Crystallization of the human, mitochondrial voltage-dependent anion-selective channel in the presence of phospholipids.
Overexpressed human voltage-dependent anion-selective channel VDAC or porin from mitochondrial outer membranes has been purified to homogeneity. Electron microscopic analysis of VDAC in detergent solution revealed a uniform particle population consisting of porin monomers. After dialysis of detergent-solubilized porin in the presence of dimyristoylphosphatidylcholine at lipid-to-protein ratios between 0.2 and 0.5 (percentage by weight), mostly multilamellar crystals were obtained. Crystals adsorbed to carbon films flattened during negative staining and air-drying and exhibited different structural features due to differences in the vertical stacking of several crystalline layers, each consisting of one membrane bilayer. Adsorbed, frozen-hydrated multilamellar membrane crystals revealed uniform diffraction patterns with sharp diffraction spots extending to 8.2 A. The surface structure of VDAC was reconstructed from freeze-dried and unidirectionally metal-shadowed crystals. Major protein protrusions were observed from two VDAC monomers present in the unit cell. Differences in the surface structural features indicate alternate orientations of VDAC molecules with respect to the lipid bilayer, allowing the simultaneous imaging of both the cytosolic and intramitochondrial surfaces. Each VDAC molecule consists of a pore lumen with a diameter of 17-20 A surrounded by a protein rim of nonuniform height, suggesting an asymmetrical distribution of protein mass around the diffusion channels.
Dolder
M
M
Institute of Cell Biology, Institute of Applied Physics, Swiss Federal Institute of Technology, ETH-Hönggerberg, Zürich, CH-8093, Switzerland. dolder@cell.biol.ethz.ch
Zeth
K
K
Tittmann
P
P
Gross
H
H
Welte
W
W
Wallimann
T
T
D000595:Amino Acid Sequence; D020285:Cryoelectron Microscopy; D003460:Crystallization; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000648:ultrastructure; D007473:Ion Channels / Q000648:ultrastructure; D008928:Mitochondria / Q000737:chemistry* / Q000648:ultrastructure*; D018272:Porins / Q000302:isolation & purification / Q000648:ultrastructure*; D050994:Voltage-Dependent Anion Channels
D007473:Ion Channels; D018272:Porins; D050994:Voltage-Dependent Anion Channels; D006639:Histidine; D004134:Dimyristoylphosphatidylcholine