Structure and function of an OmpC deletion mutant porin from Escherichia coli K-12.
Escherichia coli K-12 strain RAM122 contains a mutation in the ompC gene that results in an eight amino acid deletion, delta 103-110, in the porin protein. Since this strain is capable of growing on maltodextrins in the absence of a functional lamB gene, the mutant protein is thought to have a larger channel size. The stability and structure/function properties of the mutant OmpC porin were investigated and compared to wild-type porin. Isolated unheated RAM122 porin was characterized as a trimer on sodium dodecyl sulfate-polyacrylamide gels. The RAM122 trimer was less stable to temperature when compared to the wild-type porin. In addition, the overall enthalpy for thermal denaturation was lower for the mutant than the wild-type porin as determined by using differential scanning microcalorimetry. Both the proteins' secondary structures, monitored by circular dichroism, were high in beta-sheet content, but the spectra were slightly different in their crossover points as well as their minima. When the proteins were reconstituted and channel activity was assayed by using a liposome swelling technique, the size-exclusion limit of the mutant porin was twice that of the wild-type porin. Conductance measurements across bilayer lipid membranes showed that the mutant porin was voltage gated at much lower membrane potentials, 50 and 75 mV, than the wild-type sample. The closing events of the mutant porin were predominantly of monomer size. The channels detected by using the mutant protein were larger in size than those measured for the wild-type porin monomer. These data suggest that the OmpC mutant porin has a channel size capable of allowing maltodextrins to enter and that this channel is highly voltage regulated.
Department of Biochemistry, Michigan State University, East Lansing 48824.
W J
WJ
Rocque
E J
EJ
McGroarty
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002872:Chromosome Deletion; D004594:Electrophysiology; D004926:Escherichia coli / Q000235:genetics* / Q000502:physiology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000502:physiology; D009154:Mutation; D018272:Porins; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D001425:Bacterial Outer Membrane Proteins; D007473:Ion Channels; D018272:Porins