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In Vitro Mutagenesis Protocols

Volume 634 of the series Methods in Molecular Biology pp 343-354

Date:

Site-Directed Disulfide Cross-Linking to Probe Conformational Changes of a Transporter During Its Functional Cycle: Escherichia coli AcrB Multidrug Exporter as an Example

  • Yumiko TakatsukaAffiliated withDepartment of Molecular and Cell Biology, University of California
  • , Hiroshi NikaidoAffiliated withDepartment of Molecular and Cell Biology, University of California Email author 

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Abstract

Many proteins, especially transporters, are thought to undergo large conformational alterations during their functional cycle. Since X-ray crystallography usually gives only the most stable conformation, other methods are needed to probe this conformational change. Site-directed disulfide cross-linking is often very useful for this purpose. We illustrate this by using the Escherichia coli AcrB, a proton-motive-force-dependent multidrug efflux transporter. Crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export (functional rotation hypothesis). Site-directed disulfide cross-linking between those residues that come close to each other in only one stage in the cycle inactivated the transporter, showing that the conformational changes indeed occurred in vivo and that they are required for drug transport. A dsbA strain, which has a diminished activity to form disulfide bonds in the periplasm, was used to verify the conclusion by showing a restored transport activity in this strain. Furthermore, we describe “a real-time cross-linking experiment,” in which rapidly reacting, sulfhydryl-specific cross-linkers, methanethiosulfonates, inactivate the AcrB double-cysteine mutant expressed in dsbA cells instantaneously.

Key words

Site-directed cross-linking Conformational change Disulfide bond Sulfhydryl-specific cross-linker Methanethiosulfonate dsbA Multidrug efflux transporter AcrB Escherichia coli