Labeling of Specific Residues of the Multidrug Transporter AcrB with Its Fluorescent Substrate

Gram-negative bacteria possess resistance nodulation cell division (RND)-type drug efflux transporters in the inner membrane that confer a high degree of intrinsic resistance to various drugs. Escherichia coli expresses the most powerful RND-type drug efflux transporter, AcrB, which extrudes various chemical compounds, including antibiotics, by forming a complex with the periplasmic membrane fusion protein AcrA and the outer membrane channel TolC. To date, various reported structures of RND-type drug efflux transporters and AcrA-AcrB-TolC ternary complex have indicated substrate-binding sites and substrate translocation pathways, but their biochemical verification is insufficient.In this chapter, I describe the labeling, purification, and detection of AcrB Cys mutants using tetramethylrhodamine-5-iodoacetamide, a thiol-reactive fluorescent reagent recognized by AcrB, as a substrate. The labeling of substrate-binding sites and substrate translocation pathways of the AcrA-AcrB-TolC system using this method will be useful for elucidating the multidrug transport mechanism of this ternary complex system.