Membrane Insertion of Small Proteins

  • Andreas Kuhn
  • Natalie Stiegler
  • Anne-Kathrin Schubert
Part of the Methods in Molecular Biology book series (MIMB, volume 619)


Proteins that are less than 10 kDa in size are easily purified under denaturing conditions and can often be refolded by removal of the denaturing agents. The purified small membrane proteins are competent for membrane insertion when the denaturing agent is diluted out and a membranous system like liposomes or proteoliposomes is added. This system allows the characterization of the membrane insertion process at the molecular level. The insertion of the protein into proteoliposomes can be followed by protease digestion and Western blot analysis. Only if the antigenic region of the protein has translocated into the lumen of the proteoliposome it is protected from the protease. When combining this approach with fluorophores that are placed within the membrane protein, membrane insertion can also be followed by fluorescence correlation spectroscopy.

Key words

Pf3 coat protein M13 procoat protein YidC membrane insertase liposomes fluorescence labelling 


  1. 1.
    Wolfe, P.B., Rice, M. and Wickner, W. (1985) Effects of two sec genes on protein assembly into the plasma membrane of Escherichia coli J Biol Chem 260, 1836–1841.PubMedGoogle Scholar
  2. 2.
    Rohrer, J. and Kuhn, A. (1990) The function of a leader peptide in translocating charged amino acyl residues across a membrane Science 250, 1418–1421.CrossRefPubMedGoogle Scholar
  3. 3.
    Van Der Laan, M., Bechtluft, P., Kol, S., Nouwen, N. and Driessen, A.J. (2004) F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis J Cell Biol 165, 213–222.CrossRefPubMedGoogle Scholar
  4. 4.
    Facey, S.J., Neugebauer, S.A., Krauss, S. and Kuhn, A. (2007) The mechanosensitive channel protein MscL is targeted by the SRP to the novel YidC membrane insertion pathway of Escherichia coli J Mol Biol 365, 995–1004.CrossRefPubMedGoogle Scholar
  5. 5.
    Voss, N.R., Gerstein, M., Steitz, T.A. and Moore, P.B. (2006) The geometry of the ribosomal polypeptide exit tunnel J Mol Biol 360, 893–906.CrossRefPubMedGoogle Scholar
  6. 6.
    Kuhn, A. and Wickner, W. (1985) Conserved residues of the leader peptide are essential for cleavage by leader peptidase J Biol Chem 260, 15914–15918.PubMedGoogle Scholar
  7. 7.
    Kiefer, D. and Kuhn, A. (1999) Hydrophobic forces drive spontaneous membrane insertion of the bacteriophage Pf3 coat protein without topological control EMBO J 18, 6299–6306.CrossRefPubMedGoogle Scholar
  8. 8.
    Serek, J., Bauer-Manz, G., Struhalla, G., Van Den Berg, L., Kiefer, D., Dalbey, R. et al. (2004) Escherichia coli YidC is a membrane insertase for Sec-independent proteins EMBO J 23, 294–301.CrossRefPubMedGoogle Scholar
  9. 9.
    Samuelson, J.C., Chen, M., Jiang, F., Moller, I., Wiedmann, M., Kuhn, A. et al. (2000) YidC mediates membrane protein insertion in bacteria Nature 406, 637–641.CrossRefPubMedGoogle Scholar
  10. 10.
    Studier, F.W., Rosenberg, A.H., Dunn, J.J. and Dubendorff, J.W. (1990) Use of T7 RNA polymerase to direct expression of cloned genes Methods Enzymol 185, 60–89.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Andreas Kuhn
    • 1
  • Natalie Stiegler
    • 1
  • Anne-Kathrin Schubert
    • 1
  1. 1.Institute of Microbiology and Molecular BiologyUniversity of HohenheimStuttgartGermany

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