AMPHIPATHIC MOTIFS IN APOLIPOPROTEIN B

载脂蛋白 B 中的两亲基序

• 批准号: 6327703
• 负责人: NASSRIN DASHTI
• 金额: $ 17.62万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6327703
• 关键词: amphiphilicity; apolipoprotein B; chemical models; protein isoforms; protein structure function; site directed mutagenesis

Prioritized project goals are to: (a) test the hypothesis that the three amphipathic helical domains of apo B ( alpha 1, alpha 2, and alpha 3) act as independent structural and functional units; (b) investigate the role of amphipathic beta strands and/or antiparallel amphipathic beta sheets in the interactions of apo B with lipoprotein surfaces, and ~ in a more risky sub-project, explore the structural features of apo B that are involved in assembly of apo B-containing lipoproteins. The research strategy involves studies of the biophysical and biological properties of: 1) rationally designed apolipoprotein B fragments, created and expressed in vitro by cultured cells and in intact lipoproteins by cells in culture by Core D, and 2) shorter sequences produced by chemical peptide synthesis by Core B. WHEEL and LOCATE computer algorithms (Project 1, in collaboration with Project 2) will be used to design mutations. To achieve the above goals, two specific aims are proposed: 1) Studies of rationally designed site- directed mutants of apo B. (a) We will test the hypothesis that the three amphipathic helical domains of apo B (alpha 1, alpha 2, and alpha 3) act as independent structural and functional units and that the alpha 1 domain is ~globular~ and can be crystallized. Each domain will be expressed in cells in culture and studied. (b) We will test the hypothesis that amphipathic beta strands from the N- terminal 18-28% of apo B, in association with the alpha 1 domain, form a lipovitellin-like intermediate that contains a lipid pocket required to initiate the assembly of triglyceride-rich lipoprotein particles. ~ An additional hypothesis to be tested is that this lipovitellin-like intermediate structure is not complete without structural integration of MTP.2) Studies of lipid interactions of amphipathic beta strand and sheets. In addition to amphipathic helixes, putative amphipathic beta strands have been located in the primary structure of apo B. We propose to study the structure and lipid associating properties of native (from apo B-100) as well as de novo designed amphipathic beta strands and sheets. Both peptide synthesis and molecular biology techniques will be used to obtain these peptides.

优先的项目目标是：（a）检验以下假设。 Apo B的三个两亲性螺旋结构域（Alpha 1，Alpha 2和 alpha 3）充当独立的结构和功能单位； （b） 研究两亲β链和/或反平行的作用 Apo B与Apo B与 脂蛋白表面，〜在更风险的子项目中探索 Apo B的结构特征与组装有关 含Apo B的脂蛋白。 研究策略涉及 生物物理和生物学特性的研究：1） 创建的理性设计的载脂蛋白B片段和 由培养细胞和完整的脂蛋白在体外表达 通过核心D培养细胞，以及2）由 Core B. Wheel并定位的化学肽合成 计算机算法（项目1，与项目2合作） 将用于设计突变。 为了实现上述目标，两个 提出了具体目的：1）对理性设计的地点的研究 - ApoB的定向突变体（a）我们将检验以下假设。 Apo B的三个两亲性螺旋结构域（Alpha 1，Alpha 2和 alpha 3）充当独立的结构和功能单位， alpha 1域是〜球形〜，可以结晶。 每个 域将在培养细胞中表达并进行了研究。 （b）我们 将检验以下假设，即来自n-的两亲性β链 与Alpha 1域相关的Apo B的终端18-28％， 形成一个含有脂质口袋的Lipovitellin样中间体 需要启动富含甘油三酸酯的脂蛋白的组装 颗粒。 〜要测试的另一个假设是 没有脂维肽样的中间结构没有完整 MTP的结构整合2）脂质相互作用的研究 两亲性β链和床单。 除了两亲性 螺旋，推定的两亲β链已位于 ApoB的主要结构。我们建议研究结构 以及天然的脂质关联特性（来自APO B-100） 从头开始设计两亲性的beta链和床单。 两个都 肽合成和分子生物学技术将用于 获得这些肽。