title: In vitro unfolding and refolding of the small multidrug transporter EmrE.
abstract: The composition of the lipid bilayer is increasingly being recognised as important for the regulation of integral membrane protein folding and function, both in vivo and in vitro. The folding of only a few membrane proteins, however, has been characterised in different lipid environments. We have refolded the small multidrug transporter EmrE in vitro from a denatured state to a functional protein and monitored the influence of lipids on the folding process. EmrE is part of a multidrug resistance protein family that is highly conserved amongst bacteria and is responsible for bacterial resistance to toxic substances. We find that the secondary structure of EmrE is very stable and only small amounts are denatured even in the presence of unusually high denaturant concentrations involving a combination of 10 M urea and 5% SDS. Substrate binding by EmrE is recovered after refolding this denatured protein into dodecylmaltoside detergent micelles or into lipid vesicles. The yield of refolded EmrE decreases with lipid bilayer compositional changes that increase the lateral chain pressure within the bilayer, whilst conversely, the apparent rate of folding seems to increase. These results add further weight to the hypothesis that an increased lateral chain pressure hinders protein insertion across the bilayer. Once the protein is inserted, however, the greater pressure on the transmembrane helices accelerates correct packing and final folding. This work augments the relatively small number of biophysical folding studies in vitro on helical membrane proteins.
authors.affiliation: Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK. david.miller@bristol.ac.uk
authors.forename: David
authors.initials: D
authors.lastname: Miller
authors.forename: Kalypso
authors.initials: K
authors.lastname: Charalambous
authors.forename: Dvir
authors.initials: D
authors.lastname: Rotem
authors.forename: Shimon
authors.initials: S
authors.lastname: Schuldiner
authors.forename: Paul
authors.initials: P
authors.lastname: Curnow
authors.forename: Paula J
authors.initials: PJ
authors.lastname: Booth
mesh_terms: D017920:Antiporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020404:Glycerophospholipids / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D008563:Membrane Lipids / Q000737:chemistry; D008823:Micelles; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D014508:Urea / Q000737:chemistry
chemical_list: D017920:Antiporters; D003902:Detergents; D029968:Escherichia coli Proteins; D020404:Glycerophospholipids; D008051:Lipid Bilayers; D008081:Liposomes; D008563:Membrane Lipids; D008823:Micelles; C075436:EmrE protein, E coli; D012967:Sodium Dodecyl Sulfate; D014508:Urea