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. 2012 Aug 13;51(33):8343-7.
doi: 10.1002/anie.201202094. Epub 2012 Jul 23.

GRecon: a method for the lipid reconstitution of membrane proteins

Thorsten Althoff  1 , Karen M Davies, Sabrina Schulze, Friederike Joos, Werner Kühlbrandt
Affiliations
Free PMC article

GRecon: a method for the lipid reconstitution of membrane proteins

Thorsten Althoff et al. Angew Chem Int Ed Engl. .
Free PMC article
No abstract available

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Figures

Figure 1
Figure 1
The GRecon method: a) In the GRecon gradient the concentrations of cyclodextrin and lipid increase in parallel with the sucrose density. b) The gradient is loaded with the detergent-solubilized membrane protein. As the protein migrates into the gradient during ultracentrifugation, the detergent is gradually absorbed by increasing levels of cyclodextrin, and the protein incorporates into destabilized, preformed liposomes. Opaque proteoliposome bands are harvested manually (c) or the whole gradient can be fractionated with a peristaltic pump (d). Subsequently proteoliposomes are collected by dilution and ultracentrifugation.
Figure 2
Figure 2
Gradient reconstitution of mitochondrial supercomplexes: a) BN-PAGE (3–10 %) of purified supercomplex I1III2IV1 in ca. 0.1 % digitonin after incubation with γ-cyclodextrin (γ-CD). The protein is precipitated by 0.212–0.424 % γ-cyclodextrin. b) GRecon gradients (0.3–1.3 m sucrose, 0–0.75 mg mL−1 bovine heart polar lipids, LPR 3 (w/w), 0–0.75 mg mL−1 Triton X-100, 0–0.53 % γ-cyclodextrin) with and without supercomplex. Protein incorporation causes a shift of the opaque liposome band towards higher density. L=liposomes; PL=proteoliposomes. c) BN-PAGE of resolubilized proteoliposomes. Enzymatic in-gel assay indicates the activity of complex I. d) Freeze-fracture electron microscopic image of reconstituted supercomplex. Scale bar 200 nm. e) Slice through the tomographic volume of a proteoliposome containing reconstituted supercomplexes. Inserts show twofold enlargements of individual supercomplexes. Scale bar 50 nm. f) 3D segmentation of the vesicle shown in (e) with supercomplexes in yellow. g) Comparison of the sub-tomogram average of supercomplexes from (f) and a single-particle cryo-EM map. From the left: single-particle reconstruction at 19 Å (EMD-1876), same map filtered to 65 Å, sub-tomogram average of 110 particles, sub-tomogram average with the docked single-particle map as a yellow mesh and with the docked model of the supercomplex (PDB 2ybb) with an electron density map of the enzyme from Yarrowia lipolytica. Top row: view in the plane of the membrane, bottom row: view from the matrix. Scale bar 10 nm. The matrix domains of complex I and complex III are clearly recognizable.
Figure 3
Figure 3
Gradient reconstitution of E. coli CaiT: a) α-cyclodextrin precipitates CaiT in 0.04 % DDM at a concentration of 0.182 %. b) GRecon gradients (0.3–1.0 m sucrose, 0–1.2 mg mL−1 E. coli polar lipids, LPR 4 (w/w), 0–1.2 mg mL−1 Triton X-100, 0–0.182 % α-cyclodextrin) with and without 500 μg CaiT after ultracentrifugation. Protein incorporation causes a shift of the opaque liposome band towards higher density. L=liposomes; PL=proteoliposomes. c) Coomassie stained SDS-PAGE of GRecon proteoliposomes indicating CaiT at roughly 43 kDa. d) Freeze-fracture electron microscopic image of GRecon CaiT proteoliposomes. Scale bar 200 nm. e) Transport kinetics of CaiT reconstituted into liposomes with GRecon.
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