AMPHIPATHIC MOTIFS IN APOLIPOPROTEIN B

载脂蛋白 B 中的两亲基序

• 批准号: 6272738
• 负责人: NASSRIN DASHTI
• 金额: $ 17.69万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6272738
• 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（α 1、α 2 和 alpha 3) 作为独立的结构和功能单元； (二) 研究两亲性β链和/或反平行的作用 apo B 相互作用中的两亲性 β 片层 脂蛋白表面，并且〜在风险更大的子项目中，探索 参与组装的 apo B 的结构特征 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) 合理设计载脂蛋白B片段，创建并 在体外由培养细胞和完整脂蛋白表达 Core D 培养的细胞，以及 2) 产生的较短序列 通过 Core B. WHEEL 和 LOCATE 进行化学肽合成 计算机算法（项目 1，与项目 2 合作） 将用于设计突变。 为实现上述目标，两 提出了具体目标： 1) 研究合理设计的场地- apo B 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（α 1、α 2 和 alpha 3) 作为独立的结构和功能单元，并且 α 1 结构域是〜球状〜并且可以结晶。 每个 域将在培养的细胞中表达并进行研究。 (b) 我们 将检验来自 N- 的两亲性 β 链的假设 apo B 末端 18-28%，与 alpha 1 结构域相关， 形成含有脂质袋的类卵黄蛋白中间体 启动富含甘油三酯的脂蛋白的组装所需 颗粒。 ~ 另一个需要检验的假设是 如果没有脂质卵黄素样中间结构，则不完整 MTP的结构整合。2)脂质相互作用的研究 两亲性β链和片层。 除了两亲性 螺旋，假定的两亲性β链已位于 apo B的一级结构。我们建议研究该结构 以及天然（来自 apo B-100）的脂质相关特性 从头设计了两亲性β链和片层。 两个都 肽合成和分子生物学技术将被用于 获得这些肽。