Probing the Organization of Oligosaccharyltransferase

描述：（由申请人提供）多亚基蛋白如何聚集在一起 形成一个活跃的复合体是一个基本问题， 细胞功能特别地，寡糖基转移酶（OT）是寡糖基转移酶。 复合酶，由至少9种膜整合多肽组成， 其在生长过程中将预组装的寡糖连接到新生多肽上， 蛋白质转运进入内质网（ER）。虽然所有九个 多肽是体内最佳功能所必需的，只有四种多肽 是体外酶活性所必需的。该提案旨在采取 用生物物理学的方法来理解和描述 将OT的四个基本亚单位结合在一起的相互作用网络 形成活性复合物。这将通过准备 域单独地，以一个接一个的方式组合它们，以及 使用荧光、圆形、圆形 二向色性，质谱，分析超离心，表面等离子体 共振、非变性和SDS PAGE和凝胶过滤色谱法。 将分别对管腔和跨膜进行表征 为了解开各自的角色。在获得了一张 组织的重点将转向利用全面的 在模型膜中的长度多肽。

DESCRIPTION: (provided by applicant) How multi-subunit proteins come together to form an active complex is a fundamental question which lies at the center of cellular function. Particularly, the Oligosaccharyltransferase (OT) is a complex enzyme, comprised of at least nine integral membrane polypeptides, which attaches a preassembled oligosaccharide to nascent polypeptides during protein translocation into the endoplasmic reticulum (ER). Although all nine polypeptides are necessary for optimal function in vivo, only four polypeptides are necessary for enzymatic activity in vitro. This proposal is aimed at taking a biophysical approach to understanding and characterizing the intricate network of interactions which bring the four essential subunits of OT together to form an active complex. This will be addressed by preparing each of the domains individually, combining them in a one by one fashion, and characterizing the resulting interactions using fluorescence, circular dichroism, mass spectrometry, analytical ultracentrifugation, surface plasmon resonance, native and SDS PAGE, and gel filtration chromatography. Characterization will be done separately for both the luminal and transmembrane domains in order to disentangle the respective roles. After gaining a map of the organization, focus will turn to the reconstitution of OT using the full length polypeptides in model membranes.