Braun T, Philippsen A, Wirtz S, Borgnia M J, Agre P, Kühlbrandt W, Engel A, Stahlberg H
ME Müller Institute for Microscopy, University of Basel, Switzerland.
EMBO Rep. 2000 Aug;1(2):183-9. doi: 10.1093/embo-reports/kvd022.
GlpF, the glycerol facilitator protein of Escherichia coli, is an archetypal member of the aquaporin superfamily. To assess its structure, recombinant histidine-tagged protein was overexpressed, solubilized in octylglucoside and purified to homogeneity. Negative stain electron microscopy of solubilized GlpF protein revealed a tetrameric structure of approximately 80 A side length. Scanning transmission electron microscopy yielded a mass of 170 kDa, corroborating the tetrameric nature of GlpF. Reconstitution of GlpF in the presence of lipids produced highly ordered two-dimensional crystals, which diffracted electrons to 3.6 A resolution. Cryoelectron microscopy provided a 3.7 A projection map exhibiting a unit cell comprised of two tetramers. In projection, GlpF is similar to AQP1, the erythrocyte water channel. However, the major density minimum within each monomer is distinctly larger in GlpF than in AQP1.
GlpF是大肠杆菌的甘油转运蛋白，是水通道蛋白超家族的典型成员。为了评估其结构，重组的组氨酸标签蛋白被过量表达，溶解在辛基葡糖苷中并纯化至同质。对溶解的GlpF蛋白进行负染电子显微镜观察，发现其为边长约80埃的四聚体结构。扫描透射电子显微镜测得其质量为170 kDa，证实了GlpF的四聚体性质。在脂质存在的情况下对GlpF进行重构，产生了高度有序的二维晶体，其电子衍射分辨率达到3.6埃。冷冻电子显微镜提供了一个3.7埃的投影图，显示一个由两个四聚体组成的晶胞。在投影中，GlpF与红细胞水通道AQP1相似。然而，GlpF中每个单体内部的主要密度最小值明显大于AQP1中的。
