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In-cell NMR as a sensitive tool to monitor physiological condition of Escherichia coli

Sugiki, Toshihiko 大阪大学

2020.02.12

概要

The in-cell NMR technique offers significant insights into the structure and function of heterologous proteins in the physiological intracellular environment at an atomic resolution. Escherichia coli (E. coli) is one of the most widely used host cells for heterologous protein expression in structural biological studies as well as for in-cell NMR studies to investigate fundamental structural characteristics and the physiochemistry of certain proteins and their intermolecular interactions under physiological conditions. However, in many cases, it is not easy to obtain well-resolved in-cell NMR spectra because the detectability and resolution of these spectra are significantly influenced by intracellular factors such as nonspecific intermolecular interactions. In this study, we re-examined the experimental parameters of E. coli in-cell NMR and found that the detectability and resolution of the NMR spectra clearly depended on the growth phase of the host cells. Furthermore, the detectability and resolution of the E. coli in-cell NMR spectra correlated with the soluble fraction amounts of the expressed target protein. These results indicate that the E. coli in-cell NMR spectrum of a target protein is a useful tool for monitoring the intracellular conditions of the host cell and for establishing the appropriate cultivation conditions for protein overexpression.

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参考文献

1. Chary, K. V. R. & Govil, G. NMR in Biol System From Molecules to Humans (Eds.: R. Kaptein), Supringer, Berlin, pp. 423–52 (2008).

2. Zimmerman, S. B. & Trach, S. O. Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. J. Mol. Biol. 222, 599–620 (1991).

3. Luby-Phelps, K. Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area. Int. Rev. Cytol. 192, 189–221 (2000).

4. Hatters, D. M., Minton, A. P. & Howlett, G. J. Macromolecular crowding accelerates amyloid formation by human apolipoprotein C-II. J. Biol. Chem. 277, 7824–30 (2002).

5. Stagg, L., Zhang, S. Q., Cheung, M. S. & Wittung-Stafshede, P. Molecular crowding enhances native structure and stability of alpha/ beta protein flavodoxin. Proc. Natl. Acad. Sci. USA 104, 18976–81 (2007).

6. Schlesinger, A. P., Wang, Y., Tadeo, X., Millet, O. & Pielak, G. J. Macromolecular Crowding Fails To Fold a Globular Protein in Cells. J. Am. Chem. Soc. 133, 8082–5 (2011).

7. Wang, Y., Sarkar, M., Smith, A. E., Krois, A. S. & Pielak, G. J. Macromolecular Crowding and Protein Stability. J. Am. Chem. Soc. 134, 16614–18 (2012).

8. Reckel, S., Hansel, R., Löhr, F. & Dӧtsch, V. In-cell NMR spectroscopy. Prog. Nucl. Magn. Reson. Spectrosc. 51, 91–101 (2007).

9. Pielak, G. J. et al. Protein nuclear magnetic resonance under physiological conditions. Biochemistry 48, 226–34 (2009).

10. Maldonado, A. Y., Burz, D. S. & Shekhtman, A. In-cell NMR spectroscopy. Prog. Nucl. Magn. Reson. Spectrosc. 59, 197–212 (2011).

11. Mercatelli, E., Barbieri, L., Luchinat, E. & Banci, L. Direct structural evidence of protein redox regulation obtained by in-cell NMR.

Biochim. Biophys. Acta 1863, 198–204 (2016).

12. Inomata, K., Kamoshida, H., Ikari, M., Ito, Y. & Kigawa, T. Impact of cellular health conditions on the protein folding state in mammalian cells. Chem. Commun. 53, 11245–8 (2017).

13. Tanaka, T. et al. High-resolution protein 3D structure determination in living eukaryotic cells. Angew. Chem. Int. Ed. 58, 7284–8 (2019).

14. Nishida N., Ito, Y. & Shimada, I. In situ structural biology using in-cell NMR. Biochim. Biophys. Acta Gen. Subj. in press (2019).

15. Serber, Z., Ledwidge, R., Miller, S. M. & Dӧtsch, V. Evaluation of Parameters Critical to Observing Proteins Inside Living Escherichia coli by In-Cell NMR Spectroscopy. J. Am. Chem. Soc. 123, 8895–901 (2001).

16. Serber, Z. et al. High-Resolution Macromolecular NMR Spectroscopy Inside Living Cells. J. Am. Chem. Soc. 123, 2446–7 (2001).

17. Sakakibara, D. et al. Protein structure determination in living cells by in-cell NMR spectroscopy. Nature 458, 102–5 (2009).

18. Ikeya, T. et al. NMR protein structure determination in living E. coli cells using nonlinear sampling. Nat. Protoc. 5, 1051–60 (2010).

19. Hamatsu, J. et al. High-resolution heteronuclear multidimensional NMR of proteins in living insect cells using a baculovirus protein expression system. J. Am. Chem. Soc. 135, 1688–91 (2013).

20. Xu, G. et al. Strategies for protein NMR in Escherichia coli. Biochemistry 53, 1971–81 (2014).

21. Erbel, P. J. et al. Identification and biosynthesis of cyclic enterobacterial common antigen in Escherichia coli. J. Bacteriol. 185, 1995–2004 (2003).

22. Erbel, P. J. et al. Cyclic enterobacterial common antigen: potential contaminant of bacterially expressed protein preparations. J. Biomol. NMR 29, 199–204 (2004).

23. Studier, F. W. & Moffatt, B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113–30 (1986).

24. Rosenberg, A. H. & Studier, F. W. T7 RNA polymerase can direct expression of influenza virus cap-binding protein (PB2) in Escherichia coli. Gene 59, 191–200 (1987).

25. Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, J. W. Use of T7 RNA polymerase to direct expression of cloned genes.

Methods Enzymol. 185, 60–89 (1990).

26. Qing, G. et al. Cold-shock induced high-yield protein production in Escherichia coli. Nat. Biotechnol. 22, 877–82 (2004).

27. Schubert, M., Smalla, M., Schmieder, P. & Oschkinat, H. MUSIC in triple-resonance experiments: amino acid type-selective 1H-15N correlations. J. Magn. Reson. 141, 34–4 (1999).

28. Schubert, M., Oschkinat, H. & Schmieder, P. MUSIC, selective pulses, and tuned delays: amino acid type-selective 1H-15N correlations, II. J. Magn. Reson. 148, 61–72 (2001).

29. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–93 (1995).

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