The Key Residue for Substrate Transport (Glu14) in the EmrE Dimer Is Asymmetric

摘要: Transport proteins exhibiting broad substrate specificities are major determinants for the phenomenon of multidrug resistance. The Escherichia coli transporter EmrE, a 4-transmembrane, helical 12-kDa membrane protein, forms functional dimer to transport diverse array aromatic, positively charged substrates in proton/drug antiport fashion. Here, we report 13C chemical shifts essential residue Glu14 within binding pocket. To ensure native environment, EmrE was reconstituted into E. lipids. Experiments were carried out using one- and two-dimensional double quantum filtered solid state NMR. For an unambiguous assignment Glu14, E25A mutation introduced create single glutamate mutant. 13C-labeled cell-free expression. Purity, labeling, homogeneity, functionality probed by mass spectrometry, NMR spectroscopy, freeze fracture electron microscopy, assays. two distinct sets observed that indicates structural asymmetry pocket homodimeric EmrE. Upon addition ethidium bromide, shift changes altered line shapes observed, demonstrating coordination both dimer.