Orientation of the monomeric porin OmpG in planar lipid bilayers.
Outer membrane protein G (OmpG) is a non-selective porin from Escherichia coli. OmpG is a monomer, which makes it unusual among porins, and suggests that it may be useful in biotechnology. In planar lipid bilayers, individual OmpG pores reconstituted by insertion from detergent exhibit pronounced asymmetry in current-voltage relationships and voltage-dependent gating. Here, this asymmetry is used to deduce the orientation of OmpG in the bilayers. We introduced two cysteines into the extracellular loops of OmpG. Cleavage of the disulfide bond formed by these residues significantly increases spontaneous gating of the pore. By adding DTT to one side of the bilayer or the other, we demonstrated that pores showing a quiet trace at negative potentials have a "trans" conformation (extracellular loops on the trans side of the bilayer), while pores showing a quiet trace at positive potentials have a "cis" conformation (extracellular loops on the cis side). With this knowledge, we examined the binding of a cyclodextrin to OmpG. When the cyclodextrin was presented to the extracellular face of the pore, transient multisite interactions were observed. In contrast, when the cyclodextrin was presented to the periplasmic face, a more stable single-site interaction occurred. Because the cyclodextrin can act as a molecular adapter by binding analytes, this information serves to advance the use of OmpG as a biosensor.
Chen
Min
M
Department of Chemistry, University of Oxford, Oxford, UK.
Li
Qiu-Hong
QH
Bayley
Hagan
H
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003505:Cyclodextrins / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004229:Dithiothreitol / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D018272:Porins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D050994:Voltage-Dependent Anion Channels
D001425:Bacterial Outer Membrane Proteins; D003505:Cyclodextrins; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; C114347:OmpG protein, E coli; D018272:Porins; D050994:Voltage-Dependent Anion Channels; D003545:Cysteine; D004229:Dithiothreitol