Reconstitution and characterization of membrane proteins

“A membrane protein, cytochrome c reductase, was crystallized and examined using transmission electron microscopy. Two-dimensional crystals were prepared by two methods. In Method 1, the protein was solubilized in 50 mM Tris-Acetate (pH 6.0)/0.5 mM EDTA70.05% (w/v) Triton X-100 and reconstituted into a mixture of phosphatidylcholine and phosphatidylserine (4:1), dissolved in the same buffer. The Triton X-100 was removed from the mixture by dialysis in the presence of Bio-beads (SM-2) at approximately 25-30 °C. Method 2 was the same as Method 1, except that the vesicles were fractionated before reconstitution, and the pH and temperature for crystallization were 7.0 and 4 °C respectively. After dialysis, the two samples were prepared for transmission electron microscopy by negatively staining the specimens on copper (400 mesh) grids. Electron micrographs of the proteoliposomes and single layered crystal sheets were taken along with their diffraction patterns. The proteoliposomes contained amorphous deposits of protein. The single layered sheets were highly ordered with protein. The unit cell for Method 1 could not be solved due to aggregation and overlapping of the crystal sheets. For Method 2, the unit cell is hexagonal, with p3 symmetry”--Abstract, page iii