Pore functioning of outer membrane protein PhoE of Escherichia coli: mutagenesis of the constriction loop L3.
Each monomer of the trimeric outer membrane porin PhoE of Escherichia coli consists of a 16-stranded beta-barrel with short turns at the periplasmic side and large loops at the cell surface. One of these loops, L3, is folded inside the beta-barrel and forms a constriction within the channel. Therefore, it is assumed to play an important role in the permeability properties of this general diffusion pore. Several site-directed mutations were introduced in loop L3 to investigate its function. The loop L3 contains a short alpha-helix and, at the tip of the loop, a highly conserved PEFGG sequence. The alpha-helix was deleted and the two glycines in the PEFGG sequence were either replaced by alanines or deleted. A serine residue, supposed to play an indirect role in the anion selectivity of the pore, was removed. The mutant porins were analysed both in vitro and in vivo. The results suggest that flexibility of the third loop is important for solute passage and that this flexibility is determined by the two glycine residues in the PEFGG sequence. Furthermore, the alpha-helix is probably important for the folding of the protein. The supposed involvement of Ser115 (Ser121A in OmpF nomenclature) in anion selectivity was confirmed.
Van Gelder
P
P
Department of Molecular Cell Biology, Institute of Biomembranes, Utrecht University, The Netherlands.
Saint
N
N
van Boxtel
R
R
Rosenbusch
J P
JP
Tommassen
J
J
D000900:Anti-Bacterial Agents / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D007473:Ion Channels / Q000378:metabolism; D007769:Lactams; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed; D018272:Porins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017510:Protein Folding
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D007473:Ion Channels; D007769:Lactams; D018272:Porins; C082922:PhoE protein, E coli