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Characterizing proteins in a native bacterial environment using solid-state NMR spectroscopy

Nature Protocols volume 16, pages 893–918 (2021)Cite this article

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Abstract

For a long time, solid-state nuclear magnetic resonance (ssNMR) has been employed to study complex biomolecular systems at the detailed chemical, structural, or dynamic level. Recent progress in high-resolution and high-sensitivity ssNMR, in combination with innovative sample preparation and labeling schemes, offers novel opportunities to study proteins in their native setting irrespective of the molecular tumbling rate. This protocol describes biochemical preparation schemes to obtain cellular samples of both soluble as well as insoluble or membrane-associated proteins in bacteria. To this end, the protocol is suitable for studying a protein of interest in both whole cells and in cell envelope or isolated membrane preparations. In the first stage of the procedure, an appropriate strain of Escherichia coli (DE3) is transformed with a plasmid of interest harboring the protein of interest under the control of an inducible T7 promoter. Next, the cells are adapted to grow in minimal (M9) medium. Before the growth enters stationary phase, protein expression is induced, and shortly thereafter, the native E. coli RNA polymerase is inhibited using rifampicin for targeted labeling of the protein of interest. The cells are harvested after expression and prepared for ssNMR rotor filling. In addition to conventional 13C/15N-detected ssNMR, we also outline how these preparations can be readily subjected to multidimensional ssNMR experiments using dynamic nuclear polarization (DNP) or proton (1H) detection schemes. We estimate that the entire preparative procedure until NMR experiments can be started takes 3–5 days.

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Fig. 1: Overview of the protocol divided into three stages.
Fig. 2
Fig. 3
Fig. 4: Toolkit required for filling ssNMR rotors.
Fig. 5
Fig. 6: The (13C, 13C) correlated PARIS spin diffusion (30 ms) spectrum of uniformly [13C, 15N] labeled BamCDE in BL21 (DE3) star cell outer membranes.
Fig. 7: 1H-detected ssNMR analysis of YidC in cell envelopes.
Fig. 8: The (13C, 13C) correlated proton-driven spin diffusion (30 ms) of ubiquitin in deuterated Lemo21 cells.
Fig. 9: 3D 2Q-1Q-1Q correlated spectra of ubiquitin in deuterated Lemo21 cells.

Data availability

Source data are provided with this paper. All other data supporting the approach described in this protocol are available from the corresponding authors upon reasonable request.

References

  1. Griffin, R. G. Solid state nuclear magnetic resonance of lipid bilayers. Methods Enzymol. 72, 108–174 (1981).

    Article  CAS  PubMed  Google Scholar 

  2. Seelig, J. Deuterium magnetic resonance: theory and application to lipid membranes. Q. Rev. Biophys. 10, 353–418 (1977).

    Article  CAS  PubMed  Google Scholar 

  3. Brown, L. S. & Ladizhansky, V. Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy. Prot. Sci. 24, 1333–1346 (2015).

    Article  CAS  Google Scholar 

  4. Hong, M., Zhang, Y. & Hu, F.H. Membrane protein structure and dynamics from NMR spectroscopy. Annu. Rev. Phys. Chen., 63, (eds. Johnson, M.A. & Martinez, T.J.) 1-24 (2012).

  5. Kaplan, M., Pinto, C., Houben, K. & Baldus, M. Nuclear magnetic resonance (NMR) applied to membrane–protein complexes. Q. Rev. Biophys. 49, e15 (2016).

    Article  PubMed  Google Scholar 

  6. Herzfeld, J. & Lansing, J. C. Magnetic resonance studies of the bacteriorhodopsin pump cycle. Annu. Rev. Biophys. Biomol. Struct. 31, 73–95 (2002).

    Article  CAS  PubMed  Google Scholar 

  7. Ketchem, R., Hu, W. & Cross, T. High-resolution conformation of gramicidin A in a lipid bilayer by solid-state NMR. Science 261, 1457–1460 (1993).

    Article  CAS  PubMed  Google Scholar 

  8. Lange, A. et al. Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR. Nature 440, 959–962 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Cady, S. D. et al. Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers. Nature 463, 689–U127 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Luca, S. et al. The conformation of neurotensin bound to its G protein-coupled receptor. Proc. Natl Acad. Sci. USA 100, 10706–10711 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Goncalves, J. A., Ahuja, S., Erfani, S., Eilers, M. & Smith, S. O. Structure and function of G protein-coupled receptors using NMR spectroscopy. Prog. Nucl. Magn. Reson. Spectrosc. 57, 159–180 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Loquet, A., Habenstein, B. & Lange, A. Structural investigations of molecular machines by solid-state NMR. Acc. Chem. Res. 46, 2070–2079 (2013).

