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. Elect ron microscopic analysis of VDAC in detergent solution revealed a uniform p article population consisting of porin monomers. After dialysis of detergen t-solubilized porin in the presence of dimyristoylphosphatidylcholine at li pid-to-protein ratios between 0.2 and 0.5 (percentage by weight), mostly mu ltilamellar crystals were obtained. Crystals adsorbed to carbon films flatt ened during negative staining and air-drying and exhibited different struct ural features due to differences in the vertical stacking of several crysta lline layers, each consisting of one membrane bilayer. Adsorbed, frozen-hyd rated multilamellar membrane crystals revealed uniform diffraction patterns with sharp diffraction spots extending to 8.2 Angstrom. The surface struct ure of VDAC was reconstructed from freeze-dried and unidirectionally metal- shadowed crystals. Major protein protrusions were observed from two VDAC mo nomers present in the unit cell. Differences in the surface structural feat ures 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 wi th a diameter of 17-20 Angstrom surrounded by a protein rim of nonuniform h eight, suggesting an asymmetrical distribution of protein mass around the d iffusion channels. (C) 1999 Academic Press.