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Cell surface exposure of the outer membrane protein OmpA of Escherichia coli K-12
Abstract:
The 325-residue OmpA protein is one of the major outer membrane proteins of Escherichia coli K-12. A model, in which this protein crosses the membrane eight times in an antiparallel beta-sheet conformation and in which regions around amino acids 25, 70, 110 and 154 are exposed at the cell surface, had been proposed. Linkers were inserted into the ompA gene with the result that OmpA proteins, carrying non-OmpA sequences between residues 153 and 154 or 160 and 162, were synthesized. Intact cells possessing these proteins were treated with proteases. Insertion of 15 residues between residues 153 and 154 made the protein sensitive to proteinase K and the sizes of the two cleavage products were those expected following proteolysis at the area of the insertion. Addition of at least 17 residues between residues 160 and 162 left the protein completely refractory to protease action. Thus, the former area is cell surface exposed while the latter area appears not to be. The insertions did not cause a decrease in the concentration of the hybrid proteins as compared to that of the OmpA protein, and in neither case was synthesis of the protein deleterious to cell growth. It is suggested that this method may serve to carry peptides of practical interest to the cell surface and that it can be used to probe surface-located regions of other membrane proteins.
Insights
Researchers modified the OmpA protein in Escherichia coli by inserting new sequences. This revealed specific surface-exposed regions of the outer membrane protein, aiding in the study of bacterial cell surfaces.
Area of Science:
- Microbiology
- Molecular Biology
- Protein Engineering
Background:
- The outer membrane protein A (OmpA) of Escherichia coli is a major structural component.
- A proposed model suggests OmpA spans the outer membrane eight times with surface-exposed regions.
Purpose of the Study:
- To experimentally verify the surface exposure of specific regions within the OmpA protein.
- To develop a method for probing surface-located regions of membrane proteins and displaying peptides.
Main Methods:
- Genetic engineering of the ompA gene to introduce linker sequences at specific sites (residues 153-154 and 160-162).
- Synthesis of hybrid OmpA proteins containing non-OmpA sequences.
- Protease sensitivity assays on intact bacterial cells expressing the modified OmpA proteins.
Main Results:
- Insertion of 15 residues between amino acids 153 and 154 rendered OmpA sensitive to proteinase K, indicating surface exposure.
- Insertion of at least 17 residues between amino acids 160 and 162 resulted in protease resistance, suggesting this region is not surface-exposed.
- Hybrid protein expression did not affect cell growth or protein concentration compared to wild-type OmpA.
Conclusions:
- The region around amino acid 153-154 of OmpA is accessible on the bacterial cell surface.
- The region around amino acid 160-162 is likely located within the membrane or periplasm.
- This linker-insertion strategy is a viable method for probing membrane protein topology and displaying functional peptides on the bacterial cell surface.