    Article  CAS  PubMed  Google Scholar 

  13. Tycko, R. Solid-state NMR studies of amyloid fibril structure. Annu. Rev. Phys. Chem., 62 (eds. Leone, S.R., Cremer, P.S., Groves, J.T. & Johnson, M.A.) 279-299 (2011).

  14. Das, N., Murray, D. T. & Cross, T. A. Lipid bilayer preparations of membrane proteins for oriented and magic-angle spinning solid-state NMR samples. Nat. Protoc. 8, 2256 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Fricke, P. et al. Backbone assignment of perdeuterated proteins by solid-state NMR using proton detection and ultrafast magic-angle spinning. Nat. Protoc. 12, 764–782 (2017).

    Article  CAS  PubMed  Google Scholar 

  16. Ni, Q. Z. et al. High frequency dynamic nuclear polarization. Acc. Chem. Res. 46, 1933–1941 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ishii, Y. & Tycko, R. Sensitivity enhancement in solid state N-15 NMR by indirect detection with high-speed magic angle spinning. J. Magn. Reson. 142, 199–204 (2000).

    Article  CAS  PubMed  Google Scholar 

  18. Chevelkov, V., Rehbein, K., Diehl, A. & Reif, B. Ultrahigh resolution in proton solid-state NMR spectroscopy at high levels of deuteration. Angew. Chem. Int. Ed. 45, 3878–3881 (2006).

    Article  CAS  Google Scholar 

  19. Plitzko, J. M., Schuler, B. & Selenko, P. Structural biology outside the box—inside the cell. Curr. Opin. Struct. Biol. 46, 110–121 (2017).

    Article  CAS  PubMed  Google Scholar 

  20. Baker, L. A. et al. Combined H-1-detected solid-state NMR spectroscopy and electron cryotomography to study membrane proteins across resolutions in native environments. Structure 26, 161–170 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Narasimhan, S. et al. DNP-supported solid-state NMR spectroscopy of proteins inside mammalian cells. Angew. Chem. Int. Ed. 58, 12969–12973 (2019).

    Article  CAS  Google Scholar 

  22. Damman, R. et al. Development of in vitro-grown spheroids as a 3D tumor model system for solid-state NMR spectroscopy. J. Biomol. NMR 74, 401–412 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Thongsomboon, W. et al. Phosphoethanolamine cellulose: a naturally produced chemically modified cellulose. Science 359, 334–338 (2018).

    Article  CAS  PubMed  Google Scholar 

  24. Renault, M. et al. Solid-state NMR spectroscopy on cellular preparations enhanced by dynamic nuclear polarization. Angew. Chem. Int. Ed. 51, 2998–3001 (2012).

    Article  CAS  Google Scholar 

  25. Renault, M. et al. Cellular solid-state nuclear magnetic resonance spectroscopy. Proc. Natl Acad. Sci. USA 109, 4863–4868 (2012).

    Article  CAS  PubMed  Google Scholar 

  26. Gronenborn, A. M. & Clore, G. M. Rapid screening for structural integrity of expressed proteins by heteronuclear NMR spectroscopy. Prot. Sci. 5, 174–177 (1996).

    Article  CAS  Google Scholar 

  27. Qing, G. et al. Cold-shock induced high-yield protein production in Escherichia coli. Nat. Biotechnol. 22, 877–882 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Frederick, K. K. et al. Sensitivity-enhanced NMR reveals alterations in protein Sstructure by cellular milieus. Cell 163, 620–628 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  30. Serber, Z. et al. Methyl groups as probes for proteins and complexes in in-cell NMR experiments. J. Am. Chem. Soc. 126, 7119–7125 (2004).

    Article  CAS  PubMed  Google Scholar 

  31. Binolfi, A., Theillet, F.-X. & Selenko, P. Bacterial in-cell NMR of human α-synuclein: a disordered monomer by nature? Biochem. Soc. Trans. 40, 950–954 (2012).

    Article  CAS  PubMed  Google Scholar 

  32. Barbieri, L., Luchinat, E. & Banci, L. Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells. Nat. Protoc. 11, 1101 (2016).

