Biomolecular Perturbations in In-Cell Dynamic Nuclear Polarization Experiments

Area of Science:

• Biophysics
• Biochemistry
• Cell Biology

Background:

• In-cell Dynamic Nuclear Polarization (DNP) NMR is crucial for studying biomolecular structure and function in intact cells.
• Cellular integrity under cryogenic conditions for DNP is a key concern, with limited understanding of cryopreservation effects.

Purpose of the Study:

• To investigate cell survival, apoptosis, and DNP enhancement effects of cryopreserving agents and DNP radicals.
• To determine optimal cryopreservation methods for in-cell DNP-NMR studies.

Main Methods:

• Assessed cell survival and apoptosis in the presence of cryopreserving agents (glycerol, DMSO) and DNP radicals (AMUPol).
• Evaluated the impact of cryoprotectants on cellular DNP enhancements.
• Compared glycerol-based 'DNP juice' with DMSO for cell preservation and DNP performance.

Main Results:

• The DNP radical AMUPol did not affect membrane permeability or induce apoptosis.
• Glycerol/D2O/H2O ('DNP juice') caused rapid cell dehydration and apoptosis before freezing.
• 10% d6-DMSO preserved cell size and membrane integrity, yielding comparable DNP enhancements to glycerol.
• DMSO significantly improved post-thaw cell survival after slow-freezing.

Conclusions:

• Dimethyl sulfoxide (DMSO) is recommended as a cryoprotectant for in-cell DNP-NMR, outperforming glycerol.
• DMSO maintains cellular structural integrity and enhances cell viability for cryogenic DNP applications.
• Findings provide critical considerations for advancing in-cell DNP-NMR for high-quality structural studies.