Folding and gating of the outer membrane porin PhoE of Escherichia coli

The cell envelope of Gram-negative bacteria, such as Escherichia coli, consists of a double membrane separated by the periplasm. Due to this architecture, the cell can maintain a microenvironment essential for cell viability. Several outer membrane proteins (OMPs), such as PhoE, are implicated in providing the cell of its nutrients. PhoE functions in the outer membrane as a trimeric pore allowing passive diffusion of preferentially anionic compounds with molecular masses up to 600 Da. OMPs are synthesized in the cytoplasm as precursor proteins with an N-terminal extension, the signal sequence. These precursors are translocated across the inner membrane via the Sec machinery, and the signal sequence is cleaved off. In this thesis we addressed the question whether periplasmic intermediates exists in the biogenesis of OMPs. PhoE is used in our laboratory as a model protein to study the biogenesis of OMPs. Artificial disulfide bonds were constructed within and between PhoE monomers, based upon the known 3D-structure. Formation of these disulfide bonds required the presence of the periplamic DsbA and DsbG proteins, respectively, which catalyze disulfide bonds in unfolded and folded structures, respectively. These findings indicated that folding of the monomer and trimer occurs at least partially in the periplsm. Subsequently, it was demonstrated that the periplamic peptidyl-prolyl cis/trans isomerase SurA functions as a chaperone in the biogenesis of PhoE since folding of a PhoE mutant lacking its proline residues still required the presence of SurA. PhoE monomers are foled as a b-barrel with 16-antiparallel b-strands. These strands are connected by short turns at the periplasmic side and long loop at the surface-exposed side of the membrane. The third loop (L3) is folded into the barrel, thereby forming a constriction at half the height of the membrane. The sequence PEFGG at the tip of L3 is highly conserved in a superfamily of bacterial porins. The L3 is involved in voltage-dependent closing of the pore. In this thesis the channel characteristics of mutant PhoE porins in which either the tip of the constriction loop was connected to the barrel wall or residues within the conserved PEFGG sequence were replaced, were determined. The experiments demonstrated that pore closings are not mediated by a gross movement of L3 within the channel, but by more subtle rearrangements, involving only parts of L3 or the side chains of the charged residues wihtin the constriction zone