Mechanism of initiation of lipid binding of apolipoprotein A-I

项目摘要 载脂蛋白（apo）A-I是一种多功能蛋白质，在逆转 胆固醇运输，是心脏病的重要因素。它是主要的蛋白质 高密度脂蛋白（HDL）的组成部分，通过血浆促进循环 胆固醇流出虽然尚不清楚高分辨率的结构，但广泛的生物物理 分析表明，28 kDa蛋白质由两个结构域组成，每个结构域包含 与脂质表面结合的两亲性α-螺旋。C-末端（CT）结构域包含 启动脂质结合的螺旋片段，也是负责自缔合的位点。它 是无脂质apoA-I成熟为HDL所需蛋白质的关键部分。冲突 存在关于N-末端（NT）螺旋在此过程中的作用的数据，以及精确的 CT域的螺旋段。我们最近发现CT赖氨酸残基是 对于自我结合至关重要，并且能够创建蛋白质的单体版本。到 鉴定对脂质结合和自结合起始重要apoA-I螺旋片段， 紧密相连，我们开发了一种嵌合蛋白这只奇美拉会被用来 确定apoA-I的哪些螺旋片段，包括NT和CT α螺旋，是启动 脂质结合和自缔合。鉴定CT结构域的特定氨基酸残基 在自缔合中需要的情况下，将采用定点诱变。所有的蛋白质都将 在细菌表达系统中表达，通过亲和和大小排阻纯化 色谱法，并表征了结构和功能。这项研究的结果将导致 为了更好地理解这种关键载脂蛋白的结构域， 结构功能关系，并且还可以提供高分辨率结构的机会， 使用单体apoA-I进行分析。

Project Summary Apolipoprotein (apo) A-I is a multifunctional protein with a well-established role in reverse cholesterol transport and is an important player in heart disease. It is the main protein component of high-density lipoprotein (HDL), which circulates through plasma promoting cholesterol efflux. While a high-resolution structure is not known yet, extensive biophysical analysis has suggested that the 28 kDa protein is made of two domains, each of which contain amphipathic α-helices for association with lipid surfaces. The C-terminal (CT) domain contains helical segments that initiate lipid binding, and is also the site responsible for self-association. It is a critical part of the protein needed for maturation of lipid-free apoA-I into HDL. Conflicting data exist about the role of the N-terminal (NT) helices in this process, as well as the precise helical segments of the CT domain. We have recently discovered that CT lysine residues are critical for self-association, and were able to create a monomeric version of the protein. To identify apoA-I helical segments important for initiation of lipid binding and self-association, which are closely connected, we developed a chimeric protein. This chimera will be used to identify which helical segments of apoA-I, both NT and CT α-helices, are required for initiation of lipid binding and self-association. To identify the specific amino acid residues of the CT domain required in self-association, site-directed mutagenesis will be employed. All proteins will be expressed in a bacterial expression system, purified by affinity and size-exclusion chromatography, and characterized for structure and function. The results of this study will lead to a much better understanding in the domain organization of this critical apolipoprotein, their structure function relationship, and may also provide opportunities for high-resolution structural analysis using monomeric apoA-I.