NMR investigation of the multidrug transporter EmrE, an integral membrane protein

Eur J Biochem. 1998 Jun 15;254(3):610-9. doi: 10.1046/j.1432-1327.1998.2540610.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antiporters*
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / isolation & purification
  • Carrier Proteins / chemistry*
  • Carrier Proteins / isolation & purification
  • Chromatography, Ion Exchange
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli Proteins
  • Magnetic Resonance Spectroscopy
  • Membrane Proteins / chemistry*
  • Membrane Proteins / isolation & purification
  • Molecular Sequence Data
  • Protein Structure, Secondary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification

Substances

  • Antiporters
  • Bacterial Proteins
  • Carrier Proteins
  • Escherichia coli Proteins
  • Membrane Proteins
  • Recombinant Proteins
  • EmrE protein, E coli