AMPHIPATHIC MOTIFS IN APOLIPOPROTEIN B

按优先顺序排列的项目目标是：(A)测试假设 载脂蛋白B的三个两亲性螺旋结构域(α1、α2和 阿尔法3)作为独立的结构和功能单位；(B) 研究两亲性β链和/或反平行的作用 载脂蛋白B与载脂蛋白相互作用中的两亲β-折叠 脂蛋白表面，在一个风险更高的子项目中，探索 参与装配的载脂蛋白B的结构特征 含载脂蛋白B的脂蛋白。研究策略包括 生物物理和生物学性质的研究：1) 合理设计的载脂蛋白B片段，创建和 体外培养细胞表达和完整脂蛋白表达 核心D培养中的细胞，以及2)由 用Core B.车轮和Locate合成化学多肽 计算机算法(项目1，与项目2合作) 将被用来设计突变。为实现上述目标，有两个目标 提出了具体目标：1)研究合理设计场地-- 载脂蛋白B的定向突变体(A)我们将检验以下假设 载脂蛋白B的三个两亲性螺旋结构域(α1、α2和 阿尔法3)作为独立的结构和功能单元， α1结构域是球状的，可以结晶。每个人 结构域将在培养的细胞中表达并进行研究。(二)我们 将检验这一假设，即两亲性β链来自N- 载脂蛋白B的18-28%的末端，与α1结构域相关， 形成含有脂囊的卵黄素样中间体 启动富含甘油三酯的脂蛋白的组装 粒子。~另一个需要检验的假设是，这 没有卵黄素样的中间结构是不完整的 甲氨蝶呤的结构整合2)脂质相互作用的研究 两亲性β链和片材。除了两亲性之外 螺旋，推测的两亲性β链已经在 载脂蛋白B的一级结构我们建议研究其结构 以及天然(来自载脂蛋白B-100)的脂质缔合性质 因为从头开始设计了两亲性的贝塔线束和床单。两者都有 多肽合成和分子生物学技术将用于 获得这些多肽。

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.