Assessing Energy-Dependent Protein Conformational Changes in the TonB System

  • Ray A. Larsen
Part of the Methods in Molecular Biology book series (MIMB, volume 1615)


Changes in conformation can alter a protein’s vulnerability to proteolysis. Thus, in vivo differential proteinase sensitivity provides a means for identifying conformational changes that mark discrete states in the activity cycle of a protein. The ability to detect a specific conformational state allows for experiments to address specific protein–protein interactions and other physiological components that potentially contribute to the function of the protein. This chapter presents the application of this technique to the TonB-dependent energy transduction system of Gram-negative bacteria, a strategy that has refined our understanding of how the TonB protein is coupled to the ion electrochemical gradient of the cytoplasmic membrane.

Key words

Protein conformation Ion electrochemical gradient Proteinase sensitivity TonB Spheroplast Energy transduction 


  1. 1.
    Oldfield CJ, Dunker AK (2014) Intrinsically disordered proteins and intrinsically disordered protein regions. Annu Rev Biochem 83:553–584CrossRefPubMedGoogle Scholar
  2. 2.
    Johnson DE, Xue B, Sickmeier MD, Meng J, Cortese MS, Oldfield CJ, Gall TL, Dunker AK, Uversky VN (2012) High-throughput characterization of intrinsic disorder in proteins from the protein structure initiative. J Struct Biol 180:201–215CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Peacock RS, Weijie AM, Howard SP, Price FD, Vogel HJ (2005) The solution structure of the C-terminal domain of TonB and interaction studies with TonB box peptides. J Mol Biol 345:1185–1197CrossRefGoogle Scholar
  4. 4.
    Postle K, Larsen RA (2007) TonB dependent energy transduction between outer and cytoplasmic membranes. Biometals 20:453–465CrossRefPubMedGoogle Scholar
  5. 5.
    Larsen RA, Thomas MG, Postle K (1999) Protonmotive force, ExbB and ligand-bound FepA drive conformational changes in TonB. Mol Microbiol 31:1809–1824CrossRefPubMedGoogle Scholar
  6. 6.
    Larsen RA, Deckert G, Kastead K, Devanathan S, Keller KL, Postle K (2007) His20 provides the sole functionally significant side chain in the essential TonB transmembrane domain. J Bacteriol 189:2825–2833CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Ollis AA, Postle K (2012) Identification of functionally important TonB-ExbD periplasmic domain interactions in vivo. J Bacteriol 194:3078–3087CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ollis AA, Kumar A, Postle K (2012) The ExbD periplasmic domain contains distinct functional regions for two stages in TonB energization. J Bacteriol 194:3069–3077CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Gresock MG, Kastead KA, Postle K (2015) From homodimer to heterodimer and back: elucidating the TonB energy transduction cycle. J Bacteriol 197:3433–3445CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Fischer E, Günter K, Braun V (1989) Involvement of ExbB and TonB in transport across the outer membrane of Escherichia coli: phenotypic complementation of exbB mutants by overexpressed tonB and physical stabilization of TonB by ExbB. J Bacteriol 171:5127–5134CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Larsen RA, Thomas MG, Wood GE, Postle K (1994) Partial suppression of an Escherichia coli TonB transmembrane domain mutation (∆V17) by a missense mutation in ExbB. Mol Microbiol 13:627–640CrossRefPubMedGoogle Scholar
  12. 12.
    Randall LL, Hardy SJS (1986) Correlation of competence for export with lack of tertiary structure of the mature species: a study in vivo of maltose-binding protein in E. coli. Cell 46:921–928CrossRefPubMedGoogle Scholar
  13. 13.
    Cascales E, Gavioli M, Sturgis JN, Lloubés R (2000) Proton motive force drives the interaction of the inner membrane TolA and outer membrane pal proteins in Escherichia coli. Mol Microbiol 38:904–915CrossRefPubMedGoogle Scholar
  14. 14.
    Germon P, Ray MC, Vianney A, Lazzaroni JC (2001) Energy-dependent conformational change in the TolA protein of Escherichia coli involves its N-terminal domain, TolQ, and TolR. J Bacteriol 183:4110–41104CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Hill CW, Harnish BW (1981) Inversions between ribosomal RNA genes of Escherichia coli. Proc Natl Acad Sci U S A 78:7069–7072CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Press, Cold Spring HarborGoogle Scholar
  17. 17.
    Larsen RA, Myers PS, Skare JT, Seachord CL, Darveau RP, Postle K (1996) Identification of TonB homologs in the family Enterobacteriaceae and evidence for conservation of TonB-dependent energy transduction complexes. J Bacteriol 178:1363–1373CrossRefPubMedPubMedCentralGoogle Scholar

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© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Biological SciencesBowling Green State UniversityBowling GreenUSA

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