Apolipoprotein A-I Structure in High Density Lipoprotein

描述（由申请人提供）： 高密度脂蛋白（HDL）及其主要蛋白质成分载脂蛋白（apo）A-I可能起作用， 在预防血管中胆固醇积聚方面起着关键作用，胆固醇积聚可导致人类心血管疾病，在美国每年夺去近100万人的生命。不幸的是，人们对HDL心脏保护作用的分子基础知之甚少。详细了解这些影响的一个突出障碍是缺乏HDL中apoA-I结构的信息。我们建议测试的假设，apoA-I在球形人血浆HDL颗粒的结构是相关的，在最简单的盘状颗粒，可以在体外创建。该方法将利用重组的盘状MDL颗粒的边缘固有的几何约束，以生成这些颗粒上的apoA-I组织的高度详细的模型。使用这种结构作为基准，apoA-I区域之间的空间关系的变化将被监测，因为颗粒的复杂性从盘状系统地增加，到明确定义的重构球形颗粒，最后到分离的人HDL颗粒。两 互补的方法将用于监测颗粒上apoA-I分子内部和之间的距离参数。这些是：A）使用apoA-I的色氨酸和半胱氨酸突变体的综合电池来研究荧光能量转移，和B）应用新型质谱/肽图谱技术，该技术利用可逆硫醇交联剂来确定脂蛋白中apoA-I的各个区域的接近度。这些方法将被用来产生一个详细的“邻近地图”HDL结合apoA-I在不同的颗粒形态。这一信息将提供一个高度靶向的诱变策略，导致apoA-I的变体，可用于在体内解剖出的apoA-I的心脏保护功能的基础。

DESCRIPTION (provided by applicant): High density lipoprotein (HDL) and its major protein constituent, apolipoprotein (apo)A-I, may play critical roles in the prevention of cholesterol accumulation in blood vessels that can lead to human cardiovascular disease, which claims nearly a million lives per year in the United States. Unfortunately, relatively little is known about the molecular basis for the cardio-protective effects of HDL. A prominent obstacle in the way of a detailed understanding of these effects is the lack of information on the structure of apoA-I in HDL. We propose to test the hypothesis that the structure of apoA-I in spherical human plasma HDL particles is related to that in the simplest discoidal particles that can be created in vitro. The approach will be to take advantage of the geometric constraints inherent to the edge of reconstituted discoidal MDL particles to generate a highly detailed model of apoA-I organization on these particles. Using this structure as a benchmark, changes in spatial relationships between regions of apoA-I will then be monitored as the complexity of the particles is systematically increased from discs, to well-defined reconstituted spherical particles, and finally to isolated human HDL particles. Two complementary approaches will be used to monitor the distance parameters within and between apoA-I molecules on the particles. These are: A) the use of a comprehensive battery of tryptophan and cysteine mutants of apoA-I to study fluorescence energy transfer, and B) the application of a novel mass spectrometry/peptide mapping technique that takes advantage of reversible thiol cross-linkers to determine the proximity of various regions of apoA-I in lipoproteins. These methods will be used to generate a detailed "proximity map" of HDL-bound apoA-I in different particle morphologies. This information will provide a basis for highly targeted mutagenesis strategies resulting in apoA-I variants that can be used in vivo to dissect out the cardio-protective functions of apoA-I.