EMRE, THE SMALLEST ION-COUPLED TRANSPORTER, PROVIDES A UNIQUE PARADIGM FOR STRUCTURE-FUNCTION STUDIES

EmrE is an Escherichia coli multidrug transporter which confers resist ance to a wide variety of toxicants by actively removing them in excha nge for hydrogen ions, EmrE is a highly hydrophobic 12 kDa protein whi ch has been purified by taking advantage of its unique solubility in o rganic solvents. After solubilization and purification, the protein re tains its ability to transport as judged from the fact that it can be reconstituted in a functional form. Hydrophobicity analysis of the seq uence yielded four putative transmembrane domains of similar sizes, Re sults from transmission Fourier transform infrared measurements agree remarkably well with this hypothesis and yielded alpha-helical estimat es of 78 % and 80 % for EmrE in CHCl3:MeOH and 1,2-dimyristoyl phospho choline, respectively, Furthermore, the fact that most of the amide gr oups in the protein do not undergo amide-proton H/D exchange implies t hat most (approximately 80 %) of the residues are embedded in the bila yer, These observations are only consistent with four transmembrane he lices. A domain lined by Cys41 and Cys95 accessible only to substrates such as the organic mercurial 4-(chloromercuri)benzoic acid has been identified. Both residues are asymmetric in their location with respec t to the plane of the membrane, Cys95 being closer than Cys41 to the o utside face of the membrane, In co-reconstitution experiments of wild- type protein with three different inactive mutants, negative dominance has been observed, This phenomenon suggests that EmrE is functional a s a homo-oligomer.