Probing the Organization of Oligosaccharyltransferase

描述：(申请人提供)多亚基蛋白质如何结合在一起 形成一个活跃的复合体是一个根本性的问题，它是 细胞功能。特别是，寡糖转移酶(OT)是一种 复合酶，由至少9个完整的膜多肽组成， 它将预组装的低聚糖连接到新生多肽上 蛋白质转运到内质网(ER)。虽然这九个人 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。这项提议的目的是采取 一种生物物理方法来理解和描述复杂的 将OT的四个基本亚单位结合在一起的相互作用网络 形成一种活跃的复合体。这将通过准备每个 单独的域，以一个接一个的方式组合它们，以及 使用圆形荧光表征所产生的相互作用 二向色性、质谱学、分析超速离心法、表面等离子体激元 共振层析、天然和十二烷基硫酸钠PAGE、凝胶过滤层析。 腔内膜和跨膜膜的特性将分别进行。 域，以便理清各自的角色。在获得一张地图后 在组织方面，重点将转向使用充分的OT重组 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.