Topology of the anion-selective porin Omp32 from Comamonas acidovorans.
Limited proteolysis experiments were performed with outer membranes from Comamonas acidovorans to probe the topology of its major protein component, the anion-selective porin Omp32. Proteinase K treatment above a critical temperature of 42 degrees C cleaved the surface-exposed regions of the porin, yielding membrane-embedded fragments which were separated by SDS polyacrylamide gel electrophoresis or reversed phase chromatography. The identification of the proteinase K-sensitive sites was performed by microsequencing. This allowed us to determine six surface-exposed sites of the porin, all located in nonconserved primary structure regions. These results along with the previously determined amino acid sequence and in conjunction with some structural constraints applicable to porins allowed us to propose a chain-folding model of the Omp32 porin. The features of our model are compared with the structure of the Rhodobacter capsulatus porin, recently established by X-ray crystallography (Weiss et al., 1991) and they are used to elucidate the structural basis of the anion selectivity.
Max Planck Institut für Biochemie, Martinsried, Federal Republic of Germany.
S
S
Gerbl-Rieger
H
H
Engelhardt
J
J
Peters
M
M
Kehl
F
F
Lottspeich
W
W
Baumeister
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D001426:Bacterial Proteins; D015153:Blotting, Western; D002152:Calorimetry, Differential Scanning; D002412:Cations; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D006090:Gram-Negative Bacteria / Q000648:ultrastructure*; D007473:Ion Channels / Q000737:chemistry / Q000648:ultrastructure*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D018272:Porins; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000378:metabolism; D013055:Spectrophotometry, Infrared
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002412:Cations; D007473:Ion Channels; C115885:Omp32 protein, bacteria; D010446:Peptide Fragments; D018272:Porins; D012697:Serine Endopeptidases; D019286:Endopeptidase K