Influence of lipids on membrane protein structure and function

描述(申请人提供)：在细胞膜中，蛋白质和脂类形成一个完整的系统，以支持细胞的基本功能。天然脂双层膜蛋白的结构可以用电子结晶学进行研究。水通道蛋白0(AQPO)是晶状体纤维细胞中的主要蛋白质成分，在晶状体纤维细胞中具有双重作用：形成水孔和形成粘连连接。利用电子结晶学，我们最近确定了AQPO的结构，分辨率达到前所未有的1.9A，揭示了围绕AQPO四聚体的有序脂类壳的精确结构。因此，AQPO提供了第一个真核模型系统，在该模型系统中，可以在同一膜上研究蛋白质和脂类的结构和功能相互依赖。为此，我们将使用天然或非天然的脂类，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(来自电子结晶学)和功能(来自电生理学)，当它对pH、二价钙和其他配体做出反应时，它的打开和关闭。最后，我们还将确定当两个单独的双层上的AQPO四聚体聚集在一起形成细胞间连接时，关闭孔所需的结构重排。虽然这项提案解决了有关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.