Identification of a novel heterodimeric outer membrane protein of Porphyromonas gingivalis by two-dimensional gel electrophoresis and peptide mass fingerprinting.
Porphyromonas gingivalis is a Gram-negative, anaerobic bacterium associated with chronic periodontitis. A 2D electrophoretic analysis of the outer membrane of P. gingivalis W50 revealed a dominant train of spots at 40-41 kDa. The proteins in the train of spots were digested in-gel with trypsin and identified by MS. The train of spots represented two proteins, designated Omp40 and Omp41 that share 47% sequence identity. Preparation of outer membranes in the absence of protease inhibitors resulted in partial cleavage of Omp40 and Omp41 to produce an N-terminal and C-terminal fragment of both proteins. The N-terminal fragments displayed the same isoelectric heterogeneity as the intact proteins. Almost 100% of the amino-acid sequence of these N-terminal fragments in each 2D gel spot was verified suggesting lack of post-translational modification. Re-subjecting a single N-terminal domain spot to 2D electrophoresis resulted in the complete series of spots being reproduced, suggesting that the heterogeneity was related to conformational equilibria. Under reduced conditions and without heating, Omp40 and Omp41 migrated as 34- to 35-kDa proteins in SDS/PAGE whereas under nonreduced conditions the proteins migrated as 70-kDa proteins, suggesting the formation of dimers through intersubunit disulfide bonds. The proteins each contain two cysteine residues in the conserved sequence RPVSCPECPE. Tryptic peptides generated from the nonreduced forms of the proteins confirmed the presence of heterodimers stabilized through intersubunit disulfide bond formation. With the exception of heterodimer formation, the two proteins share several similarities with OmpA-like porins of other Gram-negative bacteria including consensus sequence, abundance, modification by heat, overall length and positioning of domains.
Veith
P D
PD
School of Dental Science, The University of Melbourne, Victoria, Australia.
Talbo
G H
GH
Slakeski
N
N
Reynolds
E C
EC
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D019281:Dimerization; D015180:Electrophoresis, Gel, Two-Dimensional; D006358:Hot Temperature; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D016966:Porphyromonas gingivalis / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D001425:Bacterial Outer Membrane Proteins; C412104:omp-40 protein, Acidithiobacillus ferrooxidans; C078167:OMPA outer membrane proteins