The reconstitution and activity of the small multidrug transporter EmrE is modulated by non-bilayer lipid composition.
The ability of multidrug transport proteins within biological membranes to recognise a diverse array of substrates is a fundamental aspect of antibiotic resistance. Detailed information on the mechanisms of recognition and transport can be provided only by in vitro studies in reconstituted bilayer systems. We describe the controlled, efficient reconstitution of the small multidrug transporter EmrE in a simple model membrane and investigate the effect of non-bilayer lipids on this process. Transport activity is impaired, in line with an increase in the lateral pressure within the bilayer. We demonstrate the potential of this lateral pressure modulation method as a general approach to the folding and assembly of membrane proteins in vitro, by recovering functional transporter from a partly denatured state. Our results highlight the importance of optimising reconstitution procedures and bilayer lipid composition in studies of membrane transporters. This is particularly pertinent for multidrug proteins, and we show that the use of a sub-optimal lipid bilayer environment or reconstitution method could lead to incorrect information on protein activity.
Curnow
Paul
P
Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, BS8 1TD, UK. curnow@lifesci.ucsb.edu
Lorch
Mark
M
Charalambous
Kalypso
K
Booth
Paula J
PJ
D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry* / Q000378:metabolism; D001692:Biological Transport; D004352:Drug Resistance, Microbial; D018432:Drug Resistance, Multiple; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D004996:Ethidium / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry; D008081:Liposomes; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
D017920:Antiporters; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D008081:Liposomes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D012967:Sodium Dodecyl Sulfate; D004996:Ethidium