Determining Ligand Path Through a Major Drug Transporter, AcrB, in Escherichia coli

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1700)

Abstract

An experimental approach to detect the path a substrate takes through a complex membrane protein is described with emphasis on technical approach and theoretical considerations. The protocols for bacterial culture preparation, membrane protein purification, fluorescent assay standardization, data collection, and data analysis are provided. Useful software tools are recommended.

Key words

Substrate path Membrane transporter Membrane channel Fluorescent assay Bodipy FL Maleimide Membrane protein purification 

Notes

Acknowledgments

I am thankful to Dr. Etsuko Sugawara for suggestions and Dr. Yumiko Takatsuka and Dr. Rajeev Misra for materials. This work was carried out in the laboratory of Dr. Hiroshi Nikaido at University of California, supported by AI-09644 grant from U.S. Public Health Service.

References

  1. 1.
    Bohnert JA, Schuster S, Seeger MA et al (2008) Site-directed mutagenesis reveals putative substrate binding residues in the Escherichia coli RND efflux pump AcrB. J Bacteriol 190:8225–8229CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Wang Z, Zhong M, Lu W et al (2015) Repressive mutations restore function-loss caused by the disruption of trimerization in Escherichia coli multidrug transporter AcrB. Front Microbiol 6:4PubMedPubMedCentralGoogle Scholar
  3. 3.
    Soparkar K, Kinana AD, Weeks JW et al (2015) Reversal of the drug binding pocket defects of the AcrB multidrug efflux pump protein of Escherichia coli. J Bacteriol 197:3255–3264CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Seeger MA (2006) Structural asymmetry of AcrB trimer suggests a peristaltic pump mechanism. Science 313:1295–1298CrossRefPubMedGoogle Scholar
  5. 5.
    Kim LHH, Murakami S (2013) Crystal structure of AcrB complexed with linezolid at 3.5 Å resolution. J Struct Funct Genom 14:71–75CrossRefGoogle Scholar
  6. 6.
    Murakami S, Nakashima R, Yamashita E et al (2006) Crystal structures of a multidrug transporter reveal a functionally rotating mechanism. Nature 443:173–179CrossRefPubMedGoogle Scholar
  7. 7.
    Vargiu AV, Nikaido H (2012) Multidrug binding properties of the AcrB efflux pump characterized by molecular dynamics simulations. Proc Natl Acad Sci U S A 109:20637–20642CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ruggerone P, Vargiu AV, Collu F et al (2013) Molecular dynamics computer simulations of multidrug RND efflux pumps. Comput Struct Biotechnol J 5:e201302008CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Husain F, Nikaido H (2010) Substrate path in the AcrB multidrug efflux pump of Escherichia coli. Mol Microbiol 78:320–330CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Husain F, Bikhchandani M, Nikaido H (2011) Vestibules are part of the substrate path in the multidrug efflux transporter AcrB of Escherichia coli. J Bacteriol 193:5847–5849CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Takatsuka Y, Nikaido H (2007) Site-directed disulfide cross-linking shows that cleft flexibility in the periplasmic domain is needed for the multidrug efflux pump AcrB of Escherichia coli. J Bacteriol 189:8677–8684CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    DeLano, WL (2002) The PyMOL user’s manual. DeLano Scientific, San Carlos, CA, 452Google Scholar
  13. 13.
    Chovancova E, Pavelka A, Benes P et al (2012) CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures. PLoS Comput Biol 8:e1002708CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  15. 15.
    Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Doctor Evidence, LLCSanta MonicaUSA
  2. 2.Department of Molecular and Cell Biology, University of CaliforniaBerkeleyUSA
  3. 3.Veterans AffairsLos AngelesUSA
  4. 4.Department of Molecular and Cell BiologyUniversity of California, BerkeleyBerkeleyUSA

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