Probing the Organization of Oligosaccharyltransferase

寡糖转移酶的组织探索

• 批准号: 6487164
• 负责人: KERNEY J GLOVER
• 金额: $ 3.66万
• 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
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6487164
• 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）多亚基蛋白是如何结合在一起的