Characterization of Multidrug Efflux Pumps

Bacterial antibiotic resistance and chemical transport has in part been realized by a large group of transport proteins. One large family of transporters, called root-nodulation division (RND) efflux pumps, exhibit a broad substrate range, including antibiotics and various aromatic hydrocarbons. Unfortunately, little is known about these proteins' mechanism and selectivity. We have developed a characterization method, that includes both simulation and experimental data.

Computer simulations enable one to gain a better understanding of the structure-function relationship in proteins. Sequences and structures from eight different efflux pumps were simulated and compared. Residues at select non-consensus sites were identified and correlations attempted between residue charge, size and substrate specificity. Regions that are identified as binding sites can be modified to eliminate or enhance the selectivity of the pump toward a specific molecule, which can aid in larger-scale bioprocesses. These regions can be confirmed experimentally, through a simulated biological membrane.

We have developed a biomimetic device to enable us to characterize the efflux pumps. This device simulates a biological membrane to test for various changes, such as pH and drug concentration, which are indicative of the pump's activity. These can be used to determine diffusivity data for individual pumps. Using this device we are able to determine the effectiveness of the pump against a variety of antibiotics. Future work will entail modifying the device for quantifying the activity of the pumps in the presence of aromatic hydrocarbons to begin productive enzymatic conversion of these aromatics.