Abstract
We have investigated the effect of lipid composition on interactions between cytochrome bo3 and ATP-synthase, and the ATP-synthesis activity driven by proton pumping. The two proteins were labeled by fluorescent probes and co-reconstituted in large (d ≅ 100 nm) or giant (d ≅ 10 µm) unilamellar lipid vesicles. Interactions were investigated using fluorescence correlation/cross-correlation spectroscopy and the activity was determined by measuring ATP production, driven by electron-proton transfer, as a function of time. We found that conditions that promoted direct interactions between the two proteins in the membrane (higher fraction DOPC lipids or labeling by hydrophobic molecules) correlated with an increased activity. These data indicate that the ATP-synthesis rate increases with decreasing distance between cytochrome bo3 and the ATP-synthase, and involves proton transfer along the membrane surface. The maximum distance for lateral proton transfer along the surface was found to be ~80 nm.
Highlights
MethodsF1Fo ATP synthase was expressed from plasmid pBWU13-βHis in E. coli strain DK8 and purified as described[48]
Results from earlier studies indicate that the membrane surface is involved in proton transfer between membrane proteins
The data presented in this work show that in GUVs direct interactions between cyt. bo[3] and ATP synthase were promoted by binding of hydrophobic fluorescent dyes
Summary
F1Fo ATP synthase was expressed from plasmid pBWU13-βHis in E. coli strain DK8 and purified as described[48]. Bo[3] was expressed from plasmid pETcyo in E. coli strain C43 and purified as described The proteins were labeled with either of the three thiol-reactive fluorophores ATTO 594, ATTO 647N (ATTO TEC GmbH), or Abberior STAR 635 (Abberior GmbH). ATP synthase was labeled with ATTO 594 by adding a 3-fold molar excess of the dye and the sample was incubated while gently shaking for 1.5 h at room temperature. Cyt. bo[3] was labeled with ATTO 647N by adding a 1.3-fold molar excess dye or with Abberior STAR 635 by adding a 5-fold molar excess of dye in the presence of 1/20 volume of NaHCO3 (pH 9.0), and incubated as described above. The proteins were stored in the same buffer at −70 °C until use
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