title: Membrane insertion stabilizes the structure of TrwB, the R388 conjugative plasmid coupling protein.
abstract: TrwB is an integral membrane protein that plays a crucial role in the conjugative process of plasmid R388. We have recently shown [Vecino et al., Biochim. Biophys. Acta 1798(11), 2160-2169 (2010)] that TrwB can be reconstituted into liposomes, and that bilayer incorporation increases its affinity for nucleotides and its specificity for ATP. In the present contribution we examine the structural effects of membrane insertion on TrwB, by comparing the protein in reconstituted form and in the form of protein/lipid/detergent mixed micelles. TrwB was reconstituted in PE:PG:CL (76.3:19.6:4.1mol ratio) with a final 99:1 lipid:protein mol ratio. This lipid mixture is intended to mimic the bacterial inner membrane composition, and allows a more efficient reconstitution than other lipid mixtures tested. The studies have been carried out mainly using infrared spectroscopy, because this technique provides simultaneously information on both the lipid and protein membrane components. Membrane reconstitution of TrwB is accompanied by a decrease in β-sheet contents and an increase in β-strand structures, probably related to protein-protein contacts in the bilayer. The predominant α-helical component remains unchanged. The bilayer-embedded protein becomes thermally more stable, and also more resistant to trypsin digestion. The properties of the bilayer lipids are also modified in the presence of TrwB, the phospholipid acyl chains are slightly ordered, and the phosphate groups at the interface become more accessible to water. In addition, we observe that the protein thermal denaturation affects the lipid thermal transition profile.
authors.affiliation: Departamento de Bioquímica, Universidad del País Vasco, 48080 Bilbao, Spain.
authors.forename: Ana J
authors.initials: AJ
authors.lastname: Vecino
authors.forename: Igor
authors.initials: I
authors.lastname: de la Arada
authors.forename: Rosa L
authors.initials: RL
authors.lastname: Segura
authors.forename: Félix M
authors.initials: FM
authors.lastname: Goñi
authors.forename: Fernando
authors.initials: F
authors.lastname: de la Cruz
authors.forename: José L R
authors.initials: JL
authors.lastname: Arrondo
authors.forename: Itziar
authors.initials: I
authors.lastname: Alkorta
mesh_terms: D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D055550:Protein Stability; D017433:Protein Structure, Secondary; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D008081:Liposomes; D008563:Membrane Lipids; D008565:Membrane Proteins; C403125:trwB protein, E coli; D014357:Trypsin