Probing the Organization of Oligosaccharyltransferase

寡糖转移酶的组织探索

• 批准号: 6729040
• 负责人: Kerney Jebrell Glover
• 金额: $ 4.73万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6729040
• 关键词: Baculoviridae; Saccharomyces cerevisiae; analytical ultracentrifugation; chemical synthesis; circular dichroism; endoplasmic reticulum; enzyme complex; enzyme model; enzyme reconstitution; fluorescence resonance energy transfer; fungal proteins; gel filtration chromatography; hexosyltransferase; intermolecular interaction; mass spectrometry; membrane proteins; model design /development; molecular assembly /self assembly; molecular site; physical model; protein purification; protein structure function; structural biology; surface plasmon resonance

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.

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，也是核心问题 细胞功能。特别地，寡糖基转移酶(OT)是 复合酶，由至少九个完整膜多肽组成， 它将预组装的寡糖附着到新生的多肽上 蛋白质易位至内质网 (ER)。虽然九个都 多肽对于体内最佳功能是必需的，只有四种多肽 是体外酶活性所必需的。该提案旨在采取 理解和表征复杂的生物物理学方法 将 OT 的四个基本亚基结合在一起的相互作用网络 形成活性复合物。这将通过准备每一项来解决 单独的域，以一一的方式组合它们，以及 使用荧光、圆形表征所产生的相互作用 二色性、质谱、分析超速离心、表面等离子体 共振、天然和 SDS PAGE 以及凝胶过滤色谱。 管腔和跨膜的表征将分别进行 域，以区分各自的角色。获得地图后 该组织的重点将转向利用完整的 OT 进行重组 模型膜中多肽的长度。