Influence of lipids on membrane protein structure and function

在膜中，蛋白质和脂质形成一个集成系统来支持基本的细胞功能。的 嵌入天然脂质双层中的膜蛋白的结构可以通过电子晶体学来研究。 水通道蛋白0（AQPO）是透镜纤维细胞中的主要蛋白质成分，在其中它起双重作用： 水的孔隙和形成粘合剂连接。利用电子晶体学，我们最近确定 AQPO的结构，以前所未有的分辨率为1.9A，揭示了一个外壳的精确结构， 有序脂质围绕AQPO四聚体。因此，AQPO提供了第一个真核生物模型系统， 这是可能的，在同一个膜，结构和功能的相互依赖性， 蛋白质和脂质。为此，我们将在脂质双层中形成AQPO的单层二维晶体 使用天然的或非天然的脂质，并在存在或不存在钙调蛋白（CaM）的情况下， AQPO末端结构域。然后，我们将关联结构（从电子晶体学）和功能 (from AQPO水通道的电生理学），因为它响应于pH值打开和关闭，二价 钙和其它配体。最后，我们还将确定孔所需的结构重排， 当分开的双层上的两个AQPO四聚体聚集在一起形成细胞间连接时，细胞会关闭。 虽然该提案解决了有关AQPO水道功能的具体问题，但许多 由此产生的见解应该普遍适用于其他通道和膜蛋白。

In membranes, proteins and lipids form an integrated system to support essential cell functions. The structure of membrane proteins embedded in native lipid bilayers can be studied by electron crystallography. Aquaporin 0 (AQPO) is the major protein constituent in lens fiber cells where it serves a dual role: creating a pore for water and forming adhesive junctions. Using electron crystallography, we have recently determined the structure of AQPO to an unprecedented resolution of 1.9A, revealing the precise structure of a shell of ordered lipids surrounding the AQPO tetramer. AQPO therefore provides the first eukaryotic model system in which it is possible to study, in the very same membrane, the structural and functional interdependence of protein and lipids. To do this, we will form single-layered two-dimensional crystals of AQPO in lipid bilayers using native or unnatural lipids and in the presence or absence of calmodulin (CaM) which binds to the C- terminal domain of AQPO. We will then correlate the structure (from electron crystallography) and function (from electrophysiology) of the AQPO water channel as it opens and closes in response to pH, divalent calcium, and other ligands. Finally, we will also determine the structural rearrangements required for pore closure when two AQPO tetramers on separate bilayers come together to form intercellular junctions. Although this proposal addresses specific questions regarding AQPO water channel function, many of the resulting insights should be generally applicable to other channels and membrane proteins.