    Article  CAS  PubMed  Google Scholar 

  33. Tabaka, M., Sun, L., Kalwarczyk, T. & Hołyst, R. Implications of macromolecular crowding for protein–protein association kinetics in the cytoplasm of living cells. Soft Matter 9, 4386–4386 (2013).

    Article  CAS  Google Scholar 

  34. Siegal, G. & Selenko, P. Cells, drugs and NMR. J. Magn. Reson. 306, 202–212 (2019).

    Article  CAS  PubMed  Google Scholar 

  35. Lee, K. M., Androphy, E. J. & Baleja, J. D. A novel method for selective isotope labeling of bacterially expressed proteins. J. Biomol. NMR 5, 93–96 (1995).

    Article  CAS  PubMed  Google Scholar 

  36. Pinto, C. et al. Studying assembly of the BAM complex in native membranes by cellular solid-state NMR spectroscopy. J. Struct. Biol. 206, 1–11 (2019).

    Article  CAS  PubMed  Google Scholar 

  37. Miao, Y. et al. M2 proton channel structural validation from full-length protein samples in synthetic bilayers and E. coli membranes. Angew. Chem. Int. Ed. 51, 8383–8386 (2012).

    Article  CAS  Google Scholar 

  38. Medeiros-Silva, J. et al. 1 H-detected solid-state NMR studies of water-inaccessible proteins in vitro and in situ. Angew. Chem. Int. Ed. 55, 13606–13610 (2016).

    Article  CAS  Google Scholar 

  39. Jacso, T. et al. Characterization of membrane proteins in isolated native cellular membranes by dynamic nuclear polarization solid-state NMR spectroscopy without purification and reconstitution. Angew. Chem. Int. Ed. 51, 432–435 (2012).

    Article  CAS  Google Scholar 

  40. Ward, M. E. et al. In situ structural studies of anabaena sensory rhodopsin in the E. coli membrane. Biophys. J. 108, 1683–1696 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Etzkorn, M. et al. Complex formation and light activation in membrane-embedded sensory rhodopsin II as seen by solid-state NMR spectroscopy. Structure 18, 293–300 (2010).

    Article  CAS  PubMed  Google Scholar 

  42. Yamamoto, K., Caporini, M. A., Im, S.-C., Waskell, L. & Ramamoorthy, A. Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization. Biochim. Biophys. Acta 1848, 342–349 (2015).

    Article  CAS  PubMed  Google Scholar 

  43. Schanda, P. et al. Atomic model of a cell-wall cross-linking enzyme in complex with an intact bacterial peptidoglycan. J. Am. Chem. Soc. 136, 17852–17860 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Reckel, S., Lopez, J. J., Loehr, F., Glaubitz, C. & Doetsch, V. In-cell solid-state NMR as a tool to study proteins in large complexes. ChemBioChem 13, 534–537 (2012).

    Article  CAS  PubMed  Google Scholar 

  45. Medeiros-Silva, J. et al. High-resolution NMR studies of antibiotics in cellular membranes. Nat. Commun. 9, 3963 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Shukla, R. et al. Mode of action of teixobactins in cellular membranes. Nat. Commun. 11, 2848 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Kaplan, M. et al. Probing a cell-embedded megadalton protein complex by DNP-supported solid-state NMR. Nat. Methods 12, 649–652 (2015).

    Article  CAS  PubMed  Google Scholar 

  48. Almeida, F. C. L. et al. Selectively labeling the heterologous protein in Escherichia coli for NMR studies: A strategy to speed up NMR spectroscopy. J. Magn. Reson. 148, 142–146 (2001).

    Article  CAS  PubMed  Google Scholar 

  49. Galvão-Botton, L. M. P. et al. High-throughput screening of structural proteomics targets using NMR. FEBS Lett. 552, 207–213 (2003).

    Article  PubMed  Google Scholar 

  50. Baker, L. A., Daniëls, M., van der Cruijsen, E. A. W., Folkers, G. E. & Baldus, M. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling. J. Biomol. NMR 62, 199–208 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Serber, Z., Ledwidge, R., Miller, S. M. & Dötsch, V. Evaluation of parameters critical to observing proteins inside living Escherichia coli by in-cell NMR spectroscopy. J. Am. Chem. Soc. 123, 8895–8901 (2001).

