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Protein structure determination in living cells by in-cell NMR spectroscopy

Nature volume 458, pages 102–105 (2009)Cite this article

Abstract

Investigating proteins ‘at work’ in a living environment at atomic resolution is a major goal of molecular biology, which has not been achieved even though methods for the three-dimensional (3D) structure determination of purified proteins in single crystals or in solution are widely used. Recent developments in NMR hardware and methodology have enabled the measurement of high-resolution heteronuclear multi-dimensional NMR spectra of macromolecules in living cells (in-cell NMR)1,2,3,4,5. Various intracellular events such as conformational changes, dynamics and binding events have been investigated by this method. However, the low sensitivity and the short lifetime of the samples have so far prevented the acquisition of sufficient structural information to determine protein structures by in-cell NMR. Here we show the first, to our knowledge, 3D protein structure calculated exclusively on the basis of information obtained in living cells. The structure of the putative heavy-metal binding protein TTHA1718 from Thermus thermophilus HB8 overexpressed in Escherichia coli cells was solved by in-cell NMR. Rapid measurement of the 3D NMR spectra by nonlinear sampling of the indirectly acquired dimensions was used to overcome problems caused by the instability and low sensitivity of living E. coli samples. Almost all of the expected backbone NMR resonances and most of the side-chain NMR resonances were observed and assigned, enabling high quality (0.96 ångström backbone root mean squared deviation) structures to be calculated that are very similar to the in vitro structure of TTHA1718 determined independently. The in-cell NMR approach can thus provide accurate high-resolution structures of proteins in living environments.

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Figure 1: Stability of E. coli cells expressing TTHA1718 under NMR measurement conditions.
Figure 2: Rapid acquisition of 3D NMR spectra of TTHA1718 in living E. coli cells.
Figure 3: Collection of NOE-derived distance restraints in TTHA1718 in living E. coli cells.
Figure 4: NMR solution structure of TTHA1718 in living E. coli cells.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates of the structures of TTHA1718 in E. coli cells and in vitro have been deposited in the Protein Data Bank under accession codes 2ROG and 2ROE, respectively. Chemical shifts have been deposited in the BioMagResBank under accession numbers 11037 and 11035.

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Acknowledgements

The authors thank S. Kuramitsu for providing the plasmid encoding TTHA1718, and D. Nietlispach for setting up 3D NMR experiments with nonlinear sampling schemes and 15N relaxation experiments, T. Anzai for assistance with NMR measurements, and H. Koyama and A. Iwasaki for sample preparations. This work was supported in part by CREST, Japan Science and Technology Agency (JST), the Molecular Ensemble Program, RIKEN, Grants-in-Aid for Scientific Research of Priority Areas from the Japanese Ministry of Education, Sports, Culture, Science, and Technology on ‘Molecular Soft Interactions Regulating Membrane Interface of Biological Systems’ and ‘Molecular Science for Supra Functional Systems – Development of Advanced Methods for Exploring Elementary Process’, and by the Volkswagen Foundation.

Author Contributions B.O.S., M.S., P.G. and Y.I. designed the research and wrote the manuscript. D.S., A.S. and T.I. conducted the research including sample preparation, data acquisition, resonance assignment and structure calculation. M.M. and M.W. helped with NMR measurements. M.M. prepared TTHA1718 mutants. J.H. and T.H. measured NMR data on TTHA1718 mutants and 15N-relaxation experiments. N.H. provided the expression vector for calmodulin. M.Y. measured NMR data on calmodulin in living E. coli cells. T.M. helped during the preparation and characterisation of TTHA1718.

Author information

Author notes

  1. Nobuhiro Hayashi

    Present address: Present address: Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 B-1, Nagatsuda-chou, Midori-ku, Yokohama, Kanagawa, 226-8501, Japan.,

  2. Daisuke Sakakibara, Atsuko Sasaki and Teppei Ikeya: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji, Tokyo 192-0397, Japan,

    Daisuke Sakakibara, Atsuko Sasaki, Teppei Ikeya, Junpei Hamatsu, Tomomi Hanashima, Masaki Mishima, Peter Güntert & Yutaka Ito

  2. CREST/Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan ,

    Daisuke Sakakibara, Atsuko Sasaki, Junpei Hamatsu, Masaki Mishima, Masahiro Shirakawa & Yutaka Ito

  3. Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, J. W. Goethe-University Frankfurt, Max-von-Laue Straße 9, 60438 Frankfurt am Main, Germany ,

    Teppei Ikeya & Peter Güntert

  4. Cellular and Molecular Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan ,

    Masatoshi Yoshimasu

  5. Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake-shi, Aichi 470-1192, Japan,

    Nobuhiro Hayashi

  6. Research Group for Bio-supramolecular Structure-Function, RIKEN, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan ,

    Tsutomu Mikawa & Yutaka Ito

  7. Bruker BioSpin, 3-21-5 Ninomiya, Tsukuba-shi, Ibaraki 305-0051, Japan ,

    Markus Wälchli

  8. Division of Molecular and Cellular Biology, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK

    Brian O. Smith

  9. Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo-Ku, Kyoto 615-8510, Japan,

    Masahiro Shirakawa

  10. Frankfurt Institute for Advanced Studies, Ruth-Moufang-Str. 1, 60438 Frankfurt am Main, Germany ,

    Peter Güntert

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  1. Daisuke Sakakibara
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Correspondence to Yutaka Ito.

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Sakakibara, D., Sasaki, A., Ikeya, T. et al. Protein structure determination in living cells by in-cell NMR spectroscopy. Nature 458, 102–105 (2009). https://doi.org/10.1038/nature07814

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