rate of ∼ 12% of the added nitroxide radical concentration per hour. We also found that electroporation delivered a consistent amount of AMUPol to intact cells and that nitroxide radicals are reduced just slightly more rapidly (∼15% per hour) by intact cells than by cell lysates. The two nitroxide radicals of AMUPol are reduced independently and this leads to considerable accumulation of the DNP-silent monoradical form of AMUPol, particularly in preparations of intact cells where nearly half of the AMUPol is already reduced to the DNP silent monoradical form at the earliest experimental time points. This confirms that the loss of the DNP-active biradical form of AMUPol is faster than the nitroxide reduction rate. Finally, we investigate the effect of adding N-ethyl maleimide, a well-known inhibitor of thiol (-SH) group-based reduction of nitroxide biradicals in cells, on AMUPol reduction, cellular viability, and DNP performance. Although pre-treatment of cells with NEM effectively inhibited the reduction of AMUPol, exposure to NEM compromised cellular viability and, surprisingly, did not improve DNP performance. Collectively, these results indicate that, currently, the most effective strategy to obtain high DNP enhancements for DNP-assisted in-cell NMR is to minimize room temperature contact times with cellular constituents and suggest that the development of bio-resistant polarization agents for DNP could considerably increase the sensitivity of DNP-assisted in-cell NMR experiments.
KW - AMUPol
KW - DNP NMR
KW - In-cell EPR
KW - In-cell NMR
KW - Polarizing agent
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U2 - 10.1016/j.jmr.2022.107150
DO - 10.1016/j.jmr.2022.107150
M3 - Article
C2 - 35151975
AN - SCOPUS:85124315763
SN - 1090-7807
VL - 336
JO - Journal of Magnetic Resonance
JF - Journal of Magnetic Resonance
M1 - 107150
ER -