novobiocin and tetraphenylphosphonium

The majority of membrane proteins function as oligomers. However, it remains largely unclear how the oligomer stability of protein complexes correlates with their function. Understanding the relationship between oligomer stability and activity is essential to protein research and to virtually all cellular processes that depend on the function of protein complexes. Proteins make lasting or transient interactions as they perform their functions. Obligate oligomeric proteins exist and function exclusively at a specific oligomeric state. Although oligomerization is clearly critical for such proteins to function, a direct correlation between oligomer affinity and biological activity has not yet been reported. Here, we used an obligate trimeric membrane transporter protein, AcrB, as a model to investigate the correlation between its relative trimer affinity and efflux activity. AcrB is a component of the major multidrug efflux system in Escherichia coli. We created six AcrB constructs with mutations at the transmembrane intersubunit interface, and we determined their activities using both a drug susceptibility assay and an ethidium bromide accumulation assay. The relative trimer affinities of these mutants in detergent micelles were obtained using blue native polyacrylamide gel electrophoresis. A correlation between the relative trimer affinity and substrate efflux activity was observed, in which a threshold trimer stability was required to maintain efflux activity. The trimer affinity of the wild-type protein was approximately 3 kcal/mol more stable than the threshold value. Once the threshold was reached, an additional increase of stability in the range observed had no observable effect on protein activity.

Topics: Amino Acid Substitution; Anti-Bacterial Agents; Biological Transport; Drug Resistance, Multiple, Bacterial; Erythromycin; Escherichia coli; Escherichia coli Proteins; Ethidium; Fluorescent Dyes; Kinetics; Microbial Sensitivity Tests; Models, Molecular; Multidrug Resistance-Associated Proteins; Mutant Proteins; Novobiocin; Onium Compounds; Organophosphorus Compounds; Protein Stability; Protein Structure, Quaternary; Protein Subunits; Recombinant Proteins; Rhodamines

AcrAB-TolC is the major constitutively expressed efflux pump system that provides resistance to a variety of antimicrobial agents and dyes in Escherichia coli. However, no systematically optimized real-time dye efflux assay has been published for the measurement of its activity and for detection of possible competition between substrates. Here, we report on the development of such an assay using a lipophilic dye, Nile Red. Energy-depleted cells were loaded with the dye in the presence of low (10 microM or less) concentrations of the proton conductor carbonyl cyanide m-chlorophenylhydrazone (CCCP). The CCCP was then removed, and efflux was triggered by energization with glucose. Various known efflux pump inhibitors and antimicrobials were checked for the ability to slow down Nile Red efflux, presumably through competition. Besides the known inhibitors Phe-Arg-beta-naphthylamide and 1-naphthyl-methylpiperazine, several tetracyclic compounds (doxorubicin, minocycline, chlortetracycline, doxycycline, and tetracycline) and tetraphenylphosphonium chloride were found to interfere with dye efflux. This inhibition could not be explained by the depletion of proton motive force. None of the other tested antimicrobials, including macrolides, fluoroquinolones, and beta-lactams, had any impact on Nile Red efflux, even at concentrations of up to 1 mM.

Topics: Anti-Bacterial Agents; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Chlortetracycline; Dipeptides; Doxorubicin; Doxycycline; Drug Resistance, Multiple, Bacterial; Escherichia coli; Escherichia coli Proteins; Minocycline; Onium Compounds; Organophosphorus Compounds; Tetracycline