Organic solvent extracted EmrE solubilized in dodecyl maltoside is monomeric and binds drug ligand.
Ethidium multidrug resistance protein (EmrE) is a member of the small multidrug resistance family of proteins and is responsible for resistance to a diverse group of lipophilic cations. To examine the multimeric state(s), size-exclusion HPLC and sedimentation velocity experiments were performed with EmrE solubilized in N-dodecyl-beta-d-maltopyranoside (DM) detergent. EmrE was purified from Escherichia coli membranes using organic extraction with a 3:1 chloroform:methanol solvent followed by LH-20 chromatography and the recovered pure protein was re-solubilized in a buffer containing 2% DM. The purified protein was analyzed by SEC-HPLC to estimate the monodispersity and to determine the amount of bound detergent. The results show that EmrE is homogeneous in DM with a Stokes radius of 3.6nm compatible with that of a monomer. Sedimentation velocity experiments indicated that the EmrE preparation was monodisperse and supports the fact that the organic extracted protein solubilized in DM is monomeric. This monomeric form of the protein analyzed here is also shown to bind substrate in the micromolar range.
Winstone
Tara L
TL
Division of Biochemistry, Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, Alta., Canada T2N 1N4.
Jidenko
Marie
M
le Maire
Marc
M
Ebel
Christine
C
Duncalf
Karen A
KA
Turner
Raymond J
RJ
D017920:Antiporters / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003902:Detergents / Q000737:chemistry; D029968:Escherichia coli Proteins; D004996:Ethidium / Q000737:chemistry; D005960:Glucosides / Q000737:chemistry*; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012995:Solubility; D012997:Solvents / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation
D017920:Antiporters; D003902:Detergents; D029968:Escherichia coli Proteins; D005960:Glucosides; D008024:Ligands; D008565:Membrane Proteins; D012997:Solvents; C075436:EmrE protein, E coli; C040358:dodecyl maltoside; D004996:Ethidium