Abstract
Transport proteins exhibiting broad substrate specificities are major determinants for the phenomenon of multidrug resistance. The Escherichia coli multidrug transporter EmrE, a 4-transmembrane, helical 12-kDa membrane protein, forms a functional dimer to transport a diverse array of aromatic, positively charged substrates in a proton/drug antiport fashion. Here, we report (13)C chemical shifts of the essential residue Glu(14) within the binding pocket. To ensure a native environment, EmrE was reconstituted into E. coli lipids. Experiments were carried out using one- and two-dimensional double quantum filtered (13)C solid state NMR. For an unambiguous assignment of Glu(14), an E25A mutation was introduced to create a single glutamate mutant. Glu(14) was (13)C-labeled using cell-free expression. Purity, labeling, homogeneity, and functionality were probed by mass spectrometry, NMR spectroscopy, freeze fracture electron microscopy, and transport assays. For Glu(14), two distinct sets of chemical shifts were observed that indicates structural asymmetry in the binding pocket of homodimeric EmrE. Upon addition of ethidium bromide, chemical shift changes and altered line shapes were observed, demonstrating substrate coordination by both Glu(14) in the dimer.
Highlights
Transporter with known three-dimensional structure is AcrB [4, 5]
To allow an unambiguous NMR analysis, the nonessential residue Glu25 was replaced with alanine (EmrE E25A) to create a single glutamate mutant
The protein was reconstituted into E. coli lipids allowing the most native environment possible
Summary
Materials—All materials were purchased from Sigma Chemie GmbH (Munich) unless indicated otherwise. A protein calibration mixture was purchased from Applied Biosystems/MDS SCIEX (Darmstadt). In vitro (soluble and precipitate) and in vivo produced EmrE was diluted into 25 ml of solubilizing buffer (100 mM NaCl, 50 mM Tris, pH 8.0, 1 mM dithiothreitol, 10 mM imidazole, 1 pill Roche mini complete protease inhibitor, and 1% (w/v) DDM) and incubated for 1 h at room temperature. Protein and lipid/detergent solutions were mixed at the required ratio and incubated for 1 h at room temperature, followed by detergent removal using pre-washed and sonicated Bio-Beads SM-2. Sample measurements were performed at 200 K with MAS spinning rate of 10,000 Ϯ 5 Hz. DQSQ correlation spectra of the INADEQUATE type [39, 40] were collected by using the SR22 pulse sequence previously described [41]. For chemical shift anisotropy line shape fitting in Topspin 2.0 the Solids Lineshape Analysis tool was used with mixed Gaussian/Lorentzian line shapes
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
