Surface topographies at subnanometer-resolution reveal asymmetry and sidedness of aquaporin-1.
Surface topographies at subnanometer-resolution reveal asymmetry and sidedness of aquaporin-1.
复制标题
亚纳米分辨率的表面形貌揭示了 aquaporin-1 的不对称性和侧面性。
DOI：
10.1006/jmbi.1996.0686
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Engel,A
中科院分区：
文献类型：
--
作者：
Walz,T;Tittmann,P;Fuchs,KH;Muller,DJ;Smith,BL;Agre,P;Gross,H;Engel,A
水通道蛋白-1（AQP 1）是人红细胞膜上丰富的蛋白质，具有特异性和组成性活性的导水孔。作为四聚体溶解和分离，当在脂质存在下重构时，其形成良好有序的二维（2D）晶体。已经确定了几个高分辨率的AQP 1投影图，但其三维（3D）质量分布的信息是稀疏的。在这里，我们提出的表面浮雕在0.9 nm的分辨率，从冷冻干燥的单向金属阴影AQP 1晶体以及记录在缓冲溶液中的原生晶体的原子力显微镜的表面形貌计算。我们的研究结果证实了负染色的AQP 1晶体的3D图，该晶体显示出四聚体，一侧有四个主要突起，另一侧有一个大的中央空腔。用羧肽酶Y消化AQP 1晶体，其切割掉5 kDa的细胞内C-末端片段，导致主要突起的减少，这表明四聚体的中心腔面向细胞的外部。为了解释结果，基于序列的结构预测作为指导。
Aquaporin-1 (AQP1) is an abundant protein in human erythrocyte membranes which functions as a specific and constitutively active water conducting pore. Solubilized and isolated as tetramer, it forms well- ordered two-dimensional (2D) crystals when reconstituted in the presence of lipids. Several high resolution projection maps of AQP1 have been determined, but information on its three-dimensional (3D) mass distribution is sparse. Here, we present surface reliefs at 0.9 nm resolution that were calculated from freeze-dried unidirectionally metal-shadowed AQP1 crystals as well as surface topographs recorded with the atomic force microscope of native crystals in buffer solution. Our results confirm the 3D map of negatively stained AQP1 crystals, which exhibited tetramers with four major protrusions on one side and a large central cavity on the other side of the membrane. Digestion of AQP1 crystals with carboxypeptidase Y, which cleaves off a 5 kDa intracellular C-terminal fragment, led to a reduction of the major protrusions, suggesting that the central cavity of the tetramer faces the outside of the cell. To interpret the results, sequence based structure predictions served as a guide.