Assessment of Multidrug Efflux Assemblies by Surface Plasmon Resonance

Abstract

Surface plasmon resonance (SPR) is a powerful tool for kinetic analyses of protein–protein interactions. Here we describe the application of this method to study interactions of membrane proteins involved in multidrug efflux in Escherichia coli.These so-called multidrug efflux pumps comprise an inner membrane transporter, a periplasmic membrane fusion protein and an outer membrane channel. The three components are assembled into a protein conduit that enables bacteria to expel multiple drugs directly into the external medium bypassing the periplasm. The “across-two-membranes” transport mechanism makes tripartite transporters poorly amenable to biochemical analyses. Two out of three components are located in different membranes and must be studied in detergents or reconstituted into lipid bilayers to retain their functionality. Furthermore, cytoplasmic and exoplasmic domains of all three components perform different functions and must be oriented in a specific way to assemble functional complexes. The SPR approach provides means to overcome some of these problems in studies of protein assemblies that function in the context of two membranes.