In vitro unfolding and refolding of the small multidrug transporter EmrE

David Miller, Kalypso Charalambous, Dvir Rotem, Shimon Schuldiner, Paul Curnow, Paula J Booth

Research output: Contribution to journalArticle (Academic Journal)peer-review

52 Citations (Scopus)

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.
Translated title of the contributionIn vitro unfolding and refolding of the small multidrug transporter ErmE
Original languageEnglish
Pages (from-to)815 - 832
Number of pages18
JournalJournal of Molecular Biology
Volume393
Issue number4
DOIs
Publication statusPublished - Nov 2009

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