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High-resolution multi-dimensional NMR spectroscopy of proteins in human cells

Abstract

In-cell NMR is an isotope-aided multi-dimensional NMR technique that enables observations of conformations and functions of proteins in living cells at the atomic level1. This method has been successfully applied to proteins overexpressed in bacteria, providing information on protein–ligand interactions2 and conformations3,4. However, the application of in-cell NMR to eukaryotic cells has been limited to Xenopus laevis oocytes5,6,7. Wider application of the technique is hampered by inefficient delivery of isotope-labelled proteins into eukaryote somatic cells. Here we describe a method to obtain high-resolution two-dimensional (2D) heteronuclear NMR spectra of proteins inside living human cells. Proteins were delivered to the cytosol by the pyrenebutyrate-mediated action of cell-penetrating peptides8 linked covalently to the proteins. The proteins were subsequently released from cell-penetrating peptides by endogenous enzymatic activity or by autonomous reductive cleavage. The heteronuclear 2D spectra of three different proteins inside human cells demonstrate the broad application of this technique to studying interactions and protein processing. The in-cell NMR spectra of FKBP12 (also known as FKBP1A) show the formation of specific complexes between the protein and extracellularly administered immunosuppressants, demonstrating the utility of this technique in drug screening programs. Moreover, in-cell NMR spectroscopy demonstrates that ubiquitin has much higher hydrogen exchange rates in the intracellular environment, possibly due to multiple interactions with endogenous proteins.

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Figure 1: In-cell NMR spectra and cellular distribution of transduced ubiquitin derivative.
Figure 2: In-cell spectra of proteins delivered by disulphide-linked CPPTat.
Figure 3: In-cell NMR observation of specific complexes of FKBP12 with extracellularly administered immunosuppressants.
Figure 4: Hydrogen exchange experiments.

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Acknowledgements

We thank M. Waelchli, A. Kidera and H. Akutsu for discussion, T. Kokubo for monkey COS-7 cells, H. Ohnishi for the plasmid for production of FKBP12 and M. Imanishi for taking gel fluorimaging. This work was supported by grants to M.S. from Japan Science and Technology Agency and the Ministry of Education, Culture, Sports, Science and Technology-Japan (MEXT), and also in part by the Global COE Program ‘International Center for Integrated Research and Advanced Education in Materials Science’ (No. B-09) of MEXT, administered by the Japan Society for the Promotion of Science. This work was partly supported by the Innovative Techno-Hub for Integrated Medical Bio-imaging Project of the Special Coordination Funds for Promoting Science and Technology, from MEXT to A.O. and M.S., and by grants from MEXT to S.F. and H.T.

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Correspondence to Hidehito Tochio or Masahiro Shirakawa.

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Inomata, K., Ohno, A., Tochio, H. et al. High-resolution multi-dimensional NMR spectroscopy of proteins in human cells. Nature 458, 106–109 (2009). https://doi.org/10.1038/nature07839

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