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Droplet interface bilayer reconstitution and activity measurement of the mechanosensitive channel of large conductance from Escherichia coli

Title: Droplet interface bilayer reconstitution and activity measurement of the mechanosensitive channel of large conductance from Escherichia coli
Authors: Barriga, HMG
Booth, P
Haylock, S
Bazin, R
Templer, RH
Ces, O
Item Type: Journal Article
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.
Issue Date: 6-Sep-2014
Date of Acceptance: 13-Jun-2014
URI: http://hdl.handle.net/10044/1/19490
DOI: 10.1098/rsif.2014.0404
ISSN: 1742-5662
Publisher: The Royal Society
Journal / Book Title: Journal of the Royal Society Interface
Volume: 11
Issue: 98
Copyright Statement: © 2014 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited.
Sponsor/Funder: Engineering & Physical Science Research Council (EPSRC)
Engineering & Physical Science Research Council (EPSRC)
Funder's Grant Number: EP/G00465X/1
EP/K039946/1
Keywords: Science & Technology
Multidisciplinary Sciences
Science & Technology - Other Topics
droplet interface bilayers
mechanosensitive channel of large conductance
1,2-dioleoyl-sn-glycero-3-phosphocholine
fluorescence
DIACYLGLYCEROL KINASE
IN-VITRO
MEMBRANE
Science & Technology
Multidisciplinary Sciences
Science & Technology - Other Topics
droplet interface bilayers
mechanosensitive channel of large conductance
1,2-dioleoyl-sn-glycero-3-phosphocholine
fluorescence
DIACYLGLYCEROL KINASE
IN-VITRO
MEMBRANE
1,2-dioleoyl-sn-glycero-3-phosphocholine
droplet interface bilayers
fluorescence
mechanosensitive channel of large conductance
Escherichia coli
Escherichia coli Proteins
Fluorescent Dyes
Glycerylphosphorylcholine
Ion Channels
Lipid Bilayers
Lipids
Mesylates
Phosphatidylcholines
Escherichia coli
Mesylates
Glycerylphosphorylcholine
Lipids
Lipid Bilayers
Phosphatidylcholines
Escherichia coli Proteins
Ion Channels
Fluorescent Dyes
General Science & Technology
Publication Status: Published
Article Number: ARTN 20140404
Online Publication Date: 2014-09-06
Appears in Collections:Chemistry
Biological and Biophysical Chemistry
Faculty of Natural Sciences



This item is licensed under a Creative Commons License Creative Commons