Rapid screening of membrane protein activity: electrophysiological analysis of OmpF reconstituted in proteoliposomes.
Solvent-free planar lipid bilayers were formed in an automatic manner by bursting of giant unilamellar vesicles (GUVs) after gentle suction application through micron-sized apertures in a borosilicate glass substrate. Incubation of GUVs with the purified ion channel protein of interest yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. This approach reduces the time-consuming, laborious and sometimes difficult protein reconstitution processes normally performed after bilayer formation. Bilayer recordings are attractive for investigations of membrane proteins not accessible to patch clamp analysis, like e.g. proteins from organelles. In the presented work, we show the example of the outer membrane protein OmpF from Escherichia coli. We reconstituted OmpF in proteoliposomes and observed the characteristic trimeric conductance levels and the typical gating induced by pH and transmembrane voltage. Moreover, OmpF is the main entrance for beta-lactam antibiotics and we investigated translocation processes of antibiotics and modulation of OmpF by spermine. We suggest that the rapid formation of porin containing lipid bilayers is of potential for the efficient electrophysiological characterization of the OmpF protein, for studying membrane permeation processes and for the rapid screening of antibiotics.
Kreir
Mohamed
M
Nanion Technologies GmbH, Erzgiessereistr., 4, 80335, Munich, Germany.
Farre
Cecilia
C
Beckler
Matthias
M
George
Michael
M
Fertig
Niels
N
D000900:Anti-Bacterial Agents / Q000032:analysis / Q000378:metabolism*; D002463:Cell Membrane Permeability; D004594:Electrophysiology / Q000379:methods*; D005898:Glass / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000737:chemistry*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D018272:Porins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D011510:Proteolipids / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry
D000900:Anti-Bacterial Agents; D008051:Lipid Bilayers; D008565:Membrane Proteins; C109237:OmpF protein; D018272:Porins; D011510:Proteolipids; D012997:Solvents; C020485:proteoliposomes