In this paper we compare electrochemical behavior of two homolog proteins, namely anterior gradient 2 (AGR2) and anterior gradient 3 (AGR3), playing an important role in cancer cell biology. The slight variation in their protein structures has an impact on protein adsorption and orientation at charged surface and also enables AGR2 and AGR3 to form heterocomplexes. We confirm interaction between AGR2 and AGR3 (i) in vitro by immunochemical and constant current chronopotentiometric stripping (CPS) analysis and (ii) in vivo by bioluminescence resonance energy transfer (BRET) assay. Mutation of AGR2 in dimerization domain (E60A) prevents development of wild type AGR2 dimers and also negatively affects interaction with wild type AGR3 as shown by CPS analysis. Beside new information about AGR2 and AGR3 protein including their joint interaction, our work introduces possible applications of CPS in bioanalysis of protein complexes, including those relatively unstable, but important in the cancer research.

Institute of Biophysics The Czech Academy of Sciences v v i Královopolská 135 61265 Brno Czech Republic

Masaryk Memorial Cancer Institute Research Centre for Applied Molecular Oncology Zluty kopec 7 65653 Brno Czech Republic

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Electrochemistry in sensing of molecular interactions of proteins and their behavior in an electric field

Characterization of the AGR2 Interactome Uncovers New Players of Protein Disulfide Isomerase Network in Cancer Cells