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Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells

Nature Protocols volume 11, pages 1101–1111 (2016)Cite this article

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Abstract

In-cell NMR spectroscopy is a unique tool for characterizing biological macromolecules in their physiological environment at atomic resolution. Recent progress in NMR instruments and sample preparation methods allows functional processes, such as metal uptake, disulfide-bond formation and protein folding, to be analyzed by NMR in living, cultured human cells. This protocol describes the necessary steps to overexpress one or more proteins of interest inside human embryonic kidney 293T (HEK293T) cells, and it explains how to set up in-cell NMR experiments. The cDNA is transiently transfected as a complex with a cationic polymer (DNA:PEI (polyethylenimine)), and protein expression is carried on for 2–3 d, after which the NMR sample is prepared. 1H and 1H–15N correlation NMR experiments (for example, using band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence (SOFAST-HMQC)) can be carried out in <2 h, ensuring cell viability. Uniform 15N labeling and amino-acid-specific (e.g., cysteine, methionine) labeling schemes are possible. The entire procedure takes 4 d from cell culture seeding to NMR data collection.

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Figure 1: Alternative timings for transfection and protein expression.
Figure 2: Subtraction of 2D 1H–15N SOFAST-HMQC in-cell NMR spectra to eliminate the background signals.
Figure 3: Expression levels of different proteins transiently transfected in HEK293T cells.
Figure 4: 1H–15N in-cell NMR spectra (black) and the corresponding in vitro NMR spectra (red).
Figure 5: 2D heteronuclear in-cell NMR spectra.

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Acknowledgements

This work was supported by iNEXT (grant agreement 653706), funded by the Horizon 2020 Programme of the European Union; by MEDINTECH: Tecnologie convergenti per aumentare la sicurezza e l'efficacia di farmaci e vaccini (grant CTN01_00177_962865); by Instruct, part of the European Strategy Forum on Research Infrastructures (ESFRI); and by national member subscriptions. Specifically, we thank the EU ESFRI Instruct Core Centre CERM-Italy.

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Authors and Affiliations

  1. Magnetic Resonance Center–CERM, University of Florence, Florence, Italy

    Letizia Barbieri, Enrico Luchinat & Lucia Banci

  2. Department of Chemistry, University of Florence, Florence, Italy

    Letizia Barbieri & Lucia Banci

  3. Department of Biomedical, Clinical and Experimental Sciences, University of Florence, Florence, Italy

    Enrico Luchinat

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  1. Letizia Barbieri
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Contributions

L. Barbieri, E.L. and L. Banci conceived the work and designed the experiments. L. Barbieri and E.L. performed the experiments. Specifically, L. Barbieri seeded and transfected the cells, performed the SDS–PAGE and western blot analysis and performed cell counts and viability tests; E.L. optimized the NMR experimental conditions, set up and acquired the NMR experiments and processed and analyzed the NMR data. L. Barbieri, E.L. and L. Banci wrote the manuscript.

Corresponding author

Correspondence to Lucia Banci.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Grx1 is not detected in the in-cell NMR spectra.

1H-15N SOFAST-HMQC NMR spectra of (a) cells expressing [U-15N]-labelled Grx1 and (b) the corresponding cell lysate. In the spectrum (a) the signals arising from Grx1 are not detected, owing to the interaction with other cellular components. The two spectra were acquired with identical parameters; background subtraction was not performed. (c) Western blot of the cell lysate analyzed in b (+) together with a control sample (–).

Supplementary Figure 2 Western blots.

Full western blots corresponding to (a) Fig. 3b and (b) Fig. 3d. An unrelated sample is marked with an asterisk.

Supplementary information

Supplementary Information

Supplementary Figures 1 and 2 (PDF 303 kb)

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Barbieri, L., Luchinat, E. & Banci, L. Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells. Nat Protoc 11, 1101–1111 (2016). https://doi.org/10.1038/nprot.2016.061

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