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完整的系统来支持基本的细胞功能。利用电子晶体学可以研究天然脂质双分子层中膜蛋白的结构。水通道蛋白0 （AQPO）是晶状体纤维细胞的主要蛋白质成分，在晶状体纤维细胞中起双重作用：为水创造一个孔并形成粘合连接。利用电子晶体学，我们最近以前所未有的1.9A分辨率确定了AQPO的结构，揭示了围绕AQPO四聚体的有序脂质壳的精确结构。因此，AQPO提供了第一个真核模型系统，可以在同一膜中研究蛋白质和脂质在结构和功能上的相互依赖性。为了做到这一点，我们将使用天然或非天然脂质，在钙调素（CaM）存在或不存在的情况下，在脂质双层中形成AQPO的单层二维晶体，CaM与AQPO的C端结构域结合。然后，我们将把AQPO水通道的结构（从电子晶体学）和功能（从电生理学）联系起来，因为它根据pH值、二价钙和其他配体打开和关闭。最后，我们还将确定当两个AQPO四聚体在单独的双层上聚集形成细胞间连接时，气孔关闭所需的结构重排。虽然这一建议解决了关于AQPO水通道功能的具体问题，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。

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.