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


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.


  1. Serber, Z. & Dotsch, V. In-cell NMR spectroscopy. Biochemistry 40, 14317–14323 (2001)

    Article  CAS  Google Scholar 

  2. Burz, D. S., Dutta, K., Cowburn, D. & Shekhtman, A. Mapping structural interactions using in-cell NMR spectroscopy (STINT-NMR). Nature Methods 3, 91–93 (2006)

    Article  CAS  Google Scholar 

  3. Dedmon, M. M., Patel, C. N., Young, G. B. & Pielak, G. J. FlgM gains structure in living cells. Proc. Natl Acad. Sci. USA 99, 12681–12684 (2002)

    Article  ADS  CAS  Google Scholar 

  4. Sakakibara, D. et al. Protein structure determination in living cells by in-cell NMR spectroscopy. Nature 10.1038/nature07814 (this issue)

  5. Sakai, T. et al. In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes. J. Biomol. NMR 36, 179–188 (2006)

    Article  CAS  Google Scholar 

  6. Selenko, P. et al. Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes. Proc. Natl Acad. Sci. USA 103, 11904–11909 (2006)

    Article  ADS  CAS  Google Scholar 

  7. Selenko, P. et al. In situ observation of protein phosphorylation by high-resolution NMR spectroscopy. Nature Struct. Mol. Biol. 15, 321–329 (2008)

    Article  CAS  Google Scholar 

  8. Takeuchi, T. et al. Direct and rapid cytosolic delivery using cell-penetrating peptides mediated by pyrenebutyrate. ACS Chem. Biol. 1, 299–303 (2006)

    Article  CAS  Google Scholar 

  9. Futaki, S. Oligoarginine vectors for intracellular delivery: design and cellular-uptake mechanisms. Biopolymers 84, 241–249 (2006)

    Article  CAS  Google Scholar 

  10. Nakase, I., Takeuchi, T., Tanaka, G. & Futaki, S. Methodological and cellular aspects that govern the internalization mechanisms of arginine-rich cell-penetrating peptides. Adv. Drug Deliv. Rev. 60, 598–607 (2008)

    Article  CAS  Google Scholar 

  11. Wender, P. A. et al. The design of guanidinium-rich transporters and their internalization mechanisms. Adv. Drug Deliv. Rev. 60, 452–472 (2008)

    Article  CAS  Google Scholar 

  12. Schwarze, S. R., Ho, A., Vocero-Akbani, A. & Dowdy, S. F. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 285, 1569–1572 (1999)

    Article  CAS  Google Scholar 

  13. Loison, F. et al. A ubiquitin-based assay for the cytosolic uptake of protein transduction domains. Mol. Ther. 11, 205–214 (2005)

    Article  CAS  Google Scholar 

  14. Carlson, N. & Rechsteiner, M. Microinjection of ubiquitin - intracellular-distribution and metabolism in Hela-cells maintained under normal physiological conditions. J. Cell Biol. 104, 537–546 (1987)

    Article  CAS  Google Scholar 

  15. O’Brien, R. & Gottlieb-Rosenkrantz, P. An automatic method for viability assay of cultured cells. J. Histochem. Cytochem. 18, 581–589 (1970)

    Article  Google Scholar 

  16. Giriat, I. & Muir, T. W. Protein semi-synthesis in living cells. J. Am. Chem. Soc. 125, 7180–7181 (2003)

    Article  CAS  Google Scholar 

  17. Hicke, L., Schubert, H. L. & Hill, C. P. Ubiquitin-binding domains. Nature Rev. Mol. Cell Biol. 6, 610–621 (2005)

    Article  CAS  Google Scholar 

  18. Itoh, S. & Navia, M. A. Structure comparison of native and mutant human recombinant FKBP12 complexes with the immunosuppressant drug FK506 (tacrolimus). Protein Sci. 4, 2261–2268 (1995)

    Article  CAS  Google Scholar 

  19. Ellis, R. J. Macromolecular crowding: obvious but underappreciated. Trends Biochem. Sci. 26, 597–604 (2001)

    Article  CAS  Google Scholar 

  20. Bai, Y. et al. Thermodynamic parameters from hydrogen exchange measurements. Methods Enzymol. 259, 344–356 (1995)

    Article  CAS  Google Scholar 

  21. Lange, O. F. et al. Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution. Science 320, 1471–1475 (2008)

    Article  ADS  CAS  Google Scholar 

  22. Delom, F., Fessart, D., Caruso, M. E. & Chevet, E. Tat-mediated protein delivery in living Caenorhabditis elegans . Biochem. Biophys. Res. Commun. 352, 587–591 (2007)

    Article  CAS  Google Scholar 

  23. Schanda, P. & Brutscher, B. Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds. J. Am. Chem. Soc. 127, 8014–8015 (2005)

    Article  CAS  Google Scholar 

  24. Laue, E. D., Mayger, M. R., Skilling, J. & Staunton, J. Reconstruction of phase-sensitive two-dimensional NMR-spectra by maximum-entropy. J. Magn. Reson. 68, 14–29 (1986)

    ADS  CAS  Google Scholar 

  25. Tenno, T. et al. Structural basis for distinct roles of Lys63- and Lys48-linked polyubiquitin chains. Genes Cells 9, 865–875 (2004)

    Article  CAS  Google Scholar 

  26. Mezo, G., Mihala, N., Andreu, D. & Hudecz, F. Conjugation of epitope peptides with SH group to branched chain polymeric polypeptides via Cys(Npys). Bioconjug. Chem. 11, 484–491 (2000)

    Article  CAS  Google Scholar 

  27. Rosen, M. K., Michnick, S. W., Karplus, M. & Schreiber, S. L. Proton and nitrogen sequential assignments and secondary structure determination of the human FK506 and rapamycin binding protein. Biochemistry 30, 4774–4789 (1991)

    Article  CAS  Google Scholar 

  28. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995)

    Article  CAS  Google Scholar 

  29. Goddard, T. D. & Kneller, D. G. SPARKY 3 (University of California, 1999)

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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).

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