PMID:9250673
| Citation |
Eppens, EF, Nouwen, N and Tommassen, J (1997) Folding of a bacterial outer membrane protein during passage through the periplasm. EMBO J. 16:4295-301 |
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| Abstract |
The transport of bacterial outer membrane proteins to their destination might be either a one-step process via the contact zones between the inner and outer membrane or a two-step process, implicating a periplasmic intermediate that inserts into the membrane. Furthermore, folding might precede insertion or vice versa. To address these questions, we have made use of the known 3D-structure of the trimeric porin PhoE of Escherichia coli to engineer intramolecular disulfide bridges into this protein at positions that are not exposed to the periplasm once the protein is correctly assembled. The mutations did not interfere with the biogenesis of the protein, and disulfide bond formation appeared to be dependent on the periplasmic enzyme DsbA, which catalyzes disulfide bond formation in the periplasm. This proves that the protein passes through the periplasm on its way to the outer membrane. Furthermore, since the disulfide bonds create elements of tertiary structure within the mutant proteins, it appears that these proteins are at least partially folded before they insert into the outer membrane. |
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| Keywords |
Blotting, Western; Cell Membrane/metabolism; Cloning, Molecular; Cysteine/genetics; Disulfides/chemistry; Disulfides/metabolism; Electrophoresis, Polyacrylamide Gel; Escherichia coli/chemistry; Escherichia coli Proteins; Isomerases/metabolism; Mutagenesis, Site-Directed; Porins/chemistry; Porins/genetics; Porins/metabolism; Protein Disulfide-Isomerases; Protein Engineering; Protein Folding; Protein Structure, Tertiary; Trypsin/metabolism |
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