Biochemical and biophysical characterization of OmpG: A monomeric porin.
A recombinant form of the porin OmpG, OmpGm, lacking the signal sequence, has been expressed in Escherichia coli. After purification under denaturing conditions, the protein was refolded in the detergent Genapol X-080, where it gained a structure rich in beta sheet as evidenced by a CD spectrum similar to that of the native form. Electrophoretic analysis and limited proteolysis experiments suggested that refolded OmpGm exists in at least three forms. Nevertheless, the recombinant protein formed uniform channels in planar bilayers with a conductance of 0.81 nS (1 M NaCl, pH 7.5). Previous biochemical studies had suggested that OmpG is a monomeric porin, rather than the usual trimer. Bilayer recordings substantiated this proposal; voltage-induced closures occurred consistently in a single step, and channel block by Gd(3+) lacked the cooperativity seen with the trimeric porin OmpF. The availability of milligram amounts of a monomeric porin will be useful both for basic studies of porin function and for membrane protein engineering.
Conlan
S
S
Department of Medical Biochemistry & Genetics, The Texas A&M University System Health Science Center, College Station, Texas 77843-1114, USA.
Zhang
Y
Y
Cheley
S
S
Bayley
H
H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics; D002463:Cell Membrane Permeability; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D005682:Gadolinium / Q000737:chemistry; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008715:Methionine / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018272:Porins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000235:genetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; C114347:OmpG protein, E coli; D018272:Porins; D011994:Recombinant Proteins; D008715:Methionine; D005682:Gadolinium