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