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Functional Refolding and Characterization of Two Tom40 Isoforms from Human Mitochondria

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Abstract

Tom40 proteins represent an essential class of molecules which facilitate translocation of unfolded proteins from the cytosol into the mitochondrial intermembrane space. They are part of a high-molecular mass complex that forms the protein-conducting channel in outer mitochondrial membranes. This study concerns the recombinant expression, purification and folding of amino-terminally truncated variants of the two human Tom40 isoforms for structural biology experiments. Both CD and FTIR secondary structure analysis revealed a dominant beta-sheet structure and a short alpha-helical part for both proteins together with a high thermal stability. Two secondary structure elements can be denatured independently. Reconstitution of the recombinant protein into planar lipid bilayers demonstrated ion channel activity similar to Tom40 purified from Neurospora crassa mitochondrial membranes, but conductivity fingerprints differ from the structurally closely related VDAC proteins.

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Acknowledgments

The authors thank Beate Nitschke for expert technical assistance and Tobias Kulschewski and Dr. Simon Stutz for their contributions at the early stage of this work. The work was supported by a Competence Network on Functional Nanostructures grant of the Baden-Württemberg Foundation and the Human Frontier Science Program to S. N. and K. Z., respectively.

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Authors and Affiliations

  1. Biophysics Department, Institute of Biology, University of Stuttgart, Pfaffenwaldring 57, 70550, Stuttgart, Germany

    Frauke Mager, Dennis Gessmann & Stephan Nussberger

  2. Department for Protein Evolution, Max Planck Institute for Developmental Biology, Spemannstr. 35-39, 72076, Tübingen, Germany

    Frauke Mager & Kornelius Zeth

Authors
  1. Frauke Mager
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  2. Dennis Gessmann
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  3. Stephan Nussberger
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  4. Kornelius Zeth
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Correspondence to Stephan Nussberger.

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Mager, F., Gessmann, D., Nussberger, S. et al. Functional Refolding and Characterization of Two Tom40 Isoforms from Human Mitochondria. J Membrane Biol 242, 11–21 (2011). https://doi.org/10.1007/s00232-011-9372-8

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  • DOI: https://doi.org/10.1007/s00232-011-9372-8

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