Thermodynamic stability and ion-conduction mechanism of the outer membrane protein A from Escherichia coli

The outer membrane protein A (OmpA) is a major outer membrane protein in Gram-negative bacteria composed of an N-terminal transmembrane (residues 1-171) and a C-terminal periplasmic domain (residues 172-325). It is involved in the maintenance of cell shape and forms an ion-channel in planar lipid bilayers. Here, I have developed a fully reversible system to measure the thermodynamic stability of OmpA from Escherichia coli in lipid bilayers. This is the first time fully reversible folding has been demonstrated for any integral membrane protein. Folding is shown to be two-state under appropriate conditions permitting data analysis with a classical folding model developed for water-soluble proteins. By systematic variation of lipid headgroup and chain composition, it is shown that elastic bilayer forces such as curvature stress and hydrophobic mismatch modulate the free energy and cooperativity of folding of this and perhaps many other membrane proteins. The gate of the OmpA ion channel includes a conserved salt-bridge tetrad composed of Glu52, Lys82, Glu128, and Arg138. In order to investigate the channel opening mechanism, the thermodynamic interaction energies of the salt-bridges are obtained using double-mutant cycles. The role of each side-chain in the ion-channel function has also been investigated by single ion channel measurements in planar lipid bilayers. The results suggest that the opening of the OmpA channel is caused by thermal motions of the gating region in mus-ms time scale facilitated by a favorable Glu52-Lys82 interaction that perturbs the obstructing salt-bridge Glu52-R138 in the closed state. The pore activity of OmpA measured in vitro is closely related to the survival of E. coli cells that lack porins under osmotic stress. The E52C/R138C mutant, which presumably forms a covalent disulfide bond in place of the salt-bridge of the wild-type, does not show any ion channel activity and E. coli cells expressing this protein fail to grow under various forms of osmotic stress. The transmembrane domain of OmpA is the minimal functional unit for the osmotic response of the protein. The results suggest that OmpA functions as a biologically active pore to protect E. coli from osmotic stress.