HIGH DENSITY LIPOPROTEIN STRUCTURE-FUNCTION CORRELATIONS

这项研究的长期目标是从分子角度阐明， 人高密度脂蛋白的结构与功能关系 (高密度脂蛋白)。本项目重点研究了网络的结构和功能 载脂蛋白A-I。高密度脂蛋白的主要蛋白质成分。主 我们想要检验的假设是载脂蛋白的三维结构 脂结合状态的A-I由两个结构域组成：(L)N-末端 结构紧凑、相对稳定但构象灵活的结构域， 它通过疏水锚定残基与脂质结合 两亲性(X-螺旋，并含有特异性的LCAT激活 区域；和(2)C-末端结构域，其在 溶液，将载脂蛋白A-I初始插入到脂质聚集体中，并保留 非特异性脂质结合和LCAT激活特性。 为了研究载脂蛋白A-I的结构和功能，我们将准备 定点突变体和缺失突变体，以及大的化学突变体 产生了血浆载脂蛋白A-I和重组载脂蛋白A-I的片段。这些 然后将根据(A)项对载脂蛋白A-I的纯化变体进行研究 它们的结构、稳定性及其在水溶液和水溶液中的齐聚 30%正丙醇(在载脂蛋白A-I中诱导出类天然结构)；(B) 它们与表面、脂质体中的磷脂相互作用的能力， 和重组高密度脂蛋白(RHDL)中；(C)它们在均相rHDL中的结构； (D)在定义的rHDL中，它们作为卵磷脂激活剂的功能 胆固醇酰基转移酶及其与细胞结合的介体 细胞胆固醇摄取和低密度脂蛋白；(E)其 有可能成为高分辨率结构研究的模型。各种各样的 这些方法将被用来实现这些目标，包括重组DNA 方法，在大肠杆菌中表达，蛋白质纯化，荧光，FAR 近紫外-环糊精光谱、脂结合动力学和平衡 重组人高密度脂蛋白的层析分离、电泳法分析 蛋白质片段化、交联度和rHDL值。功能界别 研究将包括确定LCAT的动力学。 我们实验室的反应，以及细胞结合和 胆固醇从细胞中流出。

The long-term goal of this research is to elucidate, in molecular terms, the structure-function relationships in human high density lipoproteins (HDL). This project focuses on the structure and function of apolipoprotein A-I. the major protein component of HDL. The main hypothesis we wish to test is that the three-dimensional structure of apo A-I in lipid-bound states consists of two domains: (l) an N-terminal domain which is compact, relatively stable yet conformationally flexible, which binds to lipid through hydrophobic anchoring residues in addition to the amphipathic (X-helices, and contains the specific LCAT activating region; and (2) a C-terminal domain which mediates oligomerization in solution, initial insertion of apo A-I into lipid aggregates, and retains nonspecific lipid binding and LCAT activating properties. To study the domain structure and functions of apo A-I we will prepare site-directed mutants and deletion mutants, as well as large, chemically produced fragments of plasma apo A-I and recombinant proapo A-I. These purified variants of apo A-I will then be investigated in terms of (a) their structure, stability, and oligomerization in aqueous solution and in 30% n-propanol (which induces a native-like structure in apo A-I); (b) their ability to interact with phospholipids on surfaces, in liposomes, and in reconstituted HDL (rHDL); (c) their structure in homogeneous rHDL; (d) their functions, in the defined rHDL, as activators of lecithin cholesterol acyltransferase and as mediators of binding to cells and of cholesterol uptake from cells and low density lipoproteins; (e) their potential as models for high-resolution structural studies. A variety of methods will be used to accomplish these aims, including recombinant DNA methods, expression in E. coli, protein purification, fluorescence, far and near UV-CD spectroscopy, lipid-binding kinetics and equilibrium approaches, chromatographic isolation of rHDL, electrophoretic analysis of protein fragmentation, cross-linking, and rHDL sizes. The functional studies will include the determination of the kinetics of the LCAT reaction in our laboratory, and a collaborative study of cell binding and cholesterol efflux from cells.