A NOVEL 3-DIMENSIONAL CRYSTAL OF BACTERIORHODOPSIN OBTAINED BY SUCCESSIVE FUSION OF THE VESICULAR ASSEMBLIES

When the two-dimensional crystal of bacteriorhodopsin (bR), purple mem brane, is incubated at high temperature (32 degrees C) with a small am ount of the neutral detergent octylthioglucoside in the presence of th e precipitant ammonium sulfate, a large fraction of the membrane fragm ents is converted into spherical vesicles with a diameter of 50 nm, wh ich are able to assemble into optically isotropic hexagonal crystals w hen the precipitant concentration is increased. The vesicularization o f purple membrane takes place under such a condition that the miscibil ity of the detergent to the aqueous phase becomes very low, and we sug gest that it is initiated by insertion of the detergent molecules into the membrane. At low temperature, the transformation into the vesicul ar structure is inhibited and no large crystal is produced directly fr om membrane/detergent/precipitant mixtures. When a suspension of the s pherical vesicles produced at the high temperature is cooled and conce ntrated below 15 degrees C, however, a birefringent hexagonal crystal is produced that diffracts X-rays beyond 2.5 Angstrom resolution. This new crystal belongs to the space group P622 with unit cell dimensions of a = b = 104.7 Angstrom and c = 114.1 Angstrom, and it is shown to be made up of stacked planar membranes, in each of which the bR trimer s are arranged on a honeycomb lattice and the space among the proteins is filled with the detergent molecules and native lipids. These stack ed membranes are suggested to be produced by successive fusion of the spherical vesicles. This implies that the crystallization is achieved without any step for complete solubilization of the protein. The prese nt result offers a unique crystallization method that may be applicabl e to such membrane proteins that are liable to denature in the presenc e of an excess amount of detergent. (C) 1998 Academic Press.