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Droplet interface bilayer reconstitution and activity measurement of the mechanosensitive channel of large conductance from Escherichia coli
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Droplet interface bilayer reconstitution and activity measurement of the mechanosensitive channel of large conductance from Escherichia coli..pdf | Published version | 483.76 kB | Adobe PDF | View/Open |
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