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 per iplasmic 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 funct ion, The loop L3 contains a short alpha-helix and, at the tip of the l oop, a highly conserved PEFGG sequence. The alpha-helix was deleted an d the two glycines in the PEFGG sequence were either replaced by alani nes or deleted, A serine residue, supposed to play an indirect role in the anion selectivity of the pore, was removed, The mutant porins wer e analysed both in vitro and in vivo, The results suggest that flexibi lity of the third loop is important for solute passage and that this f lexibility is determined by the two glycine residues in the PEFGG sequ ence, Furthermore, the alpha-helix is probably important for the foldi ng of the protein, The supposed involvement of Ser115 (Ser121A in OmpF nomenclature) in anion selectivity was confirmed.