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In-Cell Sensitivity-Enhanced NMR of Intact Viable Mammalian Cells

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Ghosh, Rupam, Xiao, Yiling, Kragelj, Jaka, Frederick, Kendra K.·2021·Journal of the American Chemical Society
DOI 10.1021/jacs.1c06680PMC PMC8961430

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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. 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.

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  • AsymPolPOK and other biradicals are small, water-soluble molecules that diffuse through porous cell walls without disrupting their structure.

    “AsymPolPOK and other biradicals are small (2–3 nm), water-soluble molecules that diffuse through porous cell walls without disrupting their structure, enabling widespread use in the structural analysis of plant, fungal, algal, and bacterial cell wall materials as well as intact mammalian cells84–90.”

    From Distinct echinocandin responses of Candida albicans and Candida auris cell walls revealed by solid-state NMR (2025) · cites this paper as [85]

    The Results and Discussion (page unavailable) show that AMUPol, a small molecule, was used to study intact viable mammalian cells while maintaining cellular organization. They do not describe AsymPolPOK, a 2–3 nm size, or diffusion through porous plant, fungal, algal, or bacterial cell walls. Instead, the Results say most AMUPol was likely excluded by the mammalian plasma membrane, so the proposed diffusion mechanism is not supported by this paper. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

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Cited by

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Authors
Ghosh, R., Xiao, Y., Kragelj, J., & Frederick, K. K.
Affiliations
The University of Texas Southwestern Medical Center (US)
Corresponding author
Frederick, Kendra K.
ORCID
Venue
Journal of the American Chemical Society
Publisher
American Chemical Society (ACS)
Published
2021-11-01
Volume
143
Issue
44
Pages
18454-18466
ISSN
  • 0002-7863 (ISSN-L)
  • 1520-5126
DOI
10.1021/jacs.1c06680
PMC
PMC8961430
OpenAlex
W3211287374
Cited by
66
References
2
Citations by year
2026: 8 · 2025: 14 · 2024: 16 · 2023: 12 · 2022: 15 · 2021: 1
FWCI
4.02
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of the American Chemical Society (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Institute of Neurological Disorders and Stroke (NS-111236)
  • Welch Foundation (I-1923_20170325)
  • Kinship Foundation
  • Lupe Murchison Foundation
Topics
  • Physical Sciences › Chemistry › Spectroscopy › Advanced NMR Techniques and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolomics and Mass Spectrometry Studies
  • Physical Sciences › Physics and Astronomy › Nuclear and High Energy Physics › NMR spectroscopy and applications
Keywords
in-cell NMR, dynamic nuclear polarization, sensitivity enhancement, protein structure determination, isotopically labeled proteins, cell viability
MeSH terms
Cell Survival, Humans, Magnetic Resonance Spectroscopy, Cryopreservation, HEK293 Cells, Single-Cell Analysis

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