HIGH DENSITY LIPOPROTEIN STRUCTURE-FUNCTION CORRELATIONS

这项研究的长期目标是从分子角度阐明， 人高密度脂蛋白结构与功能关系 （HDL）。本项目的重点是结构和功能 载脂蛋白A-I HDL的主要蛋白质成分。主要 我们希望检验的假设是载脂蛋白的三维结构 脂质结合态的A-I由两个结构域组成：（1）N-末端 结构域是紧凑的，相对稳定但构象灵活， 其通过疏水锚定残基结合到脂质上， 两亲性（X-螺旋，并含有特定的LCAT激活 区域;和（2）C-末端结构域，其介导细胞中的寡聚化， 溶液，apo A-I初始插入脂质聚集体，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的结构域结构和功能，我们将制备 定点突变体和缺失突变体，以及大的，化学上 产生血浆apo A-I片段和重组proapo A-I。 这些 然后将根据（a）研究apo A-I的纯化变体， 它们的结构、稳定性和在水溶液中的低聚， 30%正丙醇（在apo A-I中诱导天然样结构）;（B） 它们与表面上，脂质体中， （c）它们在均质rHDL中的结构; (d)在定义的rHDL中，它们作为卵磷脂活化剂的功能 胆固醇酰基转移酶和作为与细胞结合的介质以及 从细胞和低密度脂蛋白摄取胆固醇;（e）它们的 作为高分辨率结构研究的模型的潜力。 各种 将使用包括重组DNA在内的方法来实现这些目标 方法，在E. 大肠杆菌，蛋白纯化，荧光，远红外 和近紫外-CD光谱，脂质结合动力学和平衡 方法，rHDL的色谱分离，电泳分析， 蛋白质片段化、交联和rHDL大小。 功能 研究将包括确定LCAT的动力学 反应在我们的实验室，和细胞结合的合作研究， 胆固醇从细胞流出。

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.