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

Autor: Richard Bazin, Stuart Haylock, Richard H. Templer, Hanna M. G. Barriga, Oscar Ces, Paula J. Booth
Přispěvatelé: Engineering & Physical Science Research Council (EPSRC)
Rok vydání: 2014
Předmět:
DIACYLGLYCEROL KINASE
General Science & Technology
droplet interface bilayers
Lipid Bilayers
Biomedical Engineering
Biophysics
Bioengineering
02 engineering and technology
Biochemistry
Ion Channels
Biomaterials
03 medical and health sciences
Escherichia coli
Lipid bilayer
Ion channel
Fluorescent Dyes
030304 developmental biology
Diacylglycerol kinase
Mesylates
0303 health sciences
Science & Technology
1
2-dioleoyl-sn-glycero-3-phosphocholine
Chemistry
Escherichia coli Proteins
Bilayer
fungi
mechanosensitive channel of large conductance
technology
industry
and agriculture
Conductance
IN-VITRO
021001 nanoscience & nanotechnology
Glycerylphosphorylcholine
Lipids
Multidisciplinary Sciences
Membrane
Membrane protein
Phosphatidylcholines
Science & Technology - Other Topics
lipids (amino acids
peptides
and proteins)
Mechanosensitive channels
fluorescence
MEMBRANE
0210 nano-technology
Reports
Biotechnology
Zdroj: Journal of the Royal Society Interface
ISSN: 1742-5662
1742-5689
DOI: 10.1098/rsif.2014.0404
Popis: 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.
Databáze: OpenAIRE
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