The DNA sequence of the gene which codes for the major outer membrane porin (Omp32) of Comamonas acidovorans has been determined. The structural gene encodes a precursor consisting of 351 amino acid residues with a signal peptide of 19 amino acid residues. Comparisons with amino acid sequences of outer membrane proteins and porins from several other members of the class Proteobacteria and of the Chlamydia trachomatis porin and the Neurospora crassa mitochondrial porin revealed a motif of eight regions of local homology. The results of this analysis are discussed with regard to common structural features of porins.Comamonas acidovorans is a gram-negative eubacterium belonging to the a subdivision of the class Proteobacteria. It possesses an outer membrane which is relatively simple in protein composition and therefore amenable to a comprehensive structural and functional characterization. Tightly associated with the outer membrane is a regularly arrayed surface protein forming a simple cobbelstone pattern with p4 symmetry (7,29). The outer membrane proper contains one major polypeptide species (32 kDa) together with two minor polypeptides (37 and 21 kDa). The solubilization behavior, the temperature dependence of sodium dodecyl sulfate (SDS)-polyacrylamide gels, and the secondary structure (approximately 70% 1-sheet) of the predominant 32-kDa polypeptide (Omp32) turned out to be highly reminiscent of some bona fide porins (47). Recent black lipid membrane experiments have confirmed that Omp32 is indeed a porin which forms anion selective channels (17a with Triton X-100 and density centrifugation contained two major proteins, the surface layer protein and the 32-kDa protein (Omp32); two further minor proteins not yet characterized (approximately 37 and 21 kDa) appear to be tightly associated with such outer membrane preparations.Isolation of Omp32 and the 21-kDa protein by size exclusion high-pressure liquid chromatography. Samples were separated on a TSK 3000 SW column (LKB Instruments, Inc., Rockville, Md.) in 70% formic acid at a protein concentration of 5 ,ug/,ul. The solvent system used was 0.1% trifluoroacetic acid (TFA)-30% acetonitrile at a flow rate of 1 ml/min. Detection was performed at 206 nm. The proteincontaining fractions were lyophilized in the presence of SDS.Peptide cleavage procedures. Before cleavage, Omp32 was solubilized in 2% SDS at 85°C for 10 min. Proteolytic cleavage with LysC proteinase (Boehringer Mannheim) (26) was carried out in 50 mM phosphate-buffered saline (PBS), pH 7.8-0.5% SDS at 37°C for 40 h with an enzyme-to-protein ratio of 1:40 (wt/wt). Proteolytic cleavage with GluC proteinase (Boehringer Mannheim) (23) was performed in 50 mM PBS buffer, pH 7.8. The proteinase was added in three equal aliquots after intervals of heating the digest to 100°C for 5 min and cooling to 37°C. The final enzyme-to-protein ratio was 1:10, and the incubation was done at 30°C for 10 to 20 h.
The three-dimensional structure of the regular surface protein (p4 symmetry, lattice constant a = b = 10.5 nm) of Comamonas acidovorans has been determined to a resolution of about 1.5 nm by means of electron microscopy and image processing. Three-dimensional reconstructions were performed using native outer membranes and artificial two-dimensional crystals of the surface protein, which was selectively solubilized by deoxycholate and recrystallized on carbon films. The two-fold symmetric morphological complex is composed of two identical monomers which are in tight contact with the outer membrane and presumably anchored to it by a small hydrophobic domain.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.