Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

An Escherichia coli expression–based method for heme substitution

Nature Methods volume 4, pages 43–45 (2007)Cite this article

Abstract

Heme reconstitution with porphyrin analogs is a powerful approach toward understanding the molecular function of heme proteins; present methods, however, have not proven to be generally useful. Here we describe the development and application of an expression-based method for introducing modified porphyrins. The approach allows efficient incorporation of heme analogs using a widely available bacterial strain and offers an attractive alternative to present reconstitution methods that subject proteins to harsh, denaturing conditions.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Spectra of proteins expressed in RP523.
Figure 2: Characterization of MPA-iNOSheme and Meso-iNOSheme.

Similar content being viewed by others

References

  1. Fleischmann, G. et al. Eur. J. Biochem. 267, 5156–5167 (2000).

    Article  CAS  Google Scholar 

  2. Ghisla, S. & Massey, V. Biochem. J. 239, 1–12 (1986).

    Article  CAS  Google Scholar 

  3. Li, J.M. et al. J. Bacteriol. 170, 1021–1025 (1988).

    Article  CAS  Google Scholar 

  4. Yonetani, T. & Asakura, T. J. Biol. Chem. 244, 4580–4588 (1969).

    CAS  PubMed  Google Scholar 

  5. Mondal, M.S., Mazumdar, S. & Mitra, S. Inorg. Chem. 32, 5362–5367 (1993).

    Article  CAS  Google Scholar 

  6. Gelb, M.H., Toscano, W.a. & Sligar, S.G. Proc. Natl. Acad. Sci. USA 79, 5758–5762 (1982).

    Article  CAS  Google Scholar 

  7. Antonini, E. & Brunori, M. Hemoglobin and myoglobin in their reactions with ligands. (North-Holland Publishing Company, Amsterdam, 1971).

    Google Scholar 

  8. Duprat, A.F. et al. Biochemistry 34, 2634–2644 (1995).

    Article  CAS  Google Scholar 

  9. Romberg, R.W. & Kassner, R.J. Biochemistry 18, 5387–5392 (1979).

    Article  CAS  Google Scholar 

  10. Singh, U.P. et al. Biochim. Biophys. Acta 1384, 103–111 (1998).

    Article  CAS  Google Scholar 

  11. Tamura, M., Woodrow, G.V. & Yonetani, T. Biochim. Biophys. Acta 317, 34–49 (1973).

    Article  CAS  Google Scholar 

  12. Atamian, M. & Bocian, D.F. Biochemistry 26, 8319–8326 (1987).

    Article  CAS  Google Scholar 

  13. Wagner, G.C., Gunsalus, I.C., Wang, M.Y.R. & Hoffman, B.M. J. Biol. Chem. 256, 6266–6273 (1981).

    CAS  PubMed  Google Scholar 

  14. Wang, M.Y.R., Hoffman, B.M. & Hollenberg, P.F. J. Biol. Chem. 252, 6268–6275 (1977).

    CAS  PubMed  Google Scholar 

  15. Stojiljkovic, I., Kumar, V. & Srinivasan, N. Mol. Microbiol. 31, 429–442 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the members of the Marletta laboratory for critical reading and useful suggestions during the preparation of this manuscript. We also thank S.G. Sligar (University of Illinois) for the mouse myoglobin vector, J. Rumbley (University of Minnesota, Duluth) for the cytochrome c vector, and J.C. Niles for help in developing the reverse-phase HPLC porphyrin analysis assay. This work was supported by the Aldo DeBenedictis Fund, the Natural Sciences and Engineering Council of Canada and the Alberta Heritage Foundation for Medical Research.

Author information

Authors and Affiliations

  1. Department of Chemistry, Lawrence Berkeley National Laboratory, University of California, Berkeley, 211 Lewis Hall, Berkeley, 94720, California, USA

    Joshua J Woodward, Nathaniel I Martin & Michael A Marletta

  2. Department of Molecular and Cell Biology, Lawrence Berkeley National Laboratory, University of California, Berkeley, 211 Lewis Hall, Berkeley, 94720, California, USA

    Michael A Marletta

  3. Division of Physical Biosciences, Lawrence Berkeley National Laboratory, University of California, Berkeley, 211 Lewis Hall, Berkeley, 94720, California, USA

    Michael A Marletta

Authors
  1. Joshua J Woodward
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nathaniel I Martin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael A Marletta
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.I.M. synthesized monopropargylamide heme. J.J.W. performed all other experiments, which were developed and designed with M.A.M.

Note: Supplementary information is available on the Nature Methods website.

Corresponding author

Correspondence to Michael A Marletta.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Heme analog structures.

Supplementary Fig. 2

HPLC elution profile of heme and modified hemes.

Supplementary Fig. 3

Mass spectra of MPA-PPIX standard and MPA-iNOSheme porphyrin content.

Supplementary Table 1

Heme content of proteins expressed in RP523.

Supplementary Table 2

Myoglobin spectral characteristics.

Supplementary Table 3

iNOSheme spectral characteristics.

Supplementary Methods

Rights and permissions

Reprints and permissions

About this article

Cite this article

Woodward, J., Martin, N. & Marletta, M. An Escherichia coli expression–based method for heme substitution. Nat Methods 4, 43–45 (2007). https://doi.org/10.1038/nmeth984

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth984

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing