In vivo and in vitro studies of transmembrane beta-strand deletion, insertion or substitution mutants of the Escherichia coli K-12 maltoporin.
LamB of Escherichia coli K12, also called maltoporin, is an outer membrane protein, which specifically facilitates the diffusion of maltose and maltodextrin through the bacterial outer membrane. Each monomer is composed of an 18-stranded antiparallel beta-barrel. In the present work, on the basis of the known X-ray structure of LamB, the effects of modifications of the beta-barrel domain of maltoporin were studied in vivo and in vitro. We show that: (i) the substitution of the pair of strands beta13-beta14 of the E. coli maltoporin with the corresponding pair of strands from the functionally related maltoporin of Salmonella typhimurium yielded a protein active in vivo and in vitro; and (ii) the removal of one pair of beta-strands (deletion beta13-beta14) from the E. coli maltoporin, or its replacement by a pair of strands from the general porin OmpF of E. coli, leads to recombinant proteins that lost in vivo maltoporin activities but still kept channel formation and carbohydrate binding in vitro. We also inserted into deletion beta13-beta14 the portion of the E. coli LamB protein comprising strands beta13 to beta16. This resulted in a protein expected to have 20 beta-strands and which completely lost all LamB-specific activities in vivo and in vitro.
Charbit
A
A
Unité de Programmation Moléculaire and Toxicologie Génétique - CNRS URA1444, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France. charbit@necker.fr
Andersen
C
C
Wang
J
J
Schiffler
B
B
Michel
V
V
Benz
R
R
Hofnung
M
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D050260:Carbohydrate Metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D008320:Maltose / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism; D018272:Porins; D011485:Protein Binding; D017510:Protein Folding; D011991:Receptors, Virus / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012486:Salmonella typhimurium / Q000235:genetics; D017384:Sequence Deletion; D014312:Trisaccharides / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; D009844:Oligosaccharides; D018272:Porins; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D014312:Trisaccharides; C016532:maltoporins; C019193:maltopentaose; C008317:maltotriose; D008320:Maltose