Takeda K, Sato H, Hino T, Kono M, Fukuda K, Sakurai I, Okada T, Kouyama T
Graduate School of Science, Nagoya University, Nagoya, 464-8602, Japan.
J Mol Biol. 1998 Oct 23;283(2):463-74. doi: 10.1006/jmbi.1998.2103.
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.
当细菌视紫红质（bR）的二维晶体——紫膜，在沉淀剂硫酸铵存在的情况下，于高温（32℃）下与少量中性去污剂硫代辛基葡糖苷一起孵育时，大部分膜片段会转化为直径为50nm的球形囊泡。当沉淀剂浓度增加时，这些囊泡能够组装成光学各向同性的六方晶体。紫膜的囊泡化发生在去污剂与水相的混溶性变得非常低的条件下，我们认为这是由去污剂分子插入膜中引发的。在低温下，向囊泡结构的转变受到抑制，并且不会直接从膜/去污剂/沉淀剂混合物中产生大晶体。然而，当将高温下产生的球形囊泡悬浮液冷却并浓缩至15℃以下时，会产生一种双折射六方晶体，其X射线衍射分辨率超过2.5Å。这种新晶体属于空间群P622，晶胞尺寸为a = b = 104.7Å，c = 114.1Å，并且显示为由堆叠的平面膜组成，在每个平面膜中，bR三聚体排列在蜂窝晶格上，蛋白质之间的空间填充有去污剂分子和天然脂质。这些堆叠的膜被认为是由球形囊泡的连续融合产生的。这意味着结晶过程无需蛋白质完全溶解的任何步骤即可实现。目前的结果提供了一种独特的结晶方法，该方法可能适用于在过量去污剂存在下易于变性的膜蛋白。
