title: Deletion of a single helix from the transmembrane domain causes large changes in membrane insertion properties and secondary structure of the bacterial conjugation protein TrwB.
abstract: TrwB is an essential protein in the conjugative transfer of plasmid R388. The protein consists of a bulky cytosolic domain containing the catalytic site, and a small transmembrane domain (TMD). Our previous studies support the idea that the TMD plays an essential role in the activity, structure and stability of the protein. We have prepared a mutant, TrwBΔN50 that lacks one of the two α-helices in the TMD. The mutant has been studied both in detergent suspension and reconstituted in lipid vesicles. Deletion of a single helix from the TMD is enough to increase markedly the affinity of TrwB for ATP. The deletion changes the secondary structure of the cytosolic domain, whose infrared spectroscopy (IR) spectra become similar to those of the mutant TrwBΔN70 lacking the whole TMD. Interestingly, when TrwBΔN50 is reconstituted into lipid membranes, the cytosolic domain orients itself towards the vesicle interior, opposite to what happens for wild-type TrwB. In addition, we analyze the secondary structure of the TMD and TMD-lacking mutant TrwBΔN70, and found that the sum IR spectrum of the two protein fragments is different from that of the native protein, indicating the irreversibility of changes caused in TrwB by deletion of the TMD.
authors.affiliation: Unidad de Biofísica, Universidad del País Vasco, Aptdo. 644, 48080 Bilbao, Spain.
authors.forename: Ana Julia
authors.initials: AJ
authors.lastname: Vecino
authors.forename: Rosa de Lima
authors.initials: Rde L
authors.lastname: Segura
authors.forename: Igor
authors.initials: I
authors.lastname: de la Arada
authors.forename: Fernando
authors.initials: F
authors.lastname: de la Cruz
authors.forename: Félix M
authors.initials: FM
authors.lastname: Goñi
authors.forename: José L
authors.initials: JL
authors.lastname: Arrondo
authors.forename: Itziar
authors.initials: I
authors.lastname: Alkorta
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics*; D008051:Lipid Bilayers; D008081:Liposomes; D008563:Membrane Lipids / Q000378:metabolism*; D009154:Mutation; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D008081:Liposomes; D008563:Membrane Lipids; C006094:R388; C403125:trwB protein, E coli; D013762:Tetrahydrofolate Dehydrogenase