    Article  CAS  PubMed  Google Scholar 

  52. Chordia, S., Narasimhan, S., Lucini Paioni, A., Baldus, M. & Roelfes, G. In vivo assembly of artificial metalloenzymes and application in whole‐cell biocatalysis (ChemRxiv, 2020).

  53. White, S. W., Zheng, J., Zhang, Y. M. & Rock, C. O. The structural biology of type II fatty acid biosynthesis. Annu. Rev. Biochem. 74,, 791–831 (2005).

    Article  Google Scholar 

  54. Roelfes, G. LmrR: a privileged scaffold for artificial metalloenzymes. Acc. Chem. Res. 52, 545–556 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Mitchell, A. M. & Silhavy, T. J. Envelope stress responses: balancing damage repair and toxicity. Nat. Rev. Microbiol. 17, 417–428 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Fowler, D. M., Koulov, A. V., Balch, W. E. & Kelly, J. W. Functional amyloid – from bacteria to humans. Trends Biochem. Sci. 32, 217–224 (2007).

    Article  CAS  PubMed  Google Scholar 

  57. Otzen, D. & Riek, R. Functional amyloids. Cold Spring Harb. Perspect. Biol. 11, a:033860 (2019).

    Article  CAS  PubMed  Google Scholar 

  58. Kim, K. W. Prokaryotic cytoskeletons: in situ and ex situ structures and cellular locations. Antonie van Leeuwenhoek 112, 145–157 (2019).

    Article  CAS  PubMed  Google Scholar 

  59. Albert, B. J. et al. Dynamic nuclear polarization nuclear magnetic resonance in human cells using fluorescent polarizing agents. Biochemistry 57, 4741–4746 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Ghosh, R., Kragelj, J., Xiao, Y. & Frederick, K. K. Cryogenic sample loading into a magic angle spinning nuclear magnetic resonance spectrometer that preserves cellular viability. JoVE e61733 (2020).

  61. Noinaj, N., Gumbart, J. C. & Buchanan, S. K. The β-barrel assembly machinery in motion. Nat. Rev. Microbiol. 15, 197–204 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Pinto, C. et al. Formation of the β-barrel assembly machinery complex in lipid bilayers as seen by solid-state NMR. Nat. Commun. 9, 4135–4145 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Koers, E. J. et al. NMR-based structural biology enhanced by dynamic nuclear polarization at high magnetic field. J. Biomol. NMR 60, 157–168 (2014).

    Article  CAS  PubMed  Google Scholar 

  64. Corzilius, B., Andreas, L. B., Smith, A. A., Ni, Q. Z. & Griffin, R. G. Paramagnet induced signal quenching in MAS–DNP experiments in frozen homogeneous solutions. J. Magn. Reson. 240, 113–123 (2014).

    Article  CAS  PubMed  Google Scholar 

  65. Narasimhan, S., Folkers, G. E. & Baldus, M. When small becomes too big: expanding the use of in-cell solid-state NMR spectroscopy. ChemPlusChem 85, 760–768 (2020).

    Article  CAS  PubMed  Google Scholar 

  66. Zhai, W. et al. Postmodification via thiol-click chemistry yields hydrophilic trityl-nitroxide biradicals for biomolecular high-field dynamic nuclear polarization. J. Phys. Chem. B 124, 9047–9060 (2020).

    Article  CAS  PubMed  Google Scholar 

  67. T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco.

  68. Paioni, A. L., Renault, M. A. M. & Baldus, M. DNP and cellular solid-state NMR. eMagRes 7, 51–61 (2018).

    CAS  Google Scholar 

  69. Weingarth, M., Bodenhausen, G. & Tekely, P. Broadband magnetization transfer using moderate radio-frequency fields for NMR with very high static fields and spinning speeds. Chem. Phys. Lett. 488, 10–16 (2010).

    Article  CAS  Google Scholar 

  70. Gradmann, S. et al. Rapid prediction of multi-dimensional NMR data sets. J. Biomol. NMR 54, 377–387 (2012).

    Article  CAS  PubMed  Google Scholar 

  71. Narasimhan, S. et al. Rapid prediction of multi-dimensional NMR data sets using FANDAS. Protein NMR Methods Protoc. (ed. Ghose, R.) 111-132 (Springer, 2018).

