Membrane insertion stabilizes the structure of TrwB, the R388 conjugative plasmid coupling protein

Ana J. Vecino, Igor de la Arada, Rosa L. Segura, Félix M. Goñi, Fernando de la Cruz, José L.R. Arrondo and Itziar Alkorta

^a Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU), and Departamento de Bioquímica, Universidad del País Vasco, Aptdo. 644, 48080 Bilbao, Spain

^b Departamento de Biología Molecular, Universidad de Cantabria, C/Herrera Oria s/n, 39011 Santander, Spain 

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 (76.3:19.6:4.1 mol 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.

Research Highlights

► The α-helical component of TrwB remains unchanged when inserted into the bilayer. ► Incorporation of TrwB into lipid bilayers causes an increase in β-strand structures. ► β-strand structures are probably related to protein–protein contacts in the bilayer. ► TrwB adopts a more stable conformation when reconstituted into liposomes. ► Incorporation of TrwB into lipid bilayers causes an increase in lipid chain order.

Keywords: Membrane protein; Bacterial conjugation; Conjugative coupling protein; Protein reconstitution; R388; IR spectroscopy

Abbreviations: T4CP, type IV coupling protein; TMD, transmembrane domain; IR, infrared spectroscopy; TNP-ATP, 2′(or 3′)-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; CL, cardiolipin; OG, n-octyl β-d-glucopyranoside; DDM, n-Dodecyl-β-d-maltoside

Article Outline

1. 

Introduction

2. 

Materials and methods

2.1. Materials

2.2. Overexpression and purification of TrwB

2.3. Preparation of large unilamellar vesicles

2.4. Reconstitution of TrwB into liposomes

2.5. SDS-PAGE

2.6. Determination of total phosphorous

2.7. Measurement of nucleotide binding activity in proteoliposomes

2.8. Analysis of ATPase activity of TrwB proteoliposomes

2.9. Proteolysis by trypsin and proteinase K

2.10. Infrared spectroscopy

3. 

Results

3.1. Effect of lipid composition on membrane protein incorporation

3.2. Secondary structure of solubilized and reconstituted TrwB

3.3. TrwB stability against thermal challenge

3.4. TrwB stability against proteolytic digestion

4. 

Discussion

4.1. Structure of TrwB embedded in lipid bilayers

4.2. TrwB stability in micelles and bilayers

4.3. TrwB interaction with lipids in bilayers

Acknowledgements

References

http://dx.doi.org/10.1016/j.bbamem.2010.12.025