Folding of a Monomeric Porin, OmpG, in Detergent Solution

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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 β 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 β-sheet content on the time scale of minutes at 23 °C. This is followed by the slow acquisition of heat modifiability and protease resistance over several hours. The formation of a β 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.