A novel three-dimensional crystal of bacteriorhodopsin obtained by successive fusion of the vesicular assemblies.
When the two-dimensional crystal of bacteriorhodopsin (bR), purple membrane, is incubated at high temperature (32 degreesC) with a small amount of the neutral detergent octylthioglucoside in the presence of the precipitant ammonium sulfate, a large fraction of the membrane fragments is converted into spherical vesicles with a diameter of 50 nm, which are able to assemble into optically isotropic hexagonal crystals when the precipitant concentration is increased. The vesicularization of purple membrane takes place under such a condition that the miscibility of the detergent to the aqueous phase becomes very low, and we suggest that it is initiated by insertion of the detergent molecules into the membrane. At low temperature, the transformation into the vesicular structure is inhibited and no large crystal is produced directly from membrane/detergent/precipitant mixtures. When a suspension of the spherical vesicles produced at the high temperature is cooled and concentrated below 15 degreesC, however, a birefringent hexagonal crystal is produced that diffracts X-rays beyond 2.5 A resolution. This new crystal belongs to the space group P622 with unit cell dimensions of a=b=104.7 A and c=114.1 A, and it is shown to be made up of stacked planar membranes, in each of which the bR trimers are arranged on a honeycomb lattice and the space among the proteins is filled with the detergent molecules and native lipids. These stacked 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 present result offers a unique crystallization method that may be applicable to such membrane proteins that are liable to denature in the presence of an excess amount of detergent.
Graduate School of Science, Nagoya University, Nagoya, 464-8602, Japan.
K
K
Takeda
H
H
Sato
T
T
Hino
M
M
Kono
K
K
Fukuda
I
I
Sakurai
T
T
Okada
T
T
Kouyama
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D019617:Halobacterium salinarum; D046911:Macromolecular Substances; D008561:Membrane Fusion; D011487:Protein Conformation; D018717:Purple Membrane / Q000737:chemistry* / Q000378:metabolism; D013863:Thioglucosides / Q000737:chemistry / Q000378:metabolism
D046911:Macromolecular Substances; D013863:Thioglucosides; D001436:Bacteriorhodopsins; C042989:n-octyl-beta-D-thioglucopyranoside