NMR investigation of the multidrug transporter EmrE, an integral membrane protein.
EmrE is an Escherichia coli multidrug transport protein that confers resistance to a wide range of toxicants by active transport across the bacterial cell membrane. The highly hydrophobic polytopic integral membrane protein has been purified and studied in its full-length form by high-resolution NMR spectroscopy in a mixture of chloroform/methanol/water (6:6:1, by vol.). Full activity is maintained after reconstitution of the protein into proteoliposomes from this solvent mixture. A series of heteronuclear (1H-15N) two- and three-dimensional experiments, as well as triple resonance experiments, were applied to the 110-residue protein and led to the assignment of the 1H, 15N and a large part of the 13C backbone resonances as well as many of the sidechain resonances. A preliminary analysis of the secondary structure, based on sequential NOE connectivities, deviation of chemical shifts from random coil values and 3J(NH-H alpha) coupling constants supports a model where the protein forms four alpha-helices between residues 4-26 (TM1), 32-53 (TM2), 58-76 (TM3) and 85-106 (TM4). For the residues of helices TM2 and TM3 a significant line broadening occurs due to slow conformational processes.
Schwaiger
M
M
Institut für Organische Chemie und Biochemie, Technische Universität München, Germany.
Lebendiker
M
M
Yerushalmi
H
H
Coles
M
M
Gröger
A
A
Schwarz
C
C
Schuldiner
S
S
Kessler
H
H
D000595:Amino Acid Sequence; D017920:Antiporters; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D017920:Antiporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C075436:EmrE protein, E coli