PMID:12899633
| Citation |
Conlan, S and Bayley, H (2003) Folding of a monomeric porin, OmpG, in detergent solution. Biochemistry 42:9453-65 |
|---|---|
| Abstract |
OmpG, a porin from E. coli, has been examined in planar lipid bilayers and in detergent solution. First, bilayer recordings were used to reinforce the evidence that the functional form of OmpG is a monomer. Both pH-dependent gating and blockade by covalent modification add support to this proposal. The findings contrast with the properties of the classical porins, which function as trimers. Second, the folding of OmpG in detergent solution was examined. A water-soluble form of OmpG was obtained by dialysis from denaturant into buffer. Incubation of water-soluble OmpG in detergent results in conversion to a form that possesses the hallmarks of a beta barrel. The folding of water-soluble OmpG in detergent was monitored by circular dichroism, protease resistance, and heat modifiability. OmpG is first transformed into an intermediate with increased beta-sheet content on the time scale of minutes at 23 degrees C. This is followed by the slow acquisition of heat modifiability and protease resistance over several hours. The formation of a beta barrel during this period was demonstrated in a double cysteine mutant by using intramolecular disulfide bond formation to report N and C terminus proximity. Finally, conditions are presented for folding OmpG with greater than 90% efficiency, thereby paving the way for structural studies. |
| Links |
PubMed Online version:10.1021/bi0344228 |
| Keywords |
Anilino Naphthalenesulfonates/diagnostic use; Bacterial Outer Membrane Proteins; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Cell Membrane/chemistry; Cell Membrane/genetics; Cell Membrane Permeability; Chromatography, Gel; Circular Dichroism; Cloning, Molecular; Cysteine/genetics; Detergents/chemistry; Detergents/metabolism; Escherichia coli/chemistry; Escherichia coli Proteins; Ion Channels/chemistry; Ion Channels/genetics; Ion Channels/metabolism; Lipid Bilayers/chemistry; Lipid Bilayers/metabolism; Models, Molecular; Mutagenesis, Site-Directed; Mutation/genetics; Oxidation-Reduction; Porins/chemistry; Porins/genetics; Porins/metabolism; Protein Denaturation; Protein Folding; Protein Structure, Secondary/genetics; Recombinant Proteins/antagonists & inhibitors; Recombinant Proteins/biosynthesis; Recombinant Proteins/chemistry; Recombinant Proteins/isolation & purification; Tryptophan |
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