title: Droplet interface bilayer reconstitution and activity measurement of the mechanosensitive channel of large conductance from Escherichia coli.
abstract: Droplet interface bilayers (DIBs) provide an exciting new platform for the study of membrane proteins in stable bilayers of controlled composition. To date, the successful reconstitution and activity measurement of membrane proteins in DIBs has relied on the use of the synthetic lipid 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). We report the functional reconstitution of the mechanosensitive channel of large conductance (MscL) into DIBs composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a lipid of significantly greater biological relevance than DPhPC. MscL functionality has been demonstrated using a fluorescence-based assay, showing that dye flow occurs across the DIB when MscL is gated by the cysteine reactive chemical 2-(trimethylammonium)ethyl methane thiosulfonate bromide (MTSET). MscL has already been the subject of a number of studies investigating its interaction with the membrane. We propose that this method will pave the way for future MscL studies looking in detail at the effects of controlled composition or membrane asymmetry on MscL activity using biologically relevant lipids and will also be applicable to other lipid-protein systems, paving the way for the study of membrane proteins in DIBs with biologically relevant lipids.
authors.affiliation: Membrane Biophysics Platform, Institute of Chemical Biology and Department of Chemistry, Imperial College London, South Kensington, London SW7 2AX, UK hanna.barriga@imperial.ac.uk.
authors.forename: Hanna M G
authors.initials: HM
authors.lastname: Barriga
authors.affiliation: Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Paula
authors.initials: P
authors.lastname: Booth
authors.affiliation: Membrane Biophysics Platform, Institute of Chemical Biology and Department of Chemistry, Imperial College London, South Kensington, London SW7 2AX, UK.
authors.forename: Stuart
authors.initials: S
authors.lastname: Haylock
authors.affiliation: Pfizer Global Research and Development, Sandwich CT13 9NJ, UK.
authors.forename: Richard
authors.initials: R
authors.lastname: Bazin
authors.affiliation: Membrane Biophysics Platform, Institute of Chemical Biology and Department of Chemistry, Imperial College London, South Kensington, London SW7 2AX, UK.
authors.forename: Richard H
authors.initials: RH
authors.lastname: Templer
authors.affiliation: Membrane Biophysics Platform, Institute of Chemical Biology and Department of Chemistry, Imperial College London, South Kensington, London SW7 2AX, UK o.ces@imperial.ac.uk.
authors.forename: Oscar
authors.initials: O
authors.lastname: Ces
mesh_terms: D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D005997:Glycerylphosphorylcholine / Q000031:analogs & derivatives / Q000737:chemistry; D007473:Ion Channels / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D008055:Lipids / Q000737:chemistry; D008698:Mesylates / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D007473:Ion Channels; D008051:Lipid Bilayers; D008055:Lipids; D008698:Mesylates; C086596:MscL protein, E coli; D010713:Phosphatidylcholines; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; C021016:1,2-diphytanoylphosphatidylcholine; D005997:Glycerylphosphorylcholine; C017251:1,2-oleoylphosphatidylcholine
keywords: 1,2-dioleoyl-sn-glycero-3-phosphocholine; droplet interface bilayers; fluorescence; mechanosensitive channel of large conductance