Mitra A K, Yeager M, van Hoek A N, Wiener M C, Verkman A S
Department of Cell Biology, Scripps Research Institute, La Jolla, California.
Biochemistry. 1994 Nov 1;33(43):12735-40. doi: 10.1021/bi00209a001.
Osmotic water transport across plasma membranes in erythrocytes and several epithelial cell types is facilitated by CHIP28, a water-selective membrane channel protein. In order to examine the structure of CHIP28 in membranes, large (1.5-2.5-microns diameter), highly ordered, two-dimensional (2-D) crystals of purified and deglycosylated erythrocyte CHIP28 were generated by reconstitution of detergent-solubilized protein into synthetic lipid bilayers via detergent dialysis. Fourier transforms computed from low-dose electron micrographs of such crystals preserved in negative stain display order to 12-A resolution. The crystal lattice is tetragonal (a = b = 99.2 +/- 1.4 A) with plane group symmetry p4g. A projection density map at 12-A resolution defines the molecular boundary and organization of the CHIP28 monomers in the membrane plane. The unit cell contains four CHIP28 dimers, each composed of two oblong-shaped (37 x 25 A ) monomers with opposite orientations. The CHIP28 monomers associate to form tetrameric structures around the 4-fold axes normal to the membrane plane where stain is excluded. The 2-D crystals of CHIP28 display order extending beyond the limit typically achieved by negative staining and therefore may be amenable to high-resolution structure analysis by cryo-electron microscopy.
水通道蛋白CHIP28可促进红细胞和几种上皮细胞类型中跨质膜的渗透水转运，它是一种水选择性膜通道蛋白。为了研究CHIP28在膜中的结构，通过去污剂透析将去污剂溶解的蛋白重组到合成脂质双层中，生成了大尺寸（直径1.5 - 2.5微米）、高度有序的二维（2-D）纯化去糖基化红细胞CHIP28晶体。从保存在负染中的此类晶体的低剂量电子显微照片计算得到的傅里叶变换显示出分辨率为12埃的有序结构。晶格为四方晶格（a = b = 99.2 +/- 1.4埃），平面群对称p4g。12埃分辨率的投影密度图定义了膜平面中CHIP28单体的分子边界和组织。晶胞包含四个CHIP28二聚体，每个二聚体由两个呈相反取向的长方形（37 x 25埃）单体组成。CHIP28单体围绕垂直于膜平面且无染色剂的4重轴缔合形成四聚体结构。CHIP28的二维晶体显示出超出负染通常所能达到极限的有序性，因此可能适用于通过冷冻电子显微镜进行高分辨率结构分析。
