title: Probing the structural role of an alpha beta loop of maltose-binding protein by mutagenesis: heat-shock induction by loop variants of the maltose-binding protein that form periplasmic inclusion bodies.
abstract: The maltose-binding protein (MBP) of Escherichia coli is the periplasmic receptor of the maltose transport system. Previous studies have identified amino acid substitutions in an alpha/beta loop of the structure of MBP that are critical for the in vivo folding. To probe genetically the structural role of this surface loop, we generated a library in which the corresponding codons 32 and 33 of malE were mutagenized. The maltose phenotype, which correlates with a biologically active structure of MBP in the periplasm, indicated a considerable variability in the loop residues compatible with a correct in vivo folding pathway of the protein. By the same genetic screens, we characterized loop-variant MBPs associated with a defective periplasmic folding pathway and aggregated into inclusion bodies. Heat-shock induction with production of misfolded loop variants was examined using both lon-lacZ and htrA-lacZ fusions. We found that the extent of formation of inclusion bodies in the periplasm of E. coli, from misfolded loop variant MBPs, correlated with the level of heat-shock response regulated by the alternate heat-shock sigma factor, sigma 24.
authors.affiliation: Départment des Biotechnologies, Institut Pasteur, Paris, France.
authors.forename: J M
authors.initials: JM
authors.lastname: Betton
authors.forename: D
authors.initials: D
authors.lastname: Boscus
authors.forename: D
authors.initials: D
authors.lastname: Missiakas
authors.forename: S
authors.initials: S
authors.lastname: Raina
authors.forename: M
authors.initials: M
authors.lastname: Hofnung
mesh_terms: D018528:ATP-Binding Cassette Transporters; D049069:ATP-Dependent Proteases; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D016208:Databases, Factual; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism; D006358:Hot Temperature; D002479:Inclusion Bodies / Q000737:chemistry* / Q000648:ultrastructure; D007763:Lac Operon; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins; D033903:Periplasmic Proteins; D049070:Protease La; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D018528:ATP-Binding Cassette Transporters; C016242:AraF protein, E coli; D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; C024913:LivK protein, E coli; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D033903:Periplasmic Proteins; C028003:RbsB protein, E coli; C023550:galactose-binding protein; C018963:histidine-binding protein; C033849:lysyl-arginyl-ornithine-binding protein, bacteria; C461599:maltose transport system, E coli; C489040:sbp protein, E coli; C044773:sulfate-binding protein, bacteria; D049069:ATP-Dependent Proteases; C061682:DegP protease; D012697:Serine Endopeptidases; C483567:Lon protein, E coli; D049070:Protease La