CEEPC/IPM/CMSC - Abstrakt prezentace

(4. konference České společnosti pro hmotnostní spektrometrii - WeP-006)
Seeking of cytochrome P450 2B4:b5 interactions both in cytosol and lipid membrane combining photo-initiated cross-linking by photo-activable amino acid and high resolution mass spectrometry

Tomáš Ječmen 1,2 *, Renata Ptáčková 1,2, Věra Černá 2, Helena Dračínská 2, Petr Pompach 1, Miroslav Šulc 1,2

  1. Institute of Microbiology ASCR, v.v.i.
  2. Charles University in Prague

Abstrakt

Mixed function oxygenase system participates in organism in detoxication of exogenous substances, drug metabolism, and activation of chemical carcinogens. Structuraly diverse substrates are biotransformed by terminal oxygenases – cytochromes P450 (P450). Catalytic properties of certain P450s (e.g. studied isoforms 2B4) are altered in the presence of cytochrome b5 (cyb5) – their facultative redox partner. Both cytochromes are anchored in lipid membrane of endoplasmic reticulum by hydrophobic domains whereas their catalytic domains are exposed to cytosol.

Photo-activable methionine analog (pMet) serving as an unselective zero-length cross-linking agent was utilized to covalently fixate individual P450 2B4:cyb5 interactions in an attempt to determine their impact on catalysis. Introduction of pMet to methionine site of protein sequence was realized during recombinant expression in E. coli carried out in limit medium supplemented with the unnatural amino acid. Photo-activable cyb5 wild type1 as well as its 6 mutants each containing single methionine site2,3 were prepared to probe both interactions in cytosol and within membrane environment. P450 2B4 and a particular photo-activable cyb5 were reconstituted with lipid vesicles mimicking natural membrane and UV-irradiated. Resulting covalent complexes were electrophoretically separated, proteolytically digested and analyzed by LC-ESI-FTICR MS.

The range of amino acids known to participate in binding of P450 2B4 and cyb5 was further extended and yet unexplored interacting regions were identified by this experimental approach. At least two distinct mutual orientations of studied proteins were proposed taking all presented findings into consideration.

* Korespondující autor: tomas.jecmen@centrum.cz

Literatura

  1. Koberova M. et al.: Int. J. Electrochem. Sci. 8, 125-134 (2013).
  2. Ječmen T. et al.: Neuro Endocrinol Lett. 35(Suppl2), 114-122 (2014).
  3. Ječmen T. et al.: Methods, submitted (2015).

Poděkování:

Financial support: GAČR P207/12/0627, UNCE 204025/2012.


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