AMPHIPATHIC MOTIFS IN APOLIPOPROTEIN B

项目的优先目标是：（a）检验以下假设： 载脂蛋白B的三个两亲性螺旋结构域（α 1，α 2， α 3）作为独立的结构和功能单元;（B） 研究两亲性β链和/或反平行链的作用 载脂蛋白B与 脂蛋白表面，和~在一个更危险的子项目，探索 apo B的结构特征参与了 载脂蛋白B。 研究策略包括 生物物理和生物学特性的研究：1） 合理设计的载脂蛋白B片段， 在体外由培养的细胞表达，在完整的脂蛋白中由 细胞培养物中的核心D，和2）较短的序列产生的 通过核心B进行化学肽合成。 轮子和定位 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，两个 具体目标是：1）研究合理设计的网站- apo B的定向突变体。 (a)我们将测试假设， 载脂蛋白B的三个两亲性螺旋结构域（α 1，α 2， α 3）作为独立的结构和功能单元， α 1结构域是球状的并且可以结晶。 每个 结构域将在培养的细胞中表达并进行研究。 (b)我们 将检验一个假设，即来自N- apo B末端18-28%，与α 1结构域结合， 形成含有脂质袋的卵黄脂蛋白样中间体 启动富含磷脂酰肌醇的脂蛋白的组装所需的 粒子另一个有待检验的假设是， 卵黄脂蛋白样中间结构不完整， MTP的结构整合。2）脂质相互作用的研究 两亲性β链和片。 除了两亲性 螺旋，推定的两亲性β链已被定位在 载脂蛋白B的一级结构。 我们建议研究 以及天然（来自apo B-100）的脂质缔合性质 作为从头设计的两亲性β链和片。 两 肽合成和分子生物学技术将用于 得到这些肽。

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.