title: In vivo and in vitro studies of major surface loop deletion mutants of the Escherichia coli K-12 maltoporin: contribution to maltose and maltooligosaccharide transport and binding.
abstract: The trimeric protein LamB of Escherichia coli K-12 (maltoporin) specifically facilitates the diffusion of maltose and maltooligosaccharides through the outer membrane. Each monomer consists of an 18-stranded antiparallel beta-barrel with nine surface loops (L1 to L9). The effects on transport and binding of the deletion of some of the surface loops or of combinations of several of them were studied in vivo and in vitro. In vivo, single-, DeltaL4, DeltaL5, DeltaL6, and double-loop deletions, DeltaL4 + DeltaL5 and DeltaL5 + DeltaL6, abolished maltoporin functions, but not the double deletion DeltaL4 + DeltaL6 and the triple deletion DeltaL4 + DeltaL5 + DeltaL6. While deletion of the central variable portion of loop L9 (DeltaL9v) affected maltoporin function only moderately, the combination of DeltaL9v with the double deletion of loops L4 and L6 (triple deletion DeltaL4 + DeltaL6 + DeltaL9v) strongly impaired maltoporin function and resulted in sensitivity to large hydrophilic antibiotics without change in channel size as measured in vitro. In vitro, the carbohydrate-binding properties of the different loop mutants were studied in titration experiments using the asymmetric and symmetric addition of the mutant porins and of the carbohydrates to one or both sides of the lipid bilayer membranes. The deletion of loop L9v alone (LamBDeltaL9v), of two loops L4 and L6 (LamBDeltaL4 + DeltaL6), of three loops L4, L5 and L6 (LamBDeltaL4 + DeltaL5 + DeltaL6) or of L4, L6 and L9v (LamBDeltaL4 + DeltaL6 + DeltaL9v) had relatively little influence on the carbohydrate-binding properties of the mutant channels, and they had approximately similar binding properties for carbohydrate addition to both sides compared with only one side. The deletion of one of the loops L4 (LamBDeltaL4) or L6 (LamBDeltaL6) resulted in an asymmetric carbohydrate binding. The in vivo and in vitro results, together with those of the purification across the starch column, suggest that maltooligosaccharides enter the LamB channel from the cell surface side with the non-reducing end in advance. The absence of some of the loops leads to obstruction of the channel from the outside, which results in a considerable difference in the on-rate of carbohydrate binding from the extracellular side compared with that from the periplasmic side.
authors.affiliation: Lehrstuhl für Biotechnologie, Biozentrum der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Andersen
authors.forename: C
authors.initials: C
authors.lastname: Bachmeyer
authors.forename: H
authors.initials: H
authors.lastname: Täuber
authors.forename: R
authors.initials: R
authors.lastname: Benz
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: V
authors.initials: V
authors.lastname: Michel
authors.forename: S M
authors.initials: SM
authors.lastname: Newton
authors.forename: M
authors.initials: M
authors.lastname: Hofnung
authors.forename: A
authors.initials: A
authors.lastname: Charbit
mesh_terms: D001414:Bacitracin / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D001692:Biological Transport; D004553:Electric Conductivity; D004594:Electrophysiology; D004926:Escherichia coli / Q000235:genetics*; D007473:Ion Channels / Q000235:genetics; D007700:Kinetics; D008320:Maltose / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009844:Oligosaccharides / Q000378:metabolism; D018272:Porins / Q000235:genetics; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011991:Receptors, Virus / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D014640:Vancomycin / Q000494:pharmacology
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D007473:Ion Channels; D009844:Oligosaccharides; D018272:Porins; D011991:Receptors, Virus; C016532:maltoporins; D001414:Bacitracin; C019193:maltopentaose; D008320:Maltose; D014640:Vancomycin