Isolation and characterization of the Omp-PA porin from Porphyromonas asaccharolytica, determination of the omp-PA gene sequence and prediction of Omp-PA protein structure.
A single monomeric porin, Omp-PA (37kDa), was isolated from the outer membrane of the gram-negative anaerobic rod Porphyromonas asaccharolytica. Further characterization revealed that this porin consists of two different fractions: a heat-modifiable fraction which in its denatured form migrated on SDS-PAGE as a protein with a molecular weight of 41kDa and a heat-resistant fraction which did not change its migration on SDS-PAGE after boiling. A liposome swelling assay revealed that only the heat-resistant fraction was able to transport sugars after its incorporation into the liposomes, although it did not discriminate between differently sized sugars. We hypothesize that the heat-modifiable fraction corresponds to the "closed" conformer of Omp-PA, whereas the heat-resistant fraction corresponds to the "open" conformer of the protein. Cloning of the omp-PA gene revealed an open reading frame of 1161 bases, with a predicted protein sequence of 387 amino acids. The mature protein consists of 366 amino acids with a calculated MW of 41,102Da and an estimated pI of 7.24. The C-terminal domain of Omp-PA is homologous to the characteristic OmpA signature domain (71% similarity with the OmpA consensus domain). Sequence comparison with other anaerobes from the Bacteroides family demonstrated homology across the entire ORF. Digestion of the P. asaccharolytica outer membrane analysis of trypsin-digested Omp-PA yielded two proteins migrating with apparent molecular weights of 37 and 27kDa. These data fully supported our hypothesis that the C-terminal domain of the two-domain "closed" conformer of Omp-PA was digested by trypsin, whereas the single domain beta-barrel "open" conformer was inaccessible to trypsin.
Magalashvili
Lana
L
The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.
Pechatnikov
Izabella
I
Wexler
Hannah M
HM
Nitzan
Yeshayahu
Y
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001439:Bacteroides / Q000235:genetics; D050260:Carbohydrate Metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D007526:Isoelectric Point; D008081:Liposomes; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D018272:Porins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D017861:Porphyromonas / Q000235:genetics*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D008081:Liposomes; D018272:Porins; D014357:Trypsin