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

TY - JOUR

T1 - Droplet interface bilayer reconstitution and activity measurement of the mechanosensitive channel of large conductance from Escherichia coli

AU - Barriga, Hanna M. G.

AU - Booth, Paula

AU - Haylock, Stuart

AU - Bazin, Richard

AU - Templer, Richard H.

AU - Ces, Oscar

PY - 2014/9/6

Y1 - 2014/9/6

N2 - Droplet interface bilayers (DIBs) provide an exciting newplatformfor 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.

AB - Droplet interface bilayers (DIBs) provide an exciting newplatformfor 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.

KW - droplet interface bilayers

KW - mechanosensitive channel of large conductance

KW - 1,2-dioleoyl-sn-glycero-3-phosphocholine

KW - fluorescence

KW - MEMBRANE-PROTEIN

KW - DIACYLGLYCEROL KINASE

KW - IN-VITRO

KW - MSCL

KW - NETWORKS

KW - LIPIDS

U2 - 10.1098/rsif.2014.0404

DO - 10.1098/rsif.2014.0404

M3 - Article

SN - 1742-5689

VL - 11

JO - Journal Of The Royal Society Interface

JF - Journal Of The Royal Society Interface

IS - 98

M1 - 20140404

ER -