Influence of lipids on membrane protein structure and function

描述（由申请人提供）：在膜中，蛋白质和脂质形成一个集成系统，以支持基本细胞功能。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：为水产生孔和形成粘附连接。使用电子晶体学，我们最近确定了AQPO的结构，前所未有的分辨率为1.9A，揭示了AQPO四聚体周围有序脂质壳的精确结构。因此，AQPO提供了第一个真核生物模型系统，其中有可能在同一个膜中研究蛋白质和脂质的结构和功能相互依赖性。为了做到这一点，我们将使用天然或非天然脂质，在存在或不存在与AQPO的C-末端结构域结合的钙调蛋白（CaM）的情况下，在脂质双层中形成AQPO的单层二维晶体。然后，我们将关联AQPO水通道的结构（从电子晶体学）和功能（从电生理学），因为它打开和关闭响应pH值，二价钙，和其他配体。最后，我们还将确定孔关闭时，两个AQPO四聚体在单独的双层走到一起，形成细胞间连接所需的结构重排。虽然这项建议解决了关于AQPO水通道功能的具体问题，但由此产生的许多见解应该普遍适用于其他通道和膜蛋白。

DESCRIPTION (provided by applicant): 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.