期刊
JOURNAL OF THE ROYAL SOCIETY INTERFACE
卷 11, 期 98, 页码 -出版社
ROYAL SOC
DOI: 10.1098/rsif.2014.0404
关键词
droplet interface bilayers; mechanosensitive channel of large conductance; 1,2-dioleoyl-sn-glycero-3-phosphocholine; fluorescence
资金
- EPSRC grant [EP/G00465X/1]
- BBSRC [BB/F0131674/1]
- Pfizer Global Research and Development
- EPSRC Centre for Doctoral Training Studentship from the Institute of Chemical Biology (Imperial College London) [EP/F500076/1]
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [0943383] Funding Source: National Science Foundation
- EPSRC [EP/G00465X/1, EP/K039946/1] Funding Source: UKRI
- Engineering and Physical Sciences Research Council [1375479, EP/K039946/1, EP/G00465X/1] Funding Source: researchfish
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.
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