Reconstitution of Respiratory Complexes

The (co-)reconstitution
protocols for bo₃ oxidase and F₁F_O-ATPase into hybrids were slight modifications of our
previous protocols.^{16 (link)} Briefly, for the
reconstitution of bo₃ oxidase, octyl glucoside
at the solubilization point (R_sol) was
added to hybrids (5 mg mL^–1 LUVs, final conc. of
octyl glucoside 0.11%) for the reconstitution of F₁F_O-ATPase, sodium deoxycholate at R_sol was added to hybrids (5 mg mL^–1 LUVs, final conc.
of sodium deoxycholate 0.065%), and for the co-reconstitution of bo₃ oxidase and F₁F_O-ATPase
octyl glucoside was added to hybrids (5 mg mL^–1 LUVs,
final conc. of octyl glucoside 0.05%). Next, for individual protein
reconstitution, bo₃ oxidase was gently
added to hybrids at a final conc. of 0.72, 1.35, or 2.38 μM.
Meanwhile, the final conc. of F₁F_O-ATPase was
0.68, 0.72, or 2.38 μM. For co-reconstitution, the final conc.
of bo₃ oxidase in the reconstitution mixture
was 0.72, 0.90, or 2.38 μM and the final conc. of F₁F_O-ATPase was 0.45, 0.72, or 2.38 μM. The reconstitution
mixture was incubated at 4 °C for 30 min with mild agitation,
followed by detergent removal via Bio-Beads SN-2 (Bio-Rad). For the
preparation of 200 μL of proteohybrids, the beads were added
in 3 subsequent additions, 30 mg of beads each, followed by 30-min
incubation at 4 °C and 600 rpm in a thermo shaker. After that,
beads were pelleted and the supernatant was collected and stored at
4 °C. If the proteoLUVs were not used for the preparation of
proteoGUVs the same day (which was always the case when protein insertion
and distribution were analyzed), the vesicle suspension was frozen
in liquid N₂ and aliquots of 20 μL were stored at
−80 °C. For measurements of activity of proteins reconstituted
in GUVs, proteoLUVs were always used the same day because (1) a large
sample volume was required for measurements in a luminometer, and
(2) to avoid an increase in activity due to freezing and thawing the
samples (see Figure S3).