  72. Schägger, H. Tricine–SDS-PAGE. Nat. Protoc. 1, 16–22 (2006).

    Article  PubMed  Google Scholar 

  73. Heise, H., Seidel, K., Etzkorn, M., Becker, S. & Baldus, M. 3D NMR spectroscopy for resonance assignment and structure elucidation of proteins under MAS: novel pulse schemes and sensitivity considerations. J. Magn. Reson. 173, 64–74 (2005).

    Article  CAS  PubMed  Google Scholar 

  74. Lacabanne, D., Meier, B. H. & Böckmann, A. Selective labeling and unlabeling strategies in protein solid-state NMR spectroscopy. J. Biomol. NMR 71, 141–150 (2018).

    Article  CAS  PubMed  Google Scholar 

  75. Mandal, A., Boatz, J. C., Wheeler, T. & van der Wel, P. C. A. On the use of ultracentrifugal devices for routine sample preparation in biomolecular magic-angle-spinning NMR. J. Biomol. NMR 67, 165–178 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Knowles, T. J., McClelland, D. M., Rajesh, S., Henderson, I. R. & Overduin, M. Secondary structure and (1)H, (13)C and (15)N backbone resonance assignments of BamC, a component of the outer membrane protein assembly machinery in Escherichia coli. Biomol. NMR Assign. 3, 203–206 (2009).

    Article  CAS  PubMed  Google Scholar 

  77. Kim, K. H. et al. Structural characterization of Escherichia coli BamE, a lipoprotein component of the β-barrel assembly machinery complex. Biochemistry 50, 1081–1090 (2011).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors thank H. van Ingen for providing access to the solution-state NMR instrument, and D. Mance and Dr. Klaartje Houben for technical support and discussions. We are indebted to P. Tordo and O. Ouari (Aix-Marseille Université) for providing AMUPol for the DNP experiments S.N. was supported by the Netherlands’ Magnetic Resonance Research School (NMARRS, project number 022.005.029). Furthermore, this work was supported by the Dutch Research Council (NWO, projects 700.26.121 and 700.10.443 to M.B.) and by iNEXT-Discovery (project number 871037), a project funded by the Horizon 2020 program of the European Commission.

Author information

Author notes

  1. Siddarth Narasimhan

    Present address: Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

  2. Cecilia Pinto

    Present address: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands

Authors and Affiliations

  1. NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, the Netherlands

    Siddarth Narasimhan, Cecilia Pinto, Alessandra Lucini Paioni, Johan van der Zwan, Gert E. Folkers & Marc Baldus

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  1. Siddarth Narasimhan
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Contributions

S.N. and C.P. prepared samples and conducted ssNMR experiments. They were supervised by G.E.F. and M.B. In addition, the DNP experiments were supported by A.L.P. and J.v.d.Z. All authors contributed to writing the manuscript and approved the final version.

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Correspondence to Marc Baldus.

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Related links

Key references using this protocol:

Chordia, S. et al. Preprint at ChemRxiv (2020): https://doi.org/10.26434/chemrxiv.12485993.v1

Pinto, C. et al. J Struct Biol 206, 1−11 (2019): https://doi.org/10.1016/j.jsb.2017.11.015.

Baker, L. A. et al. Structure 26, 161−170 (2018): https://doi.org/10.1016/j.str.2017.11.011

Baker, L. A. et al. J Biomol NMR 62, 199−208 (2015): https://doi.org/10.1007/s10858-015-9936-5

Supplementary information

Supplementary Information

Supplementary Figs. 1–9, Supplementary Table 1 and Supplementary Notes 1–7.

Source Data Supplementary Fig. 1

Biorad format .1sc unprocessed gel image, where the last four lanes correspond to Supplementary Fig. 2

Source Data Supplementary Fig. 2

1D Bruker-format unprocessed spectra.

Source Data Supplementary Fig. 3

2D Bruker-format unprocessed spectra.

Source Data Supplementary Fig. 4

2D Bruker-format unprocessed spectra.

Source Data Supplementary Fig. 5

1D Bruker-format unprocessed spectra.

Source data

Source Data Fig. 8

2D Bruker format “ser” file.

Source Data Fig. 9

3D Bruker format “ser” file. This is also the raw file for Supplementary Figs. 5–8.

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Narasimhan, S., Pinto, C., Lucini Paioni, A. et al. Characterizing proteins in a native bacterial environment using solid-state NMR spectroscopy. Nat Protoc 16, 893–918 (2021). https://doi.org/10.1038/s41596-020-00439